(12) United States Patent (10) Patent No.: US 8.450,069 B2 Goix Et Al

USOO8450069B2

(12) United States Patent (10) Patent No.: US 8.450,069 B2 Goix et al. (45) Date of Patent: May 28, 2013

(54) HIGHLY SENSITIVE BIOMARKERPANELS 5,645,702 A 7, 1997 Witt et al. 5,653,859 A 8, 1997 Parton et al. 0. Y8 5,653,939 A 8, 1997 Hollis et al. (75) Inventors: E.obert Puskas, RER Manchester, v. (US);G. 5,681,7515,658.413 A 10/19978/1997 KaltenbachBegg et al. et al. John Todd, Lafayette, CA (US); 5,682,038 A 10/1997 Hoffman Richard Livingston, Webster Groves, 5,716,825 A 2/1998 Hancocket al. MO (US); Douglas Held, Ballwin, MO 37:6 A Eal (US); Sara Le, Berkeley, CA (US) 5,755,942 A 5/1998 ZanzucchiaC ( a. et al. 5,770,029 A 6/1998 Nelson et al. (73) Assignee: Singulex, Inc., Alameda, CA (US) 5,793.485 A 8, 1998 GE, al 5,795,158 A 8, 1998 Warinner (*) Notice: Subject to any disclaimer, the term of this 5,795,758 A 8/1998 Gentry et al. patent is extended or adjusted under 35 38.7 A s 3. E. tal U.S.C. 154(b) by 0 days. 5,858,195 A 1/1999 Ramsey1gen et al. 5,863,801 A 1/1999 Southgate et al. (21) Appl. No.: 12/795,414 5,925,533. A 7/1999 Doth et al. 5,949,532 A 9, 1999 Schrofetal. (22) Filed: Jun. 7, 2010 5,955,028 A 9, 1999 Chow 5,989.402 A 1 1/1999 Chow et al. O O 5.999,250 A 12/1999 Hairston et al. (65) Prior Publication Data 6,033,628 A 3/2000 Kaltenbach et al. US 2011/0003707 A1 Jan. 6, 2011 6,041,515 A 3/2000 Ally et al. 6,049,380 A 4/2000 Goodwin et al. O O 6,071.478 A 6/2000 Chow Related U.S. Application Data 6,114, 180 A 9, 2000 Doth et al. (60) Provisional application No. 61/185,194, filed on Jun. 6,131, 101 A 10/2000 Maitino et al. 8, 2009. (Continued) (51) Int. Cl FOREIGN PATENT DOCUMENTS we DE 3720844 A1 1, 1989 GOIN 33/53 (2006.01) WO WO90, 10876 A1 9, 1990 (52) U.S. Cl. USPC ...... 435/7.1:436/518 (Continued) (58) Field of Classification Search None OTHER PUBLICATIONS See application file for complete search history. Verrier et al. (Cardio. Res 1996 vol. 31, p. 181-211).* Deliargyris et al. (Am J. Cardiology 2000 vol. 86, p. 913–918).* (56) References Cited Upatnieks et al.: "A kilohertz frame rate cinemagraphic PIV system U.S. PATENT DOCUMENTS for laboratory-scale turbulent and unsteady flows.” Experiments in Fluids (2002), vol. 32, pp. 87-98. 4,071,298 A 1/1978 Falconer Van Orden et al.: "Single-molecule identification in flowing sample 4,172,227 A 10/1979 Tryer et al. 4,243,318 A 1, 1981 Stohr streams by fluorescence burst size in intraburst fluorescence decay 4,251,733. A 2f1981 Hirleman rate.” Anal. Chem. (1998) vol. 70, pp. 1444-1451. 4,452,773. A 6/1984 Molday 4,521,733. A 6/1985 Bottomley (Continued) 4,768,879 A 9, 1988 McLachlan et al. 4,770,183 A 9, 1988 Groaman et al. 4,793,705 A 12/1988 Shera Primary Examiner — Jacob Cheu 4,927,265 A 5, 1990 Brownlee 4,972,265 A 11, 1990 Tanaka et al. (74) Attorney, Agent, or Firm — McDonnell Boehnen 4,979,824 A 12/1990 Mathies et al. Hulbert & Berghoff LLP 5,002,389 A 3, 1991 Benser 5,041,733. A 8/1991 Noguchi et al. 5,094,594 A 3, 1992 Brennan (57) ABSTRACT 5,108,179 A 4/1992 Myers 5,138,170 A 8/1992 Noguichi et al. 5,209,834 A 5, 1993. Shera Cardiovascular disease, e.g., congestive heart failure, is often 5,230,997 A 7, 1993 Frenkel first diagnosed after the onset of clinical symptoms, eliminat 5,269,937 A 12/1993 Dollinger et al. ing potential for early intervention. The invention provides a 5,274,240 A 12/1993 Mathies et al. multi-marker immunoassay, including cardiac pathology and 5,290,834 A 3, 1994 Kadota et al. 5,385,707 A 1/1995 Miltenyi et al. vascular inflammation biomarkers, yielding a more sensitive 5,480,614 A 1/1996 Kamalhori assay for early detection of CHF in plasma. A panel consist 5,540,494 A 7/1996 Purvis, Jr. et al. ing of cardiac pathology (cTnI, BNP) and vascular inflam 5,543,838 A 8, 1996 Hosier et al. 5,547,849 A 8, 1996 Baer et al. mation (IL-6, TNFO, IL-17a) biomarkers provided a sensi 5,571,410 A 1 1/1996 Swedberg et al. tivity of 94% for association with CHF. 5,603,351 A 2f1997 Cherukuri et al. 5,605,662 A 2f1997 Heller et al. 5,633,503 A 5, 1997 Kosaka 19 Claims, 43 Drawing Sheets US 8,450,069 B2 Page 2

U.S. PATENT DOCUMENTS 2009/0171590 A1 7/2009 Puskas et al. 2009, O197344 A1 8/2009 Villard-Saussine et al. 6,132,580 A 10, 2000 Mathies et al. 2010/0173321 A1* 7, 2010 Hamm et al...... 435/71 6,140,048 A 10, 2000 Muller et al. 2010, O255518 A1 10, 2010 Goix et al. 6,143,152 A 11, 2000 Simpson et al. 2010/0329929 A1 12/2010 Goix et al. 6,177.277 B1 1, 2001 Soini 2011/01 11524 A1 5, 2011 Goix et al. 6,208,815 B1 3, 2001 Seidel et al. 6,211.955 B1 4, 2001 Basiji et al. FOREIGN PATENT DOCUMENTS 6,242.266 B1 6, 2001 Schleifer et al. 6,249,341 B1 6, 2001 Basiji et al. WO WO99,26067 A1 5, 1999 6,280,960 B1 8, 2001 Carr WO WO 99.40416 A1 8, 1999 6,309,886 B1 10, 2001 Ambrose et al. WO WO99,54497 A1 10, 1999 6,338,746 B1 1, 2002 Detricket al. WO WO99.55461 A1 11, 1999 6,355,420 B1 3, 2002 Chan WO WO 2004/059293 A2 T 2004 6,361,671 B1 3, 2002 Mathies et al. WO WO 2004/059293 A3 3, 2005 6,372,185 B1 4, 2002 Shumate et al. WO WO 2005/089524 9, 2005 6,386.219 B1 5, 2002 Barth et al. WO WO 2005/0895.24 A2 9, 2005 6,388,746 B1 5, 2002 Eriksson et al. WO WO 2005/0895.24 A3 4/2006 6,394,305 B1 5, 2002 Sydlosky et al. WO WO 2006/036182 A2 4/2006 6,403,338 B1 6, 2002 Knapp et al. WO WO 2008/048371 4/2008 6,403,947 B1 6, 2002 Hoyt et al. 6,473, 176 B2 10, 2002 Basiji et al. OTHER PUBLICATIONS 6,482,648 B2 11, 2002 Doth et al. 6,495,104 B1 12, 2002 Unino et al. Van Wissen et al.: “Differential hs-CRP reduction in patients with 6,506,609 B1 1, 2003 Wada et al. familial hypercholesterolemia treated with aggressive or conven 6,532,067 B1 3, 2003 Chang et al. tional statin therapy.” Atherosclerosis (Dec. 2002) vol. 165, No. 2, pp. 6,533,553 B2 3, 2003 Caren 361-366. 6,537.437 B1 3, 2003 Galambos et al. 6,554,744 B2 4, 2003 Schmidt Von Zur Muhlen et al.: "Evaluation of Urine Proteome Pattern Analy 6,582,903 B1 6, 2003 Rigler et al. sis for Its Potential to Reflect Coronary Artery Atheroscierosis in 6,599.436 B1 T/2003 Matzke et al. Symptomatic Patients' J. Proteom. Res. (2009) V.8 pp. 335-345. 6,608,680 B2 8, 2003 Basiji et al. Wabuyele et al.: "Single molecule detection of double-stranded DNA 6,623,613 B1 9, 2003 Mathies et al. in poly(methylmethacrylate) and polycarbonate microfluidic 6,624,785 B2 9, 2003 Poliak et al. devices.” Electrophoresis (2001) vol. 22, pp. 3939-3948. 6,689,323 B2 2, 2004 Fisher et al. Wallace et al.: "Serum Troponins as Biomarkers of Drug-Induced 6,710,871 B1 3, 2004 Goix 6,749,734 B1 6, 2004 Simpson et al. Cardiac Toxicity.” Toxicologic Pathology, (2004) vol. 32, pp. 106 6,766,817 B2 T/2004 Da Silva 121. 6,783,992 B2 8, 2004 Robotti et al. Willneff: "A spatio-temporal matching algorithm for 3D particle 6,802.342 B2 10, 2004 Fernandes et al. tracking velocimetry: a dissertation submitted to the Swwiss Federal 6,811,668 B1 11, 2004 Berndt et al. Institute of Technology Zurich for the degree of Doctoral of Techni 6,816,257 B2 11, 2004 Goix cal Sciences.” (Sep. 2003) Diss. Eth No. 15276 (abstract). Available 6,918.404 B2 7/2005 Da Silva at http://e-collection.ethbib.ethZ.ch/ecol-pool/diss/abstracts/p15276. 6,977,305 B2 12, 2005 Leung et al. 6,991,907 B1 1, 2006 Buechler et al. pdf. 7,066,586 B2 6, 2006 Da Silva Wu et al.: Development and preliminary clinical validation of a high 7,074,194 B2 T/2006 Crosby sensitivity assay for cardiac troponin using a capillary flow (single 2002fOO3O812 A1 3, 2002 Ortyn et al. molecule) fluorescence detector. Poster presented at Oak Ridge con 20O2/O123059 A1 9, 2002 Ho ference. Apr. 22-22, 2006; 52:2157-2159. 2002.0167665 A1 11, 2002 Yeung et al. 2003/0O2.9995 A1 2, 2003 Mullins et al. Wu et al.: “Development and Preliminary Clinical Validation of a 2003/OO78737 A1 4, 2003 Keys et al. High Sensitivity Assay for Cardiac TropoinUsing a Capillary Flow 2003.0109420 A1 6, 2003 Valkirs et al. (Single Molecule) Fluorescence Detector.” Clinical Chemistry 2003/O124592 A1 T/2003 Puskas (2006) vol. 52, pp. 2157-2159. 2003/0222007 A1 12, 2003 Gu et al. Young: Singules Developing Troponin Test for earlier detection of 2004O166514 A1 8, 2004 Puskas et al. AMI. Medical Device Daily Dec. 13, 2006. 2004/0214211 A1 10, 2004 Gilmanshin et al. Zetheli et al.:"Troponin I as a predictor of coronary heart disease and 2004/0219509 A1 11, 2004 Valkirs et al. 2004/0219604 A1 11, 2004 Eriksson et al. morality in 70-year-old mend: a community-based cohort study.” 2005, OO64523 A1 3, 2005 Wu Circulation (Feb. 28, 2006) vol. 113, No. 8, pp. 1071-1078. 2005, 0164205 A1 7/2005 Puskas Zhu et al.: “Fluorescence multiplexing with time-resolved and spec 2005/0221408 A1 10, 2005 Nalefski et al. tral discrimination using a near-IR detector.” Anal. Chem (2003) vol. 2005/0272O54 A1 12, 2005 Cargill et al. 75, pp. 2280-2291. 2006,0003333 A1 1, 2006 Puskas Zimmerli et al.: “Urinary Proteomic Biomakers in Coronary Artery 2006,0004.188 A1 1, 2006 Leung et al. 2006, OO789.15 A1 4, 2006 Fuchs et al. Disease” Mol. Cell Proteomics (Feb. 2008) vol. 7, No. 2 pp. 290-298. 2006, OO78998 A1 4, 2006 Puskas et al. First Published on Oct. 19, 2007. 2006/0099638 A1 5, 2006 Leung et al. U.S. Appl. No. 60/613,881, entitled “Continuous wave single particle 2006.0160209 A1 T/2006 Larson et al. detector.” filed Sep. 28, 2004, Puskas. 2006/02287.47 A1 10, 2006 Fuchs et al. U.S. Appl. No. 60/624,785, entitled “Sandwich assay for detection of 2007/011 1316 A1 5/2007 Shi et al. individual molecules' filed Oct. 29, 2004, Puskas. 2007/0196371 A1 8, 2007 Kuestner et al. U.S. Appl. No. 10/718,194, entitled: “Preparation of defined highly 2007/O259377 A1 11/2007 Urdea et al. 2008.OOO3685 A1 1, 2008 Goix et al. labeled probes.” filed Nov. 19, 2003, Puskas, Robert Steven. 2008, OO64113 A1 3, 2008 Goix et al. U.S. Appl. No. 10,720,047, entitled: “Charge and mass tags for 2008. O158543 A1 T/2008 Puskas et al. detection and analysis.” filed Nov. 19, 2003, Puskas, Robert Steven. 2008.0171352 A1 T/2008 Goix et al. U.S. Appl. No. 1 1/048,660, entitled: "System and Methods for 2008.O2554.71 A1* 10, 2008 Naghaviet al...... 600,549 Sample Analysis.” filed Jan. 28, 2005, Puskas, Robert S. et al. 2008/026.1242 A1 10, 2008 Goix et al. U.S. Appl. No. 1 1/767,196, entitled: “System and Method for Sample 2008.O3OO798 A1* 12, 2008 McDevitt et al...... 7O2/19 Analysis.” filed Jun. 22, 2007, Goix, Philippe J. et al. US 8,450,069 B2 Page 3

U.S. Appl. No. 1 1/784, 186, entitled "Methods and Compositions for Mair, Johannes: “Cardiac troponin I and Troponin T: Are enzymes Highly Sensitive Analysis of Markers.” filed Apr. 4, 2007, Goix, still relevant as cardiac markers?' Clinica Chimica Acta, vol. 257 Philippe J. (1997) NR. 1, pp. 99-115. U.S. Appl. No. 1 1/830,762, entitled "Methods and Compositions for Missov et al.: Circulating Cardiac Troponin I in Severe Congestive Highly Sensitive Detection of Molecules.” filed Jul. 30, 2007, Goix, Heart Failure, Circulation, 96:2953-2958 (1997). Phillippe J. Nalefski et al.: "Single-molecule counting of macromolecular com U.S. Appl. No. 1 1/838,114, entitled: "Systemand Method for Sample plexes in real time: a novel approach to quantify transcription fac Analysis.” filed Aug. 13, 2007, Puskas, Robert S. et al. tor—DNA and antibody-antigen interactions.” FASEB Journal U.S. Appl. No. 12/060,997, entitled: "Methods and Compositions for (2004) vol. 18, No. 8: C176. Highly Sensitive Analysis of Markers.” filed Apr. 2, 2008, Goix, Nguyen et al.: "Detection of single molecules of phycoerythrin in Philippe J. et al. hydrodynamically focused flows by laser-induced fluorescence.” U.S. Appl. No. 12/276.277, entitled: “System and Method for Sample Anal. Chem. (1987) vol. 59, pp. 2158-2161. Analysis.” filed Nov. 21, 2008, Puskas, Robert S. et al. Oh et al.: “E of a dual monoclonal solid phase and a polyclonal U.S. Appl. No. 12/731,500, entitled "Methods and Compositions for detector to create an immunoassay for the detection of human cardiac Highly Sensitive Detection of Molecules.” filed Mar. 25, 2010, Goix, troponin I.” Clin. Biochem. (2000) vol. 33, pp. 255-262. Phillippe J. Ohman et al.: “Cardiac troponin Tlevels for risk stratification in acute Guide to Labeling Antibodies with Alexa Fluor Dyes 24-28 (2004). mycardial ischemia.” The New England Journal of Medicine (1997) Haab et al.: "Single molecule florescence burst detection of DNA vol. 335, pp. 1333-1341. fragments separated by capillary electrophoresis.” Anal Chem. Panchuk-Voloshina et al.: "Alexa dyes a series of new fluorescent (1995) vol. 67, pp. 3523-3260. dyes that yield exceptionally bright phostotable conjugates' J. Haab et al.: "Single-molecule detection of DNA separations in Histochem Cytochem (Sep. 1999) vol. 47 No.9 pp. 1179-1188. microfabricated capillary electrophoresis chips employing focused Panteghini et al.: "Evaluation of imprecision for cardiac troponin molecular stream.” Anal Chem. (1999) vol. 71, pp. 5137-5145. assays at low range concentrations. Clinical Chemistry (2004) vol. Haugland Molecular probes handbook of fluorescent probes and 50, pp. 327-332. research product Ninth Edition Molecular Probes Inc. (Table of Con Panteghini: “Role and importance of biochemical markers in clinical tents only) 2002. cardiology.” European Heart Journal (2004) vol. 25, pp. 1187-1196. Huse et al.: "Application of a filamento phage pVll fion protein Panteghini: “The interfering component in cardiac troponin I system Suitable for efficient production, Screening and mutagenesis immunoassays: need for further experimental evidence.” Clin Chem. off(ab)antibody fragments.” Journal ofImmunology (Dec. 15, 1992) (2004) vol. 50, pp. 676-677. vol. 149, No. 12, pp.39-14-20. Park. Addressing Unmet Needs in Assay Development. Medical Hirst et al.: “Production of plasma selectively depleted in fibrinogen Device Link 1-4, Mar. 2007. by affinity chromatography.” Journal of Clinical Pathology (1991) Pecket al.: "Single-molecule fluorescence detection: autocorrelation vol. 44, pp. 306–308. criterion and experimental realization with phycoerythrim.” Proc. Hublet al.: "Evaluation of the Architect Stat Troponin-I assay.” Natl. Acad. Sci. USA (1989) vol. 86, pp. 4087-4091. Clinical Laboratory Publications (2005) vol. 51, pp. 251-255. Phillips et al.: "Application of single molecule technology to rapidly Kaiser et al.: "Capillary electrophoresis coupled to mass spectrom map long DNA and study the conformation of stretched DNA” eter for automated and robust polypeptide determination in body Nucleic Acids Research vol. 33 pp. 5829-5837 (2005). fluids for clinical use” Electrophoresis (2004) V. 25 pp. 2044-2055. Rigler: “Fluorescence correlations single molecule detection and Katus et al.: “Higher sensitivity troponin assyas: Quo vadis?” Eur large number Screening Applications in Biotechnology J. Heart J. (Jan. 2009), vol. 30, No. 2, pp. 127-128. Biotechnol. (1995) vol. 41 (2-3) pp. 177-186. Keller et al.: “Analytical applications of single-molecule detection.” Sabatine et al.: “Detection of acute changes in circulating troponin in Analytical Chemistry (2002) vol. 74, pp. 317A-324A. the setting of transient stress test-induced myocardial ischaemia ing Kemp et al.: “Biochemical markers of myocardial injury.” British an ultrasensitive assay: results from TIMI 35.” CUR Heart J. Jan. Journal of Anaesthesia (2004) vol. 93, pp. 63-73. 2009 (ePub Nov. 8, 2008). Klee: “Human anti-moe antibodies.” Arch Pathol Lab Med. (2000) Sato et al.: “Biochemical markers of myocyte injury in heart failure.” vol. 124, pp. 921-923. Heart (British Cardiac Society) (2004) vol. 90, pp. 1110-11 13. Koehnlein et al.: “Increased cardiac troponin T and C-reative protein Sauer et al.: “Detection and identification of individual antigen mol levels in end-stage renal disease are associated with obstructive sleep ecules in human serum with pulsed semiconductor lasers. Appl. apnea.” Clin Nephrol (Jan. 2007) vol. 71, No. 1, pp. 50-58. Phys. B. (1997) vol. 65, pp. 427-431. Koerbin et al. The Comparative analytical performance of four Schiffer et al.: “High resolution proteome/peptidome analysis of troponin I assays at low concentration Ann Clin. Biochem. 42:19-23 body fluids by capillary electrophoresis coupled with MS” Proteom (2005). ics (2006) V. 6 pp. 5615-5627. Larue et al.: "Cardiac-Specific Immunoenzymaometric Assay of Schulz et al.: “Cardiac troponin I: A potential marker of exercise Troponin I in the Early phase of Acute Myocardial Infarction.” Clini intolerance in patients with moderate heart failure.” American Heart cal Chemistry (1993) vol.39, pp.972-979. Journal (2002) vol. 144, pp. 351-358. LeCaptain et al.: “Two-beam fluorescence cross-correlation spec Shera et al.: “Detection of single fluorescent molecules.” Chemical troscopy in an electrophoretic mobility shift assay.” Anal. Chem. Physics Letters (1990) vol. 174, pp. 553-557. (2002) vol. 74, pp. 1171-1176. Shortreed et al.: “High-throughput single-molecule DNA screening LeCaptain et al.: "Characterization of DNA-protein complex by cap based on electrophoresis.” Anal. Chem. (2000) vol. 72, pp. 2879 illary electrophoresis—single molecule fluorescence correlation 2885. spectroscopy.” Analyst (2001) vol. 126, pp. 1279-1284. Sidransky: “Emerging molecular markers of cancer, Nature Lexington Medical Center. Mycardial infarction redefined. Reviews: Cancer (2002) vol. 2, pp. 210-219. NewsPath, May 2001. Soper et al.: “Photon burst detection of single near-infrared fluores Li et al.: “Ultrasensitive coincidence fluorescence detection of single cent molecules.” Anal. Chem. (1993) vol. 65, pp. 740-747. DNA molecules.” Anal. Chem. (2003) vol. 75, pp. 1664-1670. Soper et al.: "Single-molecule detection in the near-IR using Loscher et al.: "Counting of single protein molecules at interfaces continuos wave diode laser excitation with an avalanche photon and application of this technique in early-stage diagnosis.” Anal. detector.” Applied Spectroscopy (1998) vol. 52, pp. 1-6. Chem (1998) vol. 70, pp. 3202-3205. Stiegler et al.: “Lower cardiac tropoinin T and I results in heparin Lucey et al.: “Type 1 and type 2 cytokine dysregulation in human plasma than in serum.” Clinical Chemistry, (2000) vol. 46, pp. 1338 infectioneoplastic and inflammatory diseases.” Clinical Biology 1344. Reviews (1996) vol. 9, pp. 532-562. Tanaka et al.: “Protein and polymer analyses up to m/z. 100 000 by Ma et al.: "Single-molecule immunoassay and DNA diagnosis.” laser ionization time-of-flight mass spectrometry.” Rapid Commun. Electrophoresis (2001) vol. 22, pp. 421-426. Mass. Spect. (1988) vol. 2, pp. 151-153. US 8,450,069 B2 Page 4

Thomas, Ma et al.: "A review of troponin assay performance in Castro et al.: "Single molecule detection: applications to ultrasensi Wales: can the same (method-dependent) decision limits be used in tive biochemical analysis.” Applied Optics (1995) vol. 34, pp. 3218 different sites?” Ann. of Clinical Biochemistry, British Medical 3222. Association, London, GB, vol. 42, No. 5, Sep. 1, 2005, pp. 351-356. Castro et al.: "Single-molecule detection of specific nucleic acid Todd, J. et al.: “Ultrasensitive flow-based immunoassays ing single sequences in unamplified genomic DNA.” Anal. Chemistry (1997) molecule counting.” Clinical Chemistry, American Association for vol. 69, pp. 3915-3920. Clinical Chemistry, Washington DC, vol. 53, No. 11, Nov. 1, 2007. Castro et al.: "Single-molecule electrophoresis.” Anal. Chemistry pp. 1990-1995. (1995) vol. 67, pp. 3181-3186. Achar et al.: “Diagnosis of acute coronary syndrome.” American Castro et al.: “Ultrasensitive direct detection of a specific DNA Family Physician (2005) vol. 72, pp. 119-1265. sequence of Bacillantracis in solution.” The Analyst (2000) vol. 125, Adams et al.: "Cardiac troponin I.A. marker with high specificity for pp. 9-11. cardiac injury.” Circulation (1993) vol. 88, pp. 101-106. Cayley: "Diagnosing the case of chest pain.” American Family Phy Adams et al.: "Comparable detection of acute myocardial infarction sician (2005) vol. 72, pp. 2012-2021. by creatine kinase MB isoenzyme and cardiac troponin I. Clinical Chan et al.: “DNA mapping using microfluidic stretching and single Chemistry (1994) vol. 40, pp. 1291-1295. molecule detection of fluorescent site-specific tags. Genome Research (2004) vol. 14, pp. 1137-1146. Al-Awadhi et al. Singapore Med. J. (2007) vol. 48, pp. 847-849. Chen et al.: "Single-molecule detection in capillary electrophoresis: Alexa Fluor Succinimidyl Esters. Invitrogen. Revised Jan. 4, 2006; molecular shot noise as a fundamental limit to chemical analysis.” 1-5. Anal. Chemistry (1996) vol. 68, pp. 690-696. Alexa Fluor Dyes Handbook. Simply the Best and Brightest: Fluo Cohen et al.: “The renal TGF-beta system in the db.db mouse model rescent Dyes and Conjugates. Invitrogen. Copyright 2005. Molecular of diabetic nephropathy' Exp. Nephrol. (1998) vol. 6 pp. 226-233. Probes. 1-33. Colonna: “TREMS in the immune system and beyond.” Nature Ambrose et al.: "Single molecule fluorescence spectroscopy at ambi Reviews: Immunology (2003) vol. 3, pp. 445-453. ent temperature.” Chemical Reviews (1999) vol. 99, pp. 22929 Csiro Atralia, Image motion, tracking and registration. Available at 22956. http://www.cmis.csiro.au/IAP/Motion. Accessed Jan. 24, 2005. Anazawa et al.: “Electrophoretic quantitative of nucleic acids without D'Antoni et al. Rapid quantitative analysis using a single molecule amplification by single molecule imaging.” Anal. Chem (2002) vol. counting Anal. Chem. 352:97-109 (2006). 74, pp. 5033-5038. Diderholm et al.: “The prognostic and therapeutic implications of Antman et al.: "Cardiac-specific troponin Ilevels to predict the risk of increased troponin T levels and ST depression in unstable coronary mortality in patients with acute coronary syndromes. The New artery disease: the FRISC II invasive tropoinin Telectrocardiogram England Journal of Medicine (1996) vol. 335, pp. 1342-1349. Substudy.” American Heart Journal (2002) vol. 143, pp. 760-767. Apple et al.: "The diagnostic utility of cardiac biomarkers in detect Dovichi et al.: “Laser-induced fluorescence of flowing samples as an ing myocardial infarction.” Clinical Cornerstone (2005) vol. 7, pp. approach to single-molecule detection in liquids.” Anal. Chem. S25-S30. (1984) vol. 56, pp. 348-354. Apple et al.:"Validation of the 99th Percentile Cutoff Independent of Dunbar et al. Quantitative multiplexed detection of bacterial patho Assay Imprecision (CV) for Cardiac Troponin monitoring for Ruling gens: DNA and protein applications of Luminex LabMap system J. out Myocardial Infarction.” Clinical Chemistry (2005) vol. 51, pp. Microbiol Methods. 2003: 53(2): 245-252. 2198-2200. Eder et al.: “Transforming growth factor-beta1 and beta2 in serum Babulin et al.:"Troponin: the biomarker of choice for the detection of and urine from patients with bladder carcinoma” The J. of Urology cardiac injury.” Canadian Medical Association Journal (2005) vol. (1996) vol. 156 pp. 953-957. 173, pp. 1191-1202. Effenhauser et al.: “Integrated capillary electrophoresis on flexible Becker et al.: "Three-dimensional photogrammetric particle-track silicone microdevices: analysis of DNA restriction fragments and ing velocimetry.” Preparing for the Future (2005) vol. 5, No. 3; detection of single DNA molecules on microchips.” Anal. Chem. available at http://esapubesrinesa.it/pff pffvišn3/beckvišnc.htm(7 (1997) vol. 69, pp. 3451-3457. pages). Eryoletal. “Should Troponin-TBe Assessed During Exercise Stress Testing in Patients with Stable Angina Pectoris?” Anadolu Kardiyol Bieschke et al.: “Ultrasensitive detection of pathological prion pro Der. (2002) vol. 2, pp. 132-137. tein aggregates by dual-color scanning for intensely fluorescent tar Eskelinen et al.: "A new tumor marker MCA in breast cancer diag gets.” Pro. Natl. Acad. Sci. (2000) vol.97, pp. 5468-5473. nosis' Anticancer Res. (1988) vol.8 pp. 665-668. Borrebaeck, C. Editor. Antibody Engineering. Second Edition. 1995 Etzionietal.:"The case for early detection.” Nature Reviews: Cancer Oxford University Press, Oxford. (2003) vol. 3, pp. 243-252. Bouchon et al.: "Cutting edge: inflammatory responses can be trig Ferrieres et al.: “Human cardiac troponin I: precise identification of gered by TREM-1 a novel receptor expressed on neutrophils and antigenic epitopes and prediction of secondary structure.” Clinical monocytes.” The Journal of Immunology (2000) vol. 164, pp. 4991 Chemistry (1998) vol. 44, pp. 487-493. 4995. Fister et al.: "Counting single chromphore molecules for ultrasensi Braunwald et al.:"ACC/AHA 2002 guideline update for the manage tive analysis and separations on microchip devices.” Anal. Chem. ment of patients with unstable angina and non-ST-segment elevation (1998) vol. 70, pp. 431-437. mycardial infarction—A report of the American College of Cardiol Gaze et al.: “Cardiac Troponins as Biomarkers of Drug and Toxin ogy/American Heart Association task force on practice guidelines Induced Cardiac Toxicity and Cardioprotection.” Expert Opin. Drug (Committee on the Management of Patients with Unstable Angina).” Metabl. Toxocol. (2005) vol. 1, pp. 715-725. American College of Cardiology and the American Heart Associa Giannitsis et al.: “Admission tropoinin T level predicts clinical out tion (2002). comes, TIMI flow, and mycardial tissue perfion after primary Brinkmeier et al.: “Two-beam cross-correlation: a method to charac percutaneo intervention for acute ST-segment elevation mycoardial terize transport phenomena in micrometer-sized structures.” Anal. infarction.” Circulation (2001) vol. 104, pp. 630-635. Chem. (1999) vol. 71, pp. 609-616. Gibot et al.: “Soluble triggering receptor expressed on muyeloid cells Buisson et al.: “Biochemical markers of my cardial injury.” Available and the diagnosis of pneumonia.” The New England Journal of Medi at http://ww.ampath.co.za/Documents/biochemicalMarkers.pdf. cine (2004) vol. 350, pp. 451-458. Accessed Sep. 20, 2007. Glenn Research Center NASA particle Imaging Velocimetry. Avail Castro et al.: “Fluorescence detection and size measurement of single able at http://www.grc.nasa.gov/www/Optlinstripiv/background. DNA molecules.” Anal. Chem. (1993) vol. 65, pp. 849-852. htm and associated web pages. Accessed Jan. 26, 2005. US 8,450,069 B2 Page 5

Goix. Fulfilling the promise of biomarkers in drug discovery and Guide to Amine-Reactive Probes. Reviese Oct. 13, 2005. development, Drug Discovery + International, 6-7, Apr./May 2007. Zen et al., “Analysis of Circulating Apoptosis Mediators and Goix. Slides from presentation at clinical biomarkers Summit Proinflammatory Cytokines in Patients With Idiopathic Hypertrophic Coronado, CA, Mar. 29-31, 2006. Golde: 'Alzheimer disease therapy: can the amyloid cascade be Cardiomyopathy Comparison Between Nonobstructive and Dilated halted?” The Journal of Clinical Investigation (2003) vol. 11, pp. Phase Hypertrophic Cardiomyopathy' Int Heart J. 46(2):231-44 11-18. (2005). Guenard et al.: “Two-channel sequential single-molecule measure ment.” Anal. Chem. (1997) vol. 69, pp. 2426-2433. * cited by examiner U.S. Patent May 28, 2013 Sheet 1 of 43 US 8.450,069 B2

U.S. Patent May 28, 2013 Sheet 2 of 43 US 8.450,069 B2

3. s

U.S. Patent May 28, 2013 Sheet 3 of 43 US 8.450,069 B2

U.S. Patent May 28, 2013 Sheet 4 of 43 US 8.450,069 B2

100 urn

FG. 2B U.S. Patent May 28, 2013 Sheet 5 of 43 US 8.450,069 B2

U.S. Patent May 28, 2013 Sheet 6 of 43 US 8.450,069 B2

FIG. 3B U.S. Patent May 28, 2013 Sheet 7 of 43 US 8.450,069 B2

Physiological Markers Molecular Markers (EKG, stress test, etc) (cholesterol, etc)

Biomarkers Genetic Markers (cytokines, etc) (ApoE, etc)

1 ... 1 ...

Sensitive Detection CTn Or VEGF

Detection or Monitoring 1) Disease progression 2) Disease recurrence 3) Risk assessment 4) Therapeutic efficacy 5) Surgical efficacy 6) etc Figure 4 U.S. Patent May 28, 2013 Sheet 8 of 43 US 8.450,069 B2

Server Assay Results (e.g., trence (e.g., internet, ab) digital media) Hare

Client WorkStation (e.g. doctor's

Figure 5 U.S. Patent May 28, 2013 Sheet 9 of 43 US 8.450,069 B2

9?un61-I

U.S. Patent May 28, 2013 Sheet 10 of 43 US 8.450,069 B2

4000's s 350c toopi sailple .0D o.77 pg/ml. ye 325.8 x i. 20.94 3 R logg87

2 s

iocol St. & Fajafe LCE 3.232 girl Soo- y - 136 - 2 R - e.g.988 reserversessessessey's swww.rsssssssor rissant sessssssssssssssssssessessive-wrwrver-ray see...... -asses...wrwr. s .

y is 0.8.x + o. Rs o.99

a. 6 3. Expected pg/ml FIG. 7B U.S. Patent May 28, 2013 Sheet 11 of 43 US 8.450,069 B2

U.S. Patent May 28, 2013 Sheet 12 of 43 US 8.450,069 B2

6?unfil

tuffsid 'Nei pensee N U.S. Patent May 28, 2013 Sheet 13 of 43 US 8.450,069 B2

2% N

+§§ +|+|- 0),?un61-I

uruguodou.6d

U.S. Patent May 28, 2013 Sheet 14 of 43 US 8.450,069 B2

???Jnfil

AoLianbei U.S. Patent May 28, 2013 Sheet 15 of 43 US 8.450,069 B2

S2s

s 3 3 33 S. S O918% U.S. Patent May 28, 2013 Sheet 16 of 43 US 8.450,069 B2

i 8

set is baty. U.S. Patent May 28, 2013 Sheet 17 of 43 US 8.450,069 B2

al t E. we wif 3 &N 8N &ran Swas is eus eate U.S. Patent US 8.450,069 B2

?un61-)G?. U.S. Patent May 28, 2013 Sheet 19 of 43 US 8.450,069 B2

9),aun61-I U.S. Patent May 28, 2013 Sheet 20 of 43 US 8.450,069 B2

Strikiye A.

Sport

ow Rare is Standard we B. it y’s 42x+R. Rasts

s is tigfre

Figures 17A & B

U.S. Patent May 28, 2013 Sheet 23 of 43 US 8.450,069 B2

Sh mur w rh.. a luld all had a he math

Tuffôd[g-T]] HL),aun61-I

bar-l-m------0004 eufS NWW U.S. Patent US 8.450,069 B2

to co O SunOO pezieuoN 81eun61-)

OZ 00|| SunOO peZeuiON SunOO pezieu.JON U.S. Patent May 28, 2013 Sheet 25 of 43 US 8.450,069 B2

hVEGF 165 - Full Range Standard Curve

O O O

1CO

y = 1,03x + 0.01 10 R = 0.99

O O 1OO 1OOC Expected hVEGF pg/ml

F.G. 19A

Singulex' Human VEGF Microparticle Assay 3 5 Small Range Standard Curve

2 5

s y = 0.99x + 0.06 R* = 0.99

O 5 10 15 2O 25 3D 35 Expected hVEGF pg/ml F.G. 19B U.S. Patent May 28, 2013 Sheet 26 of 43 US 8.450,069 B2

20 Trror------ressessess career swearners------...------, y = 0.98x + 3.3 R2 = 1 1.

800 G l O E

O O OO O BOO 1OOO 12 Expected rm WEGFpg|ml

FG. 20A

7 ------...... rssssssssssssss-cross-ross---assis larvaxws riversarrass----. arris irrv wt vis's stress arrestrass

y = 0.95x + 2.1 SO R2 =

S s P L

3 C

O 10 30 A. 50 B 70 Expected m WEGF, p. giml

FG 20B U.S. Patent May 28, 2013 Sheet 27 of 43 US 8.450,069 B2

2 Singulex R&D ELISA

10 -

ETDA plasma heparin plasma

FIG 21 U.S. Patent May 28, 2013 Sheet 28 of 43 US 8.450,069 B2

MDA-MEB-231

BOO

700

SOO

5 O O

4. O O Singutex R&D 3 O O

2OO

10 2 cell lysate serun free media spent media FG. 22A

HT-29

9000 8000 OOO 5 Singulex OOO &R&D 3O O O 2OOO OOC)

cell lysate serum free media spent media

FG.22B U.S. Patent May 28, 2013 Sheet 29 of 43 US 8.450,069 B2

1200

E 800 - &% "1%% Singulex 60 % %2. E. R&D ELISA g 40 % s

E 2 O . % 3 I 2 CD1-03 CD1-04 Balbic- Balbic- Babic- C57B8- C57B8- C57B6 0. 02 O3 O O2 03 Sample

FG. 23 U.S. Patent May 28, 2013 Sheet 30 of 43 US 8.450,069 B2

Vºz?un61-i U.S. Patent May 28, 2013 Sheet 31 of 43 US 8.450,069 B2

s s g 8 O U.S. Patent May 28, 2013 Sheet 32 of 43 US 8.450,069 B2

X CD g of Y

g o CD E E CD Ol

ra 9 S g N O N C)E siaS O h .

CD s D 2n d O

O O CN uffdoAu U.S. Patent May 28, 2013 Sheet 33 of 43 US 8.450,069 B2

1: -gross or on y is 1.14x - 8 1200 R2 (). () Food

vs.

: s

*YYYYYYYraws-asers res RE K S. s Expected pg/ml. FIG. 25A

ESOc. y is 89 + 26

VEGF pg/ml. FIG. 25B U.S. Patent May 28, 2013 Sheet 34 of 43 US 8.450,069 B2

1OOOO

1OOO

100

1 O 8. MP-SMC ------Pate-SMC

-- HRP-ESA

O. 1 10 1OO OOO 10000 Expected hVEGF-AJ in pg/mL

Figure 26 U.S. Patent May 28, 2013 Sheet 35 of 43 US 8.450,069 B2

A) p40.0001 --- wV W 1OO

i O or 850 - -Yy. ELISA Lloo E. s. 31.2 pg/mL Oo c 10 C W n Oo ------Erenna Llo 3.5 pg/mL

Healthy Subjects Breast Cancer Sample Volume = 10ul of plasma B) p-0.0001 - 1000 w

W W 5 g 100 d. 92 O Wv 5 maa ------S^- A 31.2 pg/ml 2 9. O P C. g - Erenna Loc 1 C 3.5 p.gim

Normal Tissue Malignant Tissue Sample Volume a 10ul of lysate Figure 27 U.S. Patent May 28, 2013 Sheet 36 of 43 US 8.450,069 B2

E *

A.3

1.07

0. Log Concentration pg/ml) analog -- WEGF-A (LoD=3.97 pgmL) signalgna 1000 C) -

combined s to

ig 10 digital signal 0. . o Logxpected o Goncertation pigmL) B) -- WEGF-A y = 0.9986x + 0.01 R2 = .9979 .

. . o Log Concentration (pl.) -- VEGF-A (LoD=0.088 pg/ml) Figure 28

U.S. Patent May 28, 2013 Sheet 38 of 43 US 8.450,069 B2

25 YWYYYYYYY-rrattravitrawww.rawww.M.v.a.a waxaas war...... y r i.00 x + 0.26 Rs 1.oe doc

Expected ill-to) pg/ml FIG. 3OA

- y at 568x -564 12 R is o,99

bot

8co

2 .

------...--Aww.r-marrierror------...saw-wirrrrrrrrrrrr.------awww.wrwr.------,88 {3.48 0.63 tic . , Expected it.-ie) pg/ml FIG. 3OB U.S. Patent May 28, 2013 Sheet 39 of 43 US 8.450,069 B2

sc corror-o-o-o-o-c-cassass-assass-acce-acre.....screwwows...ww.www.csw-oxocorrors--

y x to x - i. R’ is o.99

So w

sdi

FIG 31 A

S3 y is 86.5x -i-SS4 3:30. R as o.gg

Sai

areer-w-s.-----' arrierrrrrrrrrrr-e-...-are------Year-error-rearrrrrrrrrrrryver"r------areer G.S. i.S. R.S. 3. 3. Expected it -13) pg/ml FIG. 31 B U.S. Patent May 28, 2013 Sheet 40 of 43 US 8.450,069 B2

y it 3.3 x - c,86 R is o. 99

wreserverwr.wrwriterrrrrrrrrrow-wrwrwrriverswast-w Six 3. 3. asso Expected L-4 pg/mL FIG. 32A

o: y - (2x +27C R. O.99 s

G-secess...arriers------taletters...rer.error------screwelr-rror------.S . . . 3. . . . Expected IL- pg/ml FIG. 32B U.S. Patent May 28, 2013 Sheet 41 of 43 US 8.450,069 B2

Rai ogg too

ce riorownrnerror8 wro-wormwresorter roarrower-to-www.morror . Expected L-6 pg/mi FIG. 33A

y s gox - if

FIG. 33B U.S. Patent May 28, 2013 Sheet 42 of 43 US 8.450,069 B2

s U.S. Patent May 28, 2013 Sheet 43 of 43 US 8.450,069 B2

scoococococo-cross------isococo

S 6 s 5 3 R2

O myxaasaxwww.r O 1. 2 3 4. 5 s More Number of Elevated Bomarkers

-ss-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-rele------as assists' asssssssssssssssssssssssss-r

FIG. 35 US 8,450,069 B2 1. 2 HIGHLY SENSITIVE BOMARKERPANELS magnetic radiation detector operably connected to the inter rogation space for determining an electromagnetic character BACKGROUND OF THE INVENTION istic of the stimulated fluorescent moiety. In some embodiments, the limit of detection of the first Cardiovascular disease is an abnormalfunction of the heart marker ranges from about 10 pg/ml to about 0.01 pg/ml. In and/or blood vessels. Included under this designation are Such some embodiments, the limit of detection of the first marker diverse medical conditions as coronary artery disease, con is less than about 10 pg/ml. In some embodiments, the limit of gestive heart failure, arrhythmia, atherosclerosis, hyperten detection of the first marker is less than about 5 pg/ml. In Sion, stroke, cerebrovascular disease, peripheral vascular dis some embodiments, the limit of detection of the first marker ease and myocardial infarction. In the United States, CVD is 10 is less than about 1 pg/ml. In some embodiments, the limit of a major cause of death. About 40 percent of all deaths in 1997, detection of the first marker is less than about 0.5 pg/ml. In or about one million people, were attributed to cardiovascular some embodiments, the limit of detection of the first marker disease. There are an estimated 62 million people with car is less than about 0.1 pg/ml. In some embodiments, the limit diovascular disease and 50 million people with hypertension of detection of the first marker is less than about 0.05 pg/ml. in this country. 15 In some embodiments, the limit of detection of the first Cardiovascular disease is a progressive process with eti marker is less than about 0.01 pg/ml. In some embodiments, ologies in both cardiac muscle (cardio-pathology) and vascu the limit of detection of the first marker is less than about lar inflammation. The disease process follows a continuum 0.005 pg/ml. In some embodiments, the limit of detection of from early onset mild vascular inflammation to severe acute the first marker is less than about 0.001 pg/ml. In some events such as acute myocardial infarction or chronic events embodiments, the coefficient of variation (CV) of the limit of such as heart failure. Patients with well recognized physical detection ranges from about 20% to about 1%. In some conditions such as hypertension, obesity, diabetes, metabolic embodiments, the coefficient of variation (CV) of the limit of syndrome, hyper-cholesterolemia are at varying degrees of detection ranges from about 100% to about 1%. In some risk for developing CVD. A challenge facing clinicians who embodiments, the coefficient of variation (CV) of the limit of have patients presenting with CVD risk factors is understand 25 detection ranges from about 75% to about 1%. In some ing their degree of risk, developing the appropriate treatment embodiments, the coefficient of variation (CV) of the limit of plan and then monitoring the patient for improvements in detection ranges from about 50% to about 1%. In some disease risk. embodiments, the coefficient of variation (CV) of the limit of Ample studies have provided compelling evidence that detection ranges from about 25% to about 1%. In some CVD is largely preventable. The causes of cardiovascular 30 embodiments, the coefficient of variation (CV) of the limit of disease range from structural defects, to infection, inflamma detection ranges from about 20% to about 1%. In some tion, environment and genetics. While some risk factors can embodiments, the coefficient of variation (CV) of the limit of not be modified (genetics, age, gender), there are a number of detection ranges from about 15% to about 1%. In some risk factors that can be addressed through lifestyle changes or embodiments, the coefficient of variation (CV) of the limit of medically. These controllable risk factors include cigarette 35 detection ranges from about 10% to about 1%. In some Smoking, high blood pressure, obesity, diabetes, physical embodiments, the coefficient of variation (CV) of the limit of inactivity, and high blood cholesterol level. By the time that detection ranges from about 5% to about 1%. In some heart problems are detected, the underlying cause (athero embodiments, the sample size ranges from about 10 ul to Sclerosis) is usually quite advanced, having progressed for about 0.1 ul. In some embodiments, the sample size ranges decades. There is therefore increased emphasis on preventing 40 from about 100 ul to about 0.1 ul. In some embodiments, the atherosclerosis by modifying risk factors, such as healthy sample size ranges from about 75ul to about 0.1 ul. In some eating, exercise and avoidance of Smoking. embodiments, the sample size ranges from about 50 ul to CVD, e.g., congestive heart failure (CHF), is often first about 0.1 ul. In some embodiments, the sample size ranges diagnosed after the onset of clinical symptoms, eliminating from about 25 ul to about 0.1 ul. In some embodiments, the potential for early intervention. There is a need for highly 45 sample size ranges from about 20 Jul to about 0.1 ul. In some sensitive detection of CVD. embodiments, the sample size ranges from about 5ul to about 0.1 ul. In some embodiments, the sample size ranges from SUMMARY OF THE INVENTION about 1 Jul to about 0.1 ul. In some embodiments, the sample size is less than about 100 ul. In some embodiments, the In one embodiment, the present invention provides a 50 sample size is less than about 75ul. In some embodiments, the method for detecting or monitoring a condition in a subject, sample size is less than about 50 ul. In some embodiments, the comprising detecting a first marker in a first sample from the sample size is less than about 25ul. In some embodiments, the Subject and detecting a second marker, wherein the first sample size is less than about 20l. In some embodiments, the marker comprises Cardiac Troponin-I (cTnI) or Vascular sample size is less than about 15ul. In some embodiments, the Endothelial Growth Factor (VEGF), and wherein the limit of 55 sample size is less than about 10 ul. In some embodiments, the detection of the first marker is less than about 20 pg/ml. In sample size is less than about 5ul. In some embodiments, the Some embodiments, the detection of at least one marker com sample size is less than about 2 ul. In some embodiments, the prises contacting the sample with a label for the marker and sample size is less than about 1 ul. In some embodiments, the detecting the presence or absence of the label, wherein detec sample size is less than about 0.5 ul. In some embodiments, tion of the presence of the label indicates the presence of the 60 the sample size is less than about 0.1 ul. In some embodi corresponding marker. In some embodiments, the label com ments, the sample size is less than about 0.05 ul. In some prises a fluorescent moiety, and the detection comprises pass embodiments, the sample size is less than about 0.01 ul. ing the label through a single molecule detector, wherein the In some embodiments, the method further comprises split single molecule detector comprises: (a) an electromagnetic ting the first sample into two or more aliquots and detecting at radiation source for stimulating the fluorescent moiety; (b) an 65 least one marker in the two or more aliquots. In some embodi interrogation space for receiving electromagnetic radiation ments, the sample comprises a plasma, serum, cell lysate, or emitted from the electromagnetic source; and (c) an electro tissue sample. In some embodiments, the sample comprises US 8,450,069 B2 3 4 bronchoalveolar lavage fluid (BAL), blood, serum, plasma, less than about 0.05 pg/ml. In some embodiments, the limit of urine, nasal Swab, cerebrospinal fluid, pleural fluid, synovial detection of the second marker is less than about 0.01 pg/ml. fluid, peritoneal fluid, amniotic fluid, gastric fluid, lymph In some embodiments, the limit of detection of the second fluid, interstitial fluid, tissue homogenate, cell extracts, marker is less than about 0.005 pg/ml. In some embodiments, saliva, sputum, stool, physiological Secretions, tears, mucus, the limit of detection of the second marker is less than about Sweat, milk, semen, seminal fluid, vaginal Secretions, fluid 0.001 pg/ml. from ulcers and other Surface eruptions, blisters, and In some embodiments, the second marker comprises abscesses, and extracts of tissues including biopsies of nor B-type natiuretic peptide, IL-1C., IL-113, IL-6, IL-8, IL-10, mal, malignant, and Suspect tissues or any other constituents TNF-C, IFN-y, cTnI, VEGF, insulin, GLP-1 (active), GLP-1 of the body which may contain the target particle of interest. 10 (total), TREM1, Leukotriene E4, Akt1, AB-40, AB-42, Fas Other similar specimens such as cell or tissue culture or ligand, or PSA. In some embodiments, the second marker is a culture broth are also of interest. cytokine. In some embodiments, the cytokine is G-CSF, MIP In some embodiments, the second marker comprises a 1C, IL-10, IL-22, IL-8, IL-5, IL-21, INF-Y, IL-15, IL-6, TNF biomarker, a physiological marker or a genetic marker. In C., IL-7, GM-CSF, IL-2, IL-4, IL-1C., IL-12, IL-17C, IL-1B. Some embodiments, the second marker comprises a protein. 15 MCP, IL-32 or RANTES. In some embodiments, the cytokine In some embodiments, at least one of the first marker and the is IL-10, IL-8, INF-y, IL-6, TNF-C., IL-7, IL-1C, or IL-1 B. In second marker are found in a sample from a normal individual Some embodiments, the second marker comprises an apoli at a concentration of less than 10 pg/ml. In some embodi poprotein, ischemia-modified albumin (IMA), fibronectin, ments, at least one of the first marker and the second marker C-reactive protein (CRP), B-type Natriuretic Peptide (BNP), are found in a sample from a normal individual at a concen or Myeloperoxidase (MPO). tration of less than 100 pg/ml. In some embodiments, at least In some embodiments, the method of the invention further one of the first marker and the second marker are found in a comprises determining a concentration for the first marker, sample from a normal individual at a concentration of less and determining a concentration for the second marker if the than 75 pg/ml. In some embodiments, at least one of the first second marker comprises a protein. In some embodiments, marker and the second marker are found in a sample from a 25 the method of the invention comprises determining a ratio of normal individual at a concentration of less than 50 pg/ml. In a concentration of the first marker compared to a concentra Some embodiments, at least one of the first marker and the tion for the second marker if the second marker comprises a second marker are found in a sample from a normal individual protein. at a concentration of less than 25 pg/ml. In some embodi In some embodiments, the second marker comprises a ments, at least one of the first marker and the second marker 30 physiological marker. In some embodiments, the physiologi are found in a sample from a normal individual at a concen cal marker comprises an electrocardiogram (EKG), stress tration of less than 20 pg/ml. In some embodiments, at least testing, nuclear imaging, ultrasound, insulin tolerance, body one of the first marker and the second marker are found in a mass index, blood pressure, age, sex, or sleep apnea. sample from a normal individual at a concentration of less In some embodiments, the second marker comprises a than 15 pg/ml. In some embodiments, at least one of the first 35 molecular marker. In some embodiments, the molecular marker and the second marker are found in a sample from a marker comprises cholesterol, LDL/HDL/Q-LDL, triglycer normal individual at a concentration of less than 10 pg/ml. In ides, uric acid, creatinine, blood glucose or vitamin-D. In Some embodiments, at least one of the first marker and the Some embodiments, the molecular marker comprises Subtrac second marker are found in a sample from a normal individual tions of LDL/HDL/Q-LDL or triglycerides. at a concentration of less than 5 pg/ml. In some embodiments, 40 In some embodiments, the second marker comprises a at least one of the first marker and the second marker are genetic marker. In some embodiments, the genetic marker found in a sample from a normal individual at a concentration comprises a variation in a gene encoding an apolipoprotein of less than 2 pg/ml. In some embodiments, at least one of the Such as ApoE. In some embodiments, the genetic marker first marker and the second marker are found in a sample from comprises a single nucleotide polymorphism (SNP). In some a normal individual at a concentration of less than 1 pg/ml. In 45 embodiments, the genetic marker comprises an insertion, Some embodiments, at least one of the first marker and the deletion, fusion or other mutation. In some embodiments, the second marker are found in a sample from a normal individual genetic marker comprises an epigenetic marker, such as DNA at a concentration of less than 0.5 pg/ml. In some embodi methylation or imprinting. ments, at least one of the first marker and the second marker In some embodiments of the method of the invention, the are found in a sample from a normal individual at a concen 50 condition comprises cardiac damage, an inflammatory dis tration of less than 0.1 pg/ml. In some embodiments, at least ease, a proliferative disorder, a metabolic disorder, angiogen one of the first marker and the second marker are found in a esis, artherosclerosis or diabetes. In some embodiments, the sample from a normal individual at a concentration of less cardiac damage comprises myocardial infarct, necrosis, myo than 0.05 pg/ml. In some embodiments, at least one of the first cardial dysfunction, unstable angina, plaques, heart failure, marker and the second marker are found in a sample from a 55 coronary artery disease, or rheumatic heart disease. In some normal individual at a concentration of less than 0.01 pg/ml. embodiments, the proliferative disorder comprises a cancer. In some embodiments, the limit of detection of the second In some embodiments, the cancer comprises a breast cancer, marker ranges from about 10 pg/ml to about 0.01 pg/ml. In a prostate cancer, or lymphoma. some embodiments, the limit of detection of the second In some embodiments, the method of the invention further marker is less than about 10 pg/ml. In some embodiments, the 60 comprises determining a change in concentration of the limit of detection of the second marker is less than about 5 markers between the first sample and a second sample from pg/ml. In some embodiments, the limit of detection of the the Subject, whereby the change is used to detect or monitor second marker is less than about 1 pg/ml. In some embodi the condition. In some embodiments, the method of the inven ments, the limit of detection of the second marker is less than tion further comprises determining a change in the ratio of the about 0.5 pg/ml. In some embodiments, the limit of detection 65 concentrations of the first marker and the second marker of the second marker is less than about 0.1 pg/ml. In some between the first sample and a second sample from the Sub embodiments, the limit of detection of the second marker is ject, whereby the change is used to detect or monitor the US 8,450,069 B2 5 6 condition. In some embodiments, a medical procedure is detecting the presence or absence of the label, wherein detec performed between acquiring the first sample and the second tion of the presence of the label indicates the presence of the sample from the Subject. In some embodiments, the medical corresponding marker. In some embodiments, the label com procedure comprises a Surgical procedure, stress testing or a prises a fluorescent moiety, and the detection comprises pass therapeutic intervention. In some embodiments, a series of 5 ing the label through a single molecule detector, wherein the samples from the subject are used to detect or monitor the single molecule detector comprises: a) an electromagnetic condition. In some embodiments, the series of samples are radiation source for stimulating the fluorescent moiety; b) an collected over time and the change of concentration in the interrogation space for receiving electromagnetic radiation series of samples is assessed. emitted from the electromagnetic source; and c) an electro In some embodiments, monitoring according to the present 10 invention comprises monitoring of a disease progression, dis magnetic radiation detector operably connected to the inter ease recurrence, risk assessment, therapeutic efficacy or Sur rogation space for determining an electromagnetic character gical efficacy. istic of the stimulated fluorescent moiety. In some In one embodiment, the present invention provides a embodiments, the limit of detection of at least one marker method for detecting a single particle in a sample, compris 15 ranges from about 10 pg/ml to about 0.01 pg/ml. ing: (a) labeling the particle, if present in the sample, with a In some embodiments, the cardiac pathology biomarkers label; and (b) detecting the presence or absence of the label, are Cardiac Troponin-I (cTnI) or B-type Natriuretic Peptide wherein detection of the presence of the label indicates the (BNP proBNP or NT-proBNP). In some embodiments, the presence of the single particle in the sample; wherein the limit vascular inflammation biomarkers are Interleukin 6 (IL-6), of detection of the single particle is less than 20 pg/ml; and Tumor Necrosis Factor alpha (TNF-C.), or Interleukin 17a wherein the single particle comprises a single molecule, frag (IL-17a). In some embodiments, the condition comprises ment, or complex of Cardiac Troponin-I (cTnI), B-type Natri congestive heart failure and the biomarkers comprise cTnI. uretic Peptide (BNP proBNP or NT-proBNP), TREM-1, BNP, IL-6, TNF-C., and IL-17a. Interleukin 1 Alpha (IL-1C), Interleukin 1 Beta (IL-1B), Inter In one embodiment, the present invention provides a leukin 4 (IL-4), Interleukin 6 (IL-6), Interleukin 8 (IL-8), 25 method to detect a cardiovascular condition in a Subject, Interleukin 10 (IL-10), Interferon gamma (IFN-Y), Tumor comprising measuring a physiological biomarker and detect Necrosis Factor alpha (TNF-C.), Glucagon-like peptide-1 ing one or more biomarkers in a blood sample from the (GLP-1), Leukotriene E4 (LTE4), Transforming Growth Fac Subject. In some embodiments, the physiological biomarker tor Beta (TGFB), Akt1, AB-40, AB-42, Fas ligand (FasL), or is a stress test and the one or more biomarkers comprise cTnI. Vascular Endothelial Growth Factor (VEGF). In some 30 In some embodiments, the physiological biomarker is a sleep embodiments, the limit of detection of the single particle test and the one or more biomarkers comprise one or more ranges between about 10 pg/ml and about 0.01 pg/ml. In some cytokines. The one or more cytokines can comprise TNF-a, embodiments, the limit of detection of the single particle is IL-6, IL-17a and/or hsCRP. In some embodiments, the physi less than about 10 pg/ml. In some embodiments, the limit of ological biomarker is Carotid intima-media thickness detection of the single particle is less than about 5 pg/ml. In 35 (CIMT) and the one or more biomarkers comprise one or Some embodiments, the limit of detection of the single par more markers of vascular inflammation. The one or more ticle is less than about 1 pg/ml. In some embodiments, the markers of vascular inflammation can comprise hsCRP, IL-6, limit of detection of the single particle is less than about 0.5 TNF-a and/or IL-1. pg/ml. In some embodiments, the limit of detection of the single particle is less than about 0.1 pg/ml. In some embodi 40 INCORPORATION BY REFERENCE ments, the limit of detection of the single particle is less than about 0.05 pg/ml. In some embodiments, the limit of detec All publications, patents, and patent applications men tion of the single particle is less than about 0.01 pg/ml. In tioned in this specification are herein incorporated by refer Some embodiments, the limit of detection of the single par ence to the same extent as if each individual publication, ticle is less than about 0.005 pg/ml. In some embodiments, the 45 patent, or patent application was specifically and individually limit of detection of the single particle is less than about 0.001 indicated to be incorporated by reference. pg/ml. In one embodiment, the present invention provides a kit BRIEF DESCRIPTION OF THE DRAWINGS comprising a composition comprising two or more antibodies to two or more biomarkers, wherein the two or more antibod 50 The novel features of the invention are set forth with par ies are attached to a fluorescent dye moiety, wherein the two ticularity in the appended claims. A better understanding of or more biomarkers comprise particles as described above, the features and advantages of the present invention will be wherein the moiety is capable of emitting at least about 200 obtained by reference to the following detailed description photons when stimulated by a laser emitting light at the exci that sets forth illustrative embodiments, in which the prin tation wavelength of the moiety, wherein the laser is focused 55 ciples of the invention are utilized, and the accompanying on a spot not less than about 5 microns in diameter that drawings of which: contains the moiety, and wherein the total energy directed at FIGS. 1A and 1B illustrate schematic diagrams of the the spot by the laser is no more than about 3 microJoules, arrangement of the components of a single particle analyzer. wherein the composition is packaged in Suitable packaging. FIG. 1A shows an analyzer that includes one electromagnetic In one embodiment, the present invention provides a 60 source and one electromagnetic detector; FIG. 1B shows an method for detecting or monitoring a cardiovascular condi analyzer that includes two electromagnetic sources and one tion in a Subject, comprising detecting two or more biomar electromagnetic detector. kers, wherein the biomarkers are cardiac pathology or vascu FIGS. 2A and 2B illustrate schematic diagrams of a capil lar inflammation biomarkers, and wherein the limit of lary flow cell for a single particle analyzer. detection of at least one marker is less than about 20 pg/ml. In 65 FIG. 2A shows the flow cell of an analyzer that includes Some embodiments, the detection of at least one marker com one electromagnetic source; and FIG. 2B shows the flow cell prises contacting the sample with a label for the marker and of an analyzer that includes two electromagnetic sources. US 8,450,069 B2 7 8 FIGS. 3A and 3B illustrate schematic diagrams showing (R&D Systems, Minneapolis, Minn.) gave a linear response the conventional (A) and confocal (B) positioning of laser and between 0.1 and 10 pg/ml. B) IL-6 standard curve below 1 detector optics of a single particle analyzer. FIG. 3A shows pg/ml. C) and D) Distribution of IL-6 C) and IL-8 D) identi the arrangement for an analyzer that has one electromagnetic fied in blood bank donor EDTA specimens. E) Range of source and one electromagnetic detector; FIG. 3B shows the 5 detection at low concentrations of any analyte can be arrangement for an analyzer that has two electromagnetic extended to higher concentrations by Switching the detection Sources and two electromagnetic detectors. of the analyzer from counting molecules (digital signal) to FIG. 4 illustrates a flow chart for multiple marker detection detecting the sum of photons (analog signal) that are gener or monitoring of a condition. ated at the higher concentrations of analyte. The single par FIG.5 illustrates a computer system wherein a client work 10 ticle analyzer has an expanded linear dynamic range of 6 logs. station receives assay results from a remote computer. Six-log range of detection based on Switching from digital to FIG. 6 illustrates a linearized standard curve for the range analog detection. F) Non-linearized standard curve showing concentrations of cTnI. range of low concentrations of IL-6 (0.1 fg/ml-10 fg/ml) FIG. 7A is a graph illustrating the analytical sensitivity of determined by counting photons emitted by individual par cTnI of a 100 ul sample and a 50 ul sample at an LoD of 15 ticles (digital signal), and higher range of concentrations of 0.1-0.2 pg/ml. FIG. 7B is a graph illustrating the low end of a IL-6 (10 fg/ml-1 pg/ml). standard curve signal. FIG. 18 illustrates a comparison of assays of the invention FIG. 8 illustrates a biological threshold (cutoff concentra with conventional assays. tion) for cTnI at a cTnI concentration of 7 pg/ml, as estab FIG. 19A is a graph illustrating the performance of a lished at the 99th percentile with a corresponding coefficient human VEGF assay: FIG. 19B is a graph of the assay perfor of variation (CV) of 10%. mance at the lowest concentrations. FIG. 9 illustrates a correlation of assay results of cTnI FIG.20A is a graph illustrating the performance of amouse determined using the analyzer System of the invention with VEGF assay: FIG.20B is a graph of the assay performance at standard measurements provided by the National Institute of the lowest concentrations. Standards and Technology (NIST) (R=0.9999). 25 FIG. 21 is a graph comparing the VEGF assays of the FIG. 10 illustrates detection of cTn in serial serum present invention and ELISA assays of human plasma. samples from patients who presented at the emergency room FIG.22A is a graph comparing the level of VEGF detected with chest pain. The measurements made with the analyzer in cell lysates and culture media using MDA-MB-231 breast system of the invention were compared to measurements adenocarcinoma cells; FIG. 22B is a graph comparing the made with a commercially available assay. 30 level of VEGF detected in cell lysates and culture media using FIG. 11 illustrates distribution of normal biological con HT-29 colon adenocarcinoma cells. centrations of cTnI and concentrations of cTnI in serum FIG. 23 is a comparison of VEGF assays of the present samples from patients presenting with chest pain. invention and ELISA assays for mouse plasma samples. FIG. 12 illustrates a competition curve for LTE4. The LOD FIG.24A is a graph illustrating the concentration of mouse was determined to be 1.5 pg/ml LTE4. 35 VEGF detected in cell lysates and culture media using B16 FIG. 13 illustrates a graph showing the standard curve for melanoma mouse cell lines; FIG. 24B is a graph illustrating concentrations of Akt1. The LOD was calculated to be 25 the concentration of mouse VEGF detected in cell lysates and pg/ml Akt1. culture media using 4T1 mammary carcinoma, FIG.24C is a FIG. 14 illustrates a graph showing the standard curve for graph illustrating the concentration of mouse VEGF detected concentrations of TGFB. The LOD was calculated to be 350 40 in cell lysates and culture media using CT26 coloncarcinoma pg/ml TGFB. cell lines. FIG. 15 illustrates a schematic representation of a kit that FIG. 25A illustrates a graph showing highly sensitive includes an analyzer system for detecting a single protein detection of VEGF. FIG. 25B illustrates the low end Standard molecule in a sample and least one label that includes a curve signal. fluorescent moiety and a binding partner for the protein mol 45 FIG. 26 illustrates the measured versus expected levels of ecule, where the analyzer includes an electromagnetic radia detection of human VEGF using three different immunoassay tion source for stimulating the fluorescent moiety; a capillary formats: 1) Magnetic Microparticle based Single Molecule flow cell for passing the label; a source of motive force for Counting (MP-SMC); 2) 384-well Plate based Single Mol moving the label in the capillary flow cell; an interrogation ecule Counting (Plate-SMC); and 3) Horseradish peroxidase space defined within the capillary flow cell for receiving 50 based Enzyme LinkedImmunosorbent Assay (HRP-ELISA). electromagnetic radiation emitted from the electromagnetic FIG. 27A illustrates the levels of human VEGF detected in Source; and an electromagnetic radiation detector operably 10 ul plasma samples from healthy and breast cancer patients. connected to the interrogation space for measuring an elec The limit of detection (LOD) using the method of the present tromagnetic characteristic of the stimulated fluorescent moi invention (Errena; LOD-3.5 pg/ml) versus a standard ELISA ety, where the fluorescent moiety is capable of emitting at 55 format (LOD=31.2 pg/ml) is shown. FIG. 27B illustrates least about 200 photons when simulated by a laser emitting similar data in 10 Jul lysate samples. light at the excitation wavelength of the moiety, where the FIG. 28A-C illustrates combined analog and digital mea laser is focused on a spot not less than about 5 microns in Surements of VEGF. diameter that contains the moiety, and where the total energy FIG. 29A illustrates a graph showing the specificity and directed at the spot by the laser is no more than about 3 60 linearity of AB-40 assay. FIG. 29B is a graph showing the microJoules. specificity and linearity of an AB-42 assay. FIG. 16 illustrates a standard curve of TREM-1 measured FIG.30A is a graph illustrating an assay curve fit for IL-1C. in a sandwich molecule immunoassay developed for the FIG.30B is a graph illustrating the low end of an IL-1C. assay single particle analyzer system. The linear range of the assay standard curve signal. is 100-1500 fM. 65 FIG. 31A is a graph illustrating an IL-1B assay curve fit. FIGS. 17A-F illustrate detection of IL-6 and IL-8. A) IL-6 FIG. 31B illustrates the low end standard curve of IL-1B standards, diluted according to a commercially available kit curve signal. US 8,450,069 B2 10 FIG.32A is a graph illustrating an IL-4 assay curve fit. FIG. I. Introduction 32B is an IL-4 assay standard curve signal at the low end. The invention provides instruments, kits, compositions, FIG.33A is a graph illustrating an IL-6 assay curve fit. FIG. and methods for the highly sensitive detection of single mol 33B is an IL-6 assay standard curve signal at the low end. ecules, and for the determination of the concentration of the FIG. 34 illustrates diagnostic specificity of the cardiovas- 5 molecules in a sample. In some embodiments, the sensitivity cular disease (CVD) panel for conjestive heart failure (CHF) and precision of the instruments, compositions, methods, and Subjects compared to age and sex matched healthy control kits of the invention can be achieved by a combination of Subjects. factors selected from, but not limited to, electromagnetic FIG. 35 illustrates the number of elevated biomarkers for Sources of appropriate wavelength and power output, appro the cardiovascular disease (CVD) panel in conjestive heart 10 priate interrogation space size, high numerical aperture failure (CHF) subjects. lenses, detectors capable of detecting single photons, and data analysis systems for counting single molecules. The instru DETAILED DESCRIPTION OF THE INVENTION ments of the invention are referred to as “single molecule detectors' or 'single particle detectors, and are also encom Outline 15 passed by the terms 'single molecule analyzers' and “single I. Introduction particle analyzers.” The sensitivity and precision of the kits II. Molecules for Sensitive Detection. By the Methods and and methods of the invention are achieved in some embodi Compositions of the Invention ments by the use of the instruments of the invention together A. General with a combination of factors selected from, but not limited B. Markers 2O to, labels for molecules that exhibit characteristics that allow III. Labels the molecules to be detected at the level of the single mol A. Binding partners ecule, and methods assaying the label in the instruments 1. Antibodies described herein. B. Fluorescent Moieties The instruments, kits, and methods of the invention are 1. Dyes 25 especially useful in the sensitive and precise detection of 2. Quantum dots single molecules or Small molecules, and for the determina C. Binding Partner-Fluorescent Moiety Compositions tion of the concentration of the molecules in a sample. IV. Highly Sensitive Analysis of Molecules The invention provides, in some embodiments, instru A. Sample ments and kits for the sensitive detection and determination of B. Sample preparation 30 concentration of molecules by detection of single molecules, C. Detection of molecule of interest and determination of labels for Such detection and determination, and methods concentration using such instruments and labels in the analysis of samples. V. Instruments and Systems Suitable for Highly Sensitive In particular, the sensitivity and precision of the instruments, Analysis of Molecules kits, and methods of the invention make possible the detection A. Apparatus/System 35 and determination of concentration of molecules, e.g., mark B. Single Particle Analyzer ers for biological states, at extremely low concentrations, e.g., 1. Electromagnetic Radiation Source concentrations below about 100, 10, 1, 0.1, 0.01, or 0.001 2. Capillary Flow Cell femtomolar. In further embodiments, the instruments and kits 3. Motive Force of the invention are capable of determining a concentration of 4. Detectors 40 a species in a sample, e.g., the concentration of a molecule, C. Sampling System over a large dynamic range of concentrations without the D. Sample preparation system need for dilution or other treatment of samples, e.g., over a E. Sample recovery concentration range of more than 10-fold, 10-fold, or 10'- VI. Methods Using Highly Sensitive Analysis of Molecules fold. A. Methods 45 The high sensitivity of the instruments, kits, and methods B. Exemplary Markers of the invention allows the use of markers, e.g., biological 1. Cardiac damage markers, which were not previously useful because of a lack 2. Infection of sensitivity of detection. The high sensitivity of the instru 3. Cytokines ments, kits, and methods of the invention also facilitate the a. Interleukin 1 50 establishment of new markers. There are numerous markers b. Interleukin 4 currently available which could be useful in determining bio c. Interleukin 6 logical states, but are not currently of practical use because of 4. Inflammatory Markers current limitations in measuring their lower concentration a. Leukotrine E4 ranges. In some cases, abnormally high levels of the marker b. TGFB 55 are detectable by current methods, but normal ranges are 5. Akt 1 unknown. In some cases, abnormally high levels of the 6. Fas ligand marker are detectable by current methods, but normal ranges 7. VEGF have not been established. In some cases, upper normal 8. Amyloid beta proteins ranges of the marker are detectable, but not lower normal C. Multiple Marker Panels 60 ranges, or levels below normal. In some cases, e.g., markers 1. Multiple Biomarker Panels of cancer or infection, any level of the marker can indicate the 2. Mixed Marker Panels presence of a biological state, and enhancing sensitivity of D. Detection and Monitoring detection is an advantage for early diagnosis. In some cases, E. Cardiovascular Biomarker Panels the rate of change, or lack of change, in the concentration of F. Clinical Methods 65 a marker over multiple time points provides the most useful VII. Kits information, but present methods of analysis do not permit VIII. Examples time point sampling in the early stages of a condition when it US 8,450,069 B2 11 12 is typically most treatable. In some cases, the marker can be polypeptides can contain amino acids other than the 20 amino detected at clinically useful levels only through the use of acids commonly referred to as the 20 naturally occurring cumbersome methods that are not practical or useful in a amino acids. Also, polypeptides can include one or more clinical setting, such as methods that require complex sample amino acids, including the terminal amino acids, which are treatment and time-consuming analysis. In addition, there are modified by any means known in the art (whether naturally or potential markers of biological states with sufficiently low non-naturally). Examples of polypeptide modifications concentration that their presence remains extremely difficult include e.g., by glycosylation, or other-post-translational or impossible to detect by current methods. modification. Modifications which may be present in The analytical methods and compositions of the present polypeptides of the present invention, include, but are not invention provide levels of sensitivity, precision, and robust 10 limited to, acetylation, acylation, ADP-ribosylation, amida ness that allow the detection of markers for biological states at tion, covalent attachment of flavin, covalent attachment of a concentrations at which the markers have been previously heme moiety, covalent attachment of a polynucleotide or undetectable, thus allowing the “repurposing of Such mark polynucleotide derivative, covalent attachment of a lipid or ers from confirmatory markers, or markers useful only in lipid derivative, covalent attachment of phosphotidylinositol, limited research settings, to diagnostic, prognostic, treat 15 cross-linking, cyclization, disulfide bond formation, dem ment-directing, or other types of markers useful in clinical ethylation, formation of covalent cross-links, formation of settings and/or in large scale clinical settings, including clini cystine, formation of pyroglutamate, formylation, gamma cal trials. Such methods allow the determination of normal carboxylation, glycation, glycosylation, GPI anchor forma and abnormal ranges for Such markers. tion, hydroxylation, iodination, methylation, myristoylation, The markers thus repurposed can be used for, e.g., detec oxidation, proteolytic processing, phosphorylation, prenyla tion of normal state (normal ranges), detection of responder/ tion, racemization, selenoylation, Sulfation, transfer-RNA non-responder (e.g., to a treatment, Such as administration of mediated addition of amino acids to proteins such as arginy a drug); detection of early disease or pathological occurrence lation, and ubiquitination. (e.g., early detection of cancer, early detection of cardiac The molecules detected by the present instruments, kits, ischemia); disease staging (e.g., cancer); disease monitoring 25 and methods may be free or may be part of a complex, e.g., an (e.g., diabetes monitoring, monitoring for cancer recurrence antibody-antigen complex, or more generally a protein-pro after treatment); study of disease mechanism; and study of tein complex, e.g., complexes of troponin or prostate specific treatment toxicity, such as toxicity of drug treatments. antigen (PSA). One of skill in the art will appreciate that when The invention thus provides methods and compositions for referring to proteins, the present invention can detect frag the sensitive detection of markers, and further methods of 30 ments, polypeptides, mutants, variants or complexes thereof. establishing values for normal and abnormal levels of mark B. Markers of Biological States ers. In further embodiments, the invention provides methods In some embodiments, the invention provides composi of diagnosis, prognosis, and/or treatment selection based on tions and methods for the sensitive detection of biological values established for the markers. The invention also pro markers, and for the use of Such markers in diagnosis, prog vides compositions for use in Such methods, e.g., detection 35 nosis, and/or determination of methods of treatment. reagents for the ultrasensitive detection of markers. Markers of the present invention may be, for example, any II. Molecules for Sensitive Detection by the Methods and composition and/or molecule or a complex of compositions Compositions of the Invention and/or molecules that is associated with a biological state of The instruments, kits and methods of the invention can be an organism (e.g., a condition Such as a disease or a non used for the sensitive detection and determination of concen 40 disease state). A marker can be, for example, a small mol tration of a number of different types of single molecules. In ecule, a polypeptide, a nucleic acid, such as DNA and RNA, particular, the instruments, kits, and methods are useful in the a lipid, such as a phospholipid or a micelle, a cellular com sensitive detection and determination of concentration of ponent such as a mitochondrion or chloroplast, etc. Markers markers of biological states. “Detection of a single molecule.” contemplated by the present invention can be previously as that term is used herein, refers to both direct and indirect 45 known or unknown. For example, in Some embodiments, the detection. For example, a single molecule may be labeled methods herein may identify novel polypeptides that can be with a fluorescent label, and the molecule-label complex used as markers for a biological state of interest or condition detected in the instruments described herein. Alternatively, a of interest, while in other embodiments, known polypeptides single molecule may be labeled with a fluorescent label, then are identified as markers for a biological state of interest or the fluorescent label is detached from the single molecule, 50 condition. Using the systems of the invention it is possible and the label detected in the instruments described herein. that one can observe those markers, e.g., polypeptides with The term detection of a single molecule encompasses both high potential use in determining the biological state of an forms of detection. organism, but that are only present at low concentrations, A. General such as those “leaked from diseased tissue. Other high Examples of molecules which can be detected using the 55 potentially useful markers or polypeptides may be those that analyzer and related methods of the present invention are related to the disease, for instance, those that are generated include: biopolymers such as proteins, nucleic acids, carbo in the tumor-host environment. Any suitable marker that pro hydrates, and Small molecules, both organic and inorganic. In vides information regarding a biological state may be used in particular, the instruments, kits, and methods described the methods and compositions of the invention. A “marker.” herein are useful in the detection of single molecules of pro 60 as that term is used herein, includes any molecule that may be teins and Small molecules in biological samples, and the detected in a sample from an organism and whose detection or determination of concentration of Such molecules in the quantitation provides information about the biological state sample. of the organism. The terms “protein.” “polypeptide.” “peptide.” and “oli Biological states include but are not limited to phenotypic gopeptide are used interchangeably herein and include any 65 states; conditions affecting an organism; States of develop composition that includes two or more amino acids joined ment; age; health; pathology; disease detection, process, or together by a peptide bond. It may be appreciated that staging; infection; toxicity; or response to chemical, environ US 8,450,069 B2 13 14 mental, or drug factors (such as drug response phenotyping, TABLE 1-continued drug toxicity phenotyping, or drug effectiveness phenotyp ing). CLASSES OF MARKERS AND EXEMPLARY MARKERS INTHE CLASSES The term “organism' as used herein refers to any living being comprised of a least one cell. An organism can be as Molar Conc. Molecules simple as a one cell organism or as complex as a mammal. An Inflammation organism of the present invention is preferably a mammal. ICAM-1 (soluble) 8.67 x 101 5.22 x 10 Such mammal can be, for example, a human or an animal RANTES 6.16 x 10 3.71 x 10' Such as a primate (e.g., a monkey, chimpanzee, etc.), a domes 10 MIP-2 9.92 x 10' 2.99 x 10' ticated animal (e.g., a dog, cat, horse, etc.), farm animal (e.g., MIP-1beta 1.98 x 1013 5.97 x 106 MIP-1 alpha 2.01 x 1013 6.05 x 106 goat, sheep, pig, cattle, etc.), or laboratory animal (e.g., MMP-3 1.75 x 1012 5.28 x 107 mouse, rat, etc.). Preferably, an organism is a human. Endocrinology In some embodiments, the methods and compositions of 17 beta-Estradiol (E2) 4.69 x 10' 2.82 x 106 the invention are directed to classes of markers, e.g., cytok 15 DHEA 4.44 x 10' 2.67 x 10'7 ines, growth factors, oncology markers, markers of inflam ACTH 1.32 x 102 7.96 x 10'7 Gastrin 2.19 x 102 1.32 x 108 mation, endocrine markers, autoimmune markers, thyroid Growth Hormone (hGH) 2.74 x 102 1.65 x 108 markers, cardiovascular markers, markers of diabetes, mark Autoimmune ers of infectious disease, neurological markers, respiratory GM-CSF 1.35 x 1013 8.15 x 106 markers, gastrointestinal markers, musculoskeletal markers, C-Reactive Protein (CRP) 3.98 x 1013 2.40 x 107 dermatological disorders, and metabolic markers. G-CSF 1.76 x 102 1.06 x 108 Table 1 provides examples of these classes of markers that Thyroid have been measured by the methods and compositions of the Cyclic AMP 9.02 x 101 5.43 x 10 invention, and provides exemplary concentrations of the 25 Calcitonin 3.25 x 10' 1.95 x 10' markers detected by the methods and compositions of the Parathyroid Hormone (PTH) 1.56 x 1013 9.37 x 106 invention and number of particles that are counted by the Cardiovascular single particle analyzer System of the invention for the par B-Natriuretic Peptide 2.86 x 10 1.72 x 107 ticular marker. NT-proBNP 2.86 x 102 8.60 x 10'7 30 C-Reactive Protein, HS 3.98 x 1013 2.40 x 107 TABLE 1. Beta-Thromboglobulin (BTG) 5.59 x 10 3.36 x 107 Diabetes CLASSES OF MARKERS AND EXEMPLARY MARKERS INTHE CLASSES C-Peptide 2.41 x 10 1.45 x 10' Leptin 1.89 x 10 1.14 x 107 Molar Conc. Molecules 35 infectious Dis. Cytokines FN-gamma 2.08 x 1013 1.25 x 107 FN-alpha 4.55 x 10 2.74 x 107 L-12 p70 2.02 x 104 6.09 x 10' Metabolism L-10 5.36 x 104 1.61 x 10' L-1 alpha 5.56x 10 1.67 x 10' Bio-IntactPTH (1-84) 1.59 x 1012 1.44 x 108 L-3 5.85 x 10 1.76 x 10' 40 PTH 1.05 x 10 9.51 x 10' L-12p40 6.07 x 104 1.83 x 106 L-8 6.12 x 104 1.84 x 10' L-12 8.08 x 10 2.44 x 10" 1. Cytokines L-6 9.53 x 10 2.87 x 10' L-4 1.15 x 1013 3.47 x 106 For both research and diagnostics, cytokines are useful as L-18 1.80 x 1013 5.43 x 106 45 markers of a number of conditions, diseases, pathologies, and P-10 1.88 x 10 1.13 x 107 the like, and the compositions and methods of the invention L-5 1.99 x 10 5.98 x 10' include labels for detection and quantitation of cytokines and Eotaxin 2.06 x 1013 1.24 x 107 L-16 3.77 x 10-13 1.14 x 107 methods using Such labels to determine normal and abnormal MIG 3.83 x 10 1.15 x 107 levels of cytokines, as well as methods of diagnosis, progno L-8 4.56 x 1013 1.37 x 107 50 sis, and/or determination of treatment based on Such levels. L-17 5.18 x 1013 1.56 x 107 L-7 5.97 x 10-13 1.80 x 107 There are currently over 100 cytokines/chemokines whose L-15 6.13 x 10 1.84 x 107 coordinate or discordant regulation is of clinical interest. In L-13 8.46 x 10 2.55 x 10-7 order to correlate a specific disease process with changes in L-2R (soluble) 8.89 x 1013 2.68 x 107 cytokine levels, the ideal approach requires analyzing a L-2 8.94 x 10 2.69 x 107 55 sample for a given cytokine, or multiple cytokines, with high LIFEHILDA 9.09 x 10 5.47 x 107 L-1 beta 1.17 x 1012 3.51 x 10-7 sensitivity. Exemplary cytokines that are presently used in Fas/CD95/Apo-1 1.53 x 1012 9.24 x 107 marker panels and that may be used in methods and compo MCP-1 2.30 x 102 6.92 x 107 sitions of the invention include, but are not limited to, BDNF, Oncology CREB pS133, CREB Total, DR-5, EGFENA-78, Eotaxin, EGF 4.75 x 1014 2.86 x 10 60 Fatty Acid Binding Protein, FGF-basic, granulocyte colony TNF-alpha 6.64 x 10' 8.00 x 10' stimulating factor (G-CSF), GCP-2, Granulocyte-macroph PSA (3rd generation) 1.15 x 10 6.92 x 10' age Colony-stimulating Factor GM-CSF (GM-CSF), growth VEGF 2.31 x 1013 6.97 x 106 TGF-beta1 2.42 x 1013 3.65 x 107 related oncogene-keratinocytes (GRO-KC), HGF, ICAM-1, FGFb. 2.81 x 10 1.69 x 107 IFN-alpha, IFN-gamma, the interleukins IL-10, IL-11, IL-12, TRAIL 5.93 x 10 3.57 x 107 65 IL-12 p40, IL-12 p40/p70, IL-12 p70, IL-13, IL-15, IL-16, TNF-RI (p55) 2.17 x 1012 2.62 x 108 IL-17, IL-18, IL-1alpha, IL-1beta, IL-1 ra, IL-1 ra/IL-1F3, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, interferon

US 8,450,069 B2 17 18 Chem R23. Further markers of inflammation include Tumor include Complement markers such as C3d, C1q, C5, C4d, C4 necrosis factors (TNFs), such as TNF-alpha, 4-1BB Ligand/ bp, and C5a-C9. Further markers of inflammation include TNFSF9, LIGHT/TNFSF14, APRIL/TNFSF13, Lympho Major histocompatibility complex (MHC) glycoproteins, toxin, BAFF/TNFSF13B, Lymphotoxin beta/TNFSF3, such as HLA-DR and HLA-A.D.C. Further markers of CD27 Ligand/TNFSF7, OX40 Ligand/TNFSF4, CD30 5 inflammation include Microglial markers, such as CR3 Ligand/TNFSF8, TL1A/TNFSF15, CD40 Ligand/TNFSF5, receptor, MHC I, MHC II, CD 31, CD11a, CD11b, CD11c, TNF-alpha/TNFSF1A, EDA, TNF-beta/TNFSF1B, EDA CD68, CD45RO, CD45RD, CD18, CD59, CR4, CD45, A2, TRAIL/TNFSF10, Fas Ligand/TNFSF6, TRANCE/TN CD64, and CD44. Further markers of inflammation include FSF11, GITR Ligand/TNFSF18, TWEAK/TNFSF12. Fur alpha.2 macroglobulin receptor, Fibroblast growth factor, Fc ther markers of inflammation include TNF Superfamily 10 gamma RI, Fc gamma RII, CD8, LCA (CD45), CD18, CD59, Receptors such as 4-1BB/TNFRSF9, NGF R/TNFRSF16, Apo J., clusterin, type 2 plasminogen activator inhibitor, BAFF R/TNFRSF13C, Osteoprotegerin/TNFRSF11B, CD44, Macrophage colony stimulating factor receptor, BCMA/TNFRSF17, OX40/TNFRSF4, CD27/TNFRSF7, MRP14, 27E10, 4-hydroxynonenal-protein conjugates, RANK/TNFRSF11A, CD30/TNFRSF8, RELTA I.kappa.B, NFkappa.B., cELA.sub.2, COX-2, Matrix metal TNFRSF19L, CD40/TNFRSF5, TACI/TNFRSF13B, DcR3/ 15 loproteinases, Membrane lipid peroxidation, and ATPase TNFRSF6B, TNF R1/TNFRSF1A, DcTRAIL R1/TN activity. HSPC228, EMP1, CDC42, TLE3, SPRY2, p40BBP. FRSF23, TNFR11/TNFRSF1B, DcTRAIL R2/TNFRSF22, HSPC060 or NAB2, or a down-regulation of HSPA1A, TRAIL R1/TNFRSF 10A DR3/TNFRSF25, TRAIL HSPA1B, MAPRE2 and OAS1 expression, TACE/ADAM17, R2/TNFRSF10B, DR6/TNFRSF21, TRAIL alpha-1-Acid Glycoprotein, Angiopoietin-1, MIF, Angiopoi R3/TNFRSF10C, EDAR, TRAILR4/TNFRSF10D, Fas/TN etin-2, CD14, beta-Defensin 2, MMP-2, ECF-L/CHI3L3, FRSF6, TROY/TNFRSF19, GITR/TNFRSF18, TWEAK MMP-7, EGF, MMP-9, EMAP-II, MSP EN-RAGE, Nitric R/TNFRSF12, HVEM/TNFRSF14, XEDAR. Further mark Oxide, Endothelin-1, Osteoactivin/GPNMB, FPR1, PDGF, ers of inflammation include TNF Superfamily Regulators FPRL1, Pentraxin 3/TSG-14, FPRL2, Gas6, PLUNC, GM such as FADD, TRAF-2, RIP1, TRAF-3, TRADD, TRAF-4, CSF, RAGE, S100A10, S100A8, S100A9, HIF-1alpha, Sub TRAF-1, TRAF-6. Further markers of inflammation include 25 stance P, TFPI, TGF-beta 1, TIMP-1, TIMP-2, TIMP-3, Acute-phase reactants and acute phase proteins. Further TIMP-4, TLR4, LBP TREM-1, Leukotriene A4, Hydrolase markers of inflammation include TGF-beta superfamily TSG-6, Lipocalin-1, uPA, M-CSF, and VEGF. ligands such as Activins, Activin A, Activin B. Activin AB, 4. Miscellaneous Markers Activin C, BMPs (Bone Morphogenetic Proteins), BMP-2, Oncology markers include EGF, TNF-alpha, PSA, VEGF, BMP-7, BMP-3, BMP-8, BMP-3b/GDF-10, BMP-9, BMP 30 TGF-beta1, FGFb, TRAIL, and TNF-R1 (p55). 4, BMP-10, BMP-5, BMP-15/GDF-9B, BMP-6, Decapen Markers of endocrine function include 17 beta-estradiol taplegic, Growth/Differentiation Factors (GDFs), GDF-1, (E2), DHEA, ACTH, gastrin, and growth hormone (hGH). GDF-8, GDF-3, GDF-9 GDF-5, GDF-11, GDF-6, GDF-15, Markers of autoimmunity include GM-CSF, C-Reactive GDF-7, GDNF Family Ligands, Artemin, Neurturin, GDNF, Protein, and G-CSF. Persephin, TGF-beta, TGF-beta, TGF-beta 3, TGF-beta 1, 35 Markers of thyroid function include cyclicAMP, calcito TGF-beta 5, LAP (TGF-beta1), LatentTGF-beta bp 1, Latent nin, and parathyroid hormone. TGF-beta 1, Latent TGF-beta bp2, TGF-beta 1.2, Latent Cardiovascular markers include cardiac troponin I, cardiac TGF-beta bp4, TGF-beta 2, Lefty, MIS/AMH, Lefty-1, troponin T. B-natriuretic peptide, NT-proBNP. C-Reactive Nodal, Lefty-A, Activin RIA/ALK-2, GFR alpha-1/GDNFR Protein HS, and beta-thromboglobulin. alpha-1, Activin RIB/ALK-4, GFR alpha-2/GDNF Ralpha 40 Markers of diabetes include C-peptide and leptin. 2, Activin RIIA, GFR alpha-3/GDNF Ralpha-3, Activin Markers of infectious disease include IFN-gamma and RIIB, GFR alpha-4/GDNF Ralpha-4, ALK-1, MIS R11, IFN-alpha. ALK-7, Ret, BMPR-IA/ALK-3, TGF-beta RI/ALK-5, Markers of metabolism include Bio-intact PTH (1-84) and BMPR-IB/ALK-6, TGF-beta RII, BMPR-II, TGF-beta RIIb, PTH. Endoglin/CD105, TGF-beta RIII. Further markers of inflam 45 5. Markers of Biological States mation include TGF-beta superfamily Modulators such as Markers may indicate the presence of a particular pheno Amnionless, NCAM -1/CD56, BAMBI/NMA, Noggin, typic state of interest. Examples of phenotypic states include, BMP-1/PCP, NOMO, Caronte, PRDC, Cerberus 1, SKI, phenotypes resulting from an altered environment, drug treat Chordin, Smad1, Chordin-Like 1, Smad3, Chordin-Like 2, ment, qgenetic manipulations or mutations, injury, change in Smad3, COCO, Smada, CRIM1, Smad3, Cripto, Smad 7, 50 diet, aging, or any other characteristic(s) of a single organism Crossveinless-2, Smad3, Cryptic, SOST, DAN, Latent TGF or a class or Subclass of organisms. beta bp1, Decorin, Latent TGF-beta bp2, FLRG, Latent TGF In some embodiments, a phenotypic state of interest is a beta bp4. Follistatin, TMEFF1/Tomoregulin-1, Follistatin clinically diagnosed disease state. Such disease states like 1, TMEFF2, GASP-1/WFIKKNRP, TSG, GASP-2/ include, for example, cancer, cardiovascular disease, inflam WFIKKN, TSK, Gremlin, Vasorin. Further markers of 55 matory disease, autoimmune disease, neurological disease, inflammation include EGF Ligands Such as Amphiregulin, infectious disease and pregnancy related disorders. Alterna LRIG3, Betacellulin, Neuregulin-1/NRG1, EGF, Neuregu tively, states of health can be detected using markers. lin-3/NRG3, Epigen, TGF-alpha, Epiregulin, TMEFF1/To Cancer phenotypes are included in some aspects of the moregulin-1, HB-EGF, TMEFF2, LRIG1. Further markers of invention. Examples of cancer include, but are not limited to: inflammation include EGFR/ErbB Receptor Family, such as 60 breast cancer, skin cancer, bone cancer, prostate cancer, liver EGFR, ErbB3, ErbB2, ErbB4. Further markers of inflamma cancer, lung cancer, brain cancer, cancer of the larynx, gall tion include Fibrinogen. Further markers of inflammation bladder, pancreas, rectum, parathyroid, thyroid, adrenal, neu include SAA. Further markers of inflammation include glial ral tissue, head and neck, colon, stomach, bronchi, kidneys, markers, such as alpha.1-antitrypsin, C-reactive protein basal cell carcinoma, squamous cell carcinoma of both ulcer (CRP), alpha.2-macroglobulin, glial fibrillary acidic protein 65 ating and papillary type, metastatic skin carcinoma, osteo (GFAP), Mac-1, F4/80. Further markers of inflammation sarcoma, Ewing's sarcoma, Veticulum cell sarcoma, include myeloperoxidase. Further markers of inflammation myeloma, giant cell tumor, Small-cell lung tumor, non-small US 8,450,069 B2 19 20 cell lung carcinoma gallstones, islet cell tumor, primary brain In other embodiments, the cancer comprises Cerebellar tumor, acute and chronic lymphocytic and granulocytic Astrocytoma. In other embodiments, the cancer comprises tumors, hairy-cell tumor, adenoma, hyperplasia, medullary Cerebral Astrocytoma/Malignant Glioma. In other embodi carcinoma, pheochromocytoma, mucosal neuromas, intesti ments, the cancer comprises Cervical Cancer. In other nal ganglloneuromas, hyperplastic corneal nerve tumor, embodiments, the cancer comprises Childhood Cancers. In marfanoid habitus tumor, Wilm's tumor, seminoma, ovarian other embodiments, the cancer comprises Chordoma. In other tumor, leiomyomater tumor, cervical dysplasia and in situ embodiments, the cancer comprises Chronic Lymphocytic carcinoma, neuroblastoma, retinoblastoma, Soft tissue sar Leukemia. In other embodiments, the cancer comprises coma, malignant carcinoid, topical skin lesion, mycosis fun Chronic Myelogenous Leukemia. In other embodiments, the goide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic 10 cancer comprises Chronic Myeloproliferative Disorders. In and other sarcoma, malignant hypercalcemia, renal cell other embodiments, the cancer comprises Colon Cancer. In tumor, polycythermia Vera, adenocarcinoma, glioblastoma other embodiments, the cancer comprises Colorectal Cancer. multiforma, leukemias, lymphomas, malignant melanomas, In other embodiments, the cancer comprises Craniopharyn epidermoid carcinomas, and other carcinomas and sarcomas. gioma. In other embodiments, the cancer comprises Cutane The present invention provides methods to detect cancers. 15 ous T-Cell Lymphoma, including Mycosis Fungoides and In some embodiments, the cancer comprises Acute Lympho Sezary Syndrome. In other embodiments, the cancer com blastic Leukemia. In other embodiments, the cancer com prises Central Nervous System Embryonal Tumors. In other prises Acute Myeloid Leukemia. In other embodiments, the embodiments, the cancer comprises Endometrial Cancer. In cancer comprises Adrenocortical Carcinoma. In other other embodiments, the cancer comprises Ependymoblas embodiments, the cancer comprises an AIDS-Related Can toma. In other embodiments, the cancer comprises Ependy cer. In other embodiments, the cancer comprises AIDS-Re moma. In other embodiments, the cancer comprises Esoph lated Lymphoma. In other embodiments, the cancer com ageal Cancer. In other embodiments, the cancer comprises the prises Anal Cancer. In other embodiments, the cancer Ewing Family of Tumors. In other embodiments, the cancer comprises Appendix Cancer. In other embodiments, the can comprises Extracranial Germ Cell Tumor. In other embodi cer comprises Childhood Cerebellar Astrocytoma. In other 25 ments, the cancer comprises Extragonadal Germ Cell Tumor. embodiments, the cancer comprises Childhood Cerebral In other embodiments, the cancer comprises Extrahepatic Astrocytoma. In other embodiments, the cancer comprises a Bile Duct Cancer. In other embodiments, the cancer com Central Nervous System Atypical Teratoid/Rhabdoid Tumor. prises Intraocular Melanoma Eye Cancer. In other embodi In other embodiments, the cancer comprises Basal Cell Car ments, the cancer comprises Retinoblastoma Eye Cancer. In cinoma, or other Skin Cancer (Nonmelanoma). In other 30 other embodiments, the cancer comprises Gallbladder Can embodiments, the cancer comprises Extrahepatic Bile Duct cer. In other embodiments, the cancer comprises Gastric Cancer. In other embodiments, the cancer comprises Bladder (Stomach) Cancer. In other embodiments, the cancer com Cancer. In other embodiments, the cancer comprises Bone prises Gastrointestinal Carcinoid Tumor. In other embodi Cancer, such as Osteosarcoma or Malignant Fibrous Histio ments, the cancer comprises Gastrointestinal Stromal Tumor cytoma. In other embodiments, the cancer comprises Brain 35 (GIST). In other embodiments, the cancer comprises Gas Stem Glioma. In other embodiments, the cancer comprises an trointestinal Stromal Cell Tumor. In other embodiments, the Adult Brain Tumor. In other embodiments, the cancer com cancer comprises Extracranial Germ Cell Tumor. In other prises Brain Tumor comprising Central Nervous System embodiments, the cancer comprises Extragonadal Germ Cell Atypical Teratoid/Rhabdoid Tumor. In other embodiments, Tumor. In other embodiments, the cancer comprises Ovarian the cancer comprises a Brain Tumor comprising Cerebral 40 Germ. Cell Tumor. In other embodiments, the cancer com Astrocytoma/Malignant Glioma. In other embodiments, the prises Gestational Trophoblastic Tumor. In other embodi cancer comprises a Craniopharyngioma Brain Tumor. In ments, the cancer comprises Glioma. In other embodiments, other embodiments, the cancer comprises a Ependymoblas the cancer comprises Brain Stem Glioma. In other embodi toma Brain Tumor. In other embodiments, the cancer com ments, the cancer comprises Cerebral Astrocytoma Glioma. prises a Ependymoma Brain Tumor. In other embodiments, 45 In other embodiments, the cancer comprises Visual Pathway the cancer comprises a Medulloblastoma Brain Tumor. In or Hypothalamic Glioma. In other embodiments, the cancer other embodiments, the cancer comprises a Medulloepithe comprises Hairy Cell Leukemia. In other embodiments, the lioma Brain Tumor. In other embodiments, the cancer com cancer comprises Head and Neck Cancer. In other embodi prises Brain Tumors including Pineal Parenchymal Tumors ments, the cancer comprises Hepatocellular (Liver) Cancer. of Intermediate Differentiation. In other embodiments, the 50 In other embodiments, the cancer comprises Hodgkin Lym cancer comprises Brain Tumors including Supratentorial phoma. In other embodiments, the cancer comprises Primitive Neuroectodermal Tumors and Pineoblastoma. In Hypopharyngeal Cancer. In other embodiments, the cancer other embodiments, the cancer comprises a Brain Tumor comprises Intraocular Melanoma. In other embodiments, the including Visual Pathway and Hypothalamic Glioma. In other cancer comprises Islet Cell Tumors (Endocrine Pancreas). In embodiments, the cancer comprises Brain and Spinal Cord 55 other embodiments, the cancer comprises Kaposi Sarcoma. Tumors. In other embodiments, the cancer comprises Breast In other embodiments, the cancer comprises Kidney (Renal Cancer. In other embodiments, the cancer comprises Bron Cell) Cancer. In other embodiments, the cancer comprises chial Tumors. In other embodiments, the cancer comprises Laryngeal Cancer. In other embodiments, the cancer com Burkitt Lymphoma. In other embodiments, the cancer com prises Acute Lymphoblastic Leukemia. In other embodi prises Carcinoid Tumor. In other embodiments, the cancer 60 ments, the cancer comprises Acute Myeloid Leukemia. In comprises Gastrointestinal Carcinoid Tumor. In other other embodiments, the cancer comprises Chronic Lympho embodiments, the cancer comprises Carcinoma of Unknown cytic Leukemia. In other embodiments, the cancer comprises Primary Origin. In other embodiments, the cancer comprises Chronic Myelogenous Leukemia. In other embodiments, the Central Nervous System Atypical Teratoid/Rhabdoid Tumor. cancer comprises Hairy Cell Leukemia. In other embodi In other embodiments, the cancer comprises Central Nervous 65 ments, the cancer comprises Lip Cancer. In other embodi System Embryonal Tumors. In other embodiments, the can ments, the cancer comprises Oral Cavity Cancer. In other cer comprises Primary Central Nervous System Lymphoma. embodiments, the cancer comprises Primary Liver Cancer. In US 8,450,069 B2 21 22 other embodiments, the cancer comprises Non-Small Cell embodiments, the cancer comprises Pituitary Tumor. In other Lung Cancer. In other embodiments, the cancer comprises embodiments, the cancer comprises Plasma Cell Neoplasm/ Small Cell Lung Cancer. In other embodiments, the cancer Multiple Myeloma. In other embodiments, the cancer com comprises AIDS-Related Lymphoma. In other embodiments, prises Pleuropulmonary Blastoma. In other embodiments, the the cancer comprises Burkitt Lymphoma. In other embodi cancer comprises Primary Central Nervous System Lym ments, the cancer comprises Cutaneous T-Cell Lymphoma. In phoma. In other embodiments, the cancer comprises Prostate other embodiments, the cancer comprises Mycosis Fun Cancer. In other embodiments, the cancer comprises Rectal goides and Sézary Syndrome. In other embodiments, the Cancer. In other embodiments, the cancer comprises Renal cancer comprises Hodgkin Lymphoma. In other embodi Cell (Kidney) Cancer. In other embodiments, the cancer.com ments, the cancer comprises Non-Hodgkin Lymphoma. In 10 prises Renal Pelvis and Ureter, Transitional Cell Cancer. In other embodiments, the cancer comprises Primary Central other embodiments, the cancer comprises Respiratory Tract Nervous System Lymphoma. In other embodiments, the can Carcinoma Involving the NUT Gene on Chromosome 15. In cer comprises Waldenström Macroglobulinemia. In other other embodiments, the cancer comprises Retinoblastoma. In embodiments, the cancer comprises Malignant Fibrous His other embodiments, the cancer comprises Rhabdomyosar tiocytoma of Bone or Osteosarcoma. In other embodiments, 15 coma. In other embodiments, the cancer comprises Salivary the cancer comprises Medulloepithelioma. In other embodi Gland Cancer. In other embodiments, the cancer comprises ments, the cancer comprises Melanoma. In other embodi Sarcoma of the Ewing Family of Tumors. In other embodi ments, the cancer comprises Intraocular (Eye) Melanoma. In ments, the cancer comprises Kaposi Sarcoma. In other other embodiments, the cancer comprises Merkel Cell Car embodiments, the cancer comprises Soft Tissue Sarcoma. In cinoma. In other embodiments, the cancer comprises other embodiments, the cancer comprises Uterine Sarcoma. Mesothelioma. In other embodiments, the cancer comprises In other embodiments, the cancer comprises Sezary Syn Metastatic Squamous Neck Cancer with Occult Primary. In drome. In other embodiments, the cancer comprises Non other embodiments, the cancer comprises Mouth Cancer. In melanoma Skin Cancer. In other embodiments, the cancer other embodiments, the cancer comprises Multiple Endo comprises Melanoma Skin Cancer. In other embodiments, the crine Neoplasia Syndrome. In other embodiments, the cancer 25 cancer comprises Merkel Cell Skin Carcinoma. In other comprises Multiple Myeloma/Plasma Cell Neoplasm. In embodiments, the cancer comprises Small Cell Lung Cancer. other embodiments, the cancer comprises Mycosis Fun In other embodiments, the cancer comprises Small Intestine goides. In other embodiments, the cancer comprises Myelo Cancer. In other embodiments, the cancer comprises Squa dysplastic Syndromes. In other embodiments, the cancer mous Cell Carcinoma, e.g., Nonmelanoma Skin Cancer. In comprises Myelodysplastic or Myeloproliferative Diseases. 30 other embodiments, the cancer comprises Metastatic Squa In other embodiments, the cancer comprises Chronic Myel mous Neck Cancer with Occult Primary. In other embodi ogenous Leukemia. In other embodiments, the cancer com ments, the cancer comprises Stomach (Gastric) Cancer. In prises Acute Myeloid Leukemia. In other embodiments, the other embodiments, the cancer comprises Supratentorial cancer comprises Multiple Myeloma. In other embodiments, Primitive Neuroectodermal Tumors. In other embodiments, the cancer comprises Chronic Myeloproliferative Disorders. 35 the cancer comprises Cutaneous T-Cell Lymphoma, e.g., In other embodiments, the cancer comprises Nasal Cavity or Mycosis Fungoides and Sézary Syndrome. In other embodi Paranasal Sinus Cancer. In other embodiments, the cancer ments, the cancer comprises Testicular Cancer. In other comprises Nasopharyngeal Cancer. In other embodiments, embodiments, the cancer comprises Throat Cancer. In other the cancer comprises Nasopharyngeal Cancer. In other embodiments, the cancer comprises Thymoma or Thymic embodiments, the cancer comprises Neuroblastoma. In other 40 Carcinoma. In other embodiments, the cancer comprises embodiments, the cancer comprises Non-Hodgkin Lym Thyroid Cancer. In other embodiments, the cancer comprises phoma. In other embodiments, the cancer comprises Non Transitional Cell Cancer of the Renal Pelvis and Ureter. In Small Cell Lung Cancer. In other embodiments, the cancer other embodiments, the cancer comprises Gestational Tro comprises Oral Cancer. In other embodiments, the cancer phoblastic Tumor. In other embodiments, the cancer com comprises Oral Cavity Cancer. In other embodiments, the 45 prises a Carcinoma of Unknown Primary Site. In other cancer comprises Oropharyngeal Cancer. In other embodi embodiments, the cancer comprises an Unusual Cancer of ments, the cancer comprises Osteosarcoma. In other embodi Childhood. In other embodiments, the cancer comprises Ure ments, the cancer comprises Malignant Fibrous Histiocytoma ter and Renal Pelvis Transitional Cell Cancer. In other of Bone. In other embodiments, the cancer comprises Ovarian embodiments, the cancer comprises Urethral Cancer. In other Cancer. In other embodiments, the cancer comprises Ovarian 50 embodiments, the cancer comprises Endometrial Uterine Epithelial Cancer. In other embodiments, the cancer com Cancer. In other embodiments, the cancer comprises Uterine prises Ovarian Germ Cell Tumor. In other embodiments, the Sarcoma. In other embodiments, the cancer comprises Vagi cancer comprises Ovarian Low Malignant Potential Tumor. nal Cancer. In other embodiments, the cancer comprises In other embodiments, the cancer comprises Pancreatic Can Visual Pathway and Hypothalamic Glioma. In other embodi cer. In other embodiments, the cancer comprises Islet Cell 55 ments, the cancer comprises Vulvar Cancer. In other embodi Tumor Pancreatic Cancer. In other embodiments, the cancer ments, the cancer comprises Waldenström Macroglobuline comprises Papillomatosis. In other embodiments, the cancer mia. In other embodiments, the cancer comprises Wilms comprises Paranasal Sinus Cancer. In other embodiments, the Tumor. In other embodiments, the cancer comprises Wom cancer comprises Nasal Cavity Cancer. In other embodi en's Cancers. ments, the cancer comprises Parathyroid Cancer. In other 60 Cardiovascular disease may be included in other applica embodiments, the cancer comprises Penile Cancer. In other tions of the invention. Examples of cardiovascular disease embodiments, the cancer comprises Pharyngeal Cancer. In include, but are not limited to, congestive heart failure, high other embodiments, the cancer comprises Pheochromocy blood pressure, arrhythmias, atherosclerosis, cholesterol, toma. In other embodiments, the cancer comprises Pineal Wolff-Parkinson-White Syndrome, long QT syndrome, Parenchymal Tumors of Intermediate Differentiation. In 65 angina pectoris, tachycardia, bradycardia, atrial fibrillation, other embodiments, the cancer comprises Pineoblastoma or ventricular fibrillation, congestive heart failure, myocardial Supratentorial Primitive Neuroectodermal Tumors. In other ischemia, myocardial infarction, cardiac tamponade, myo US 8,450,069 B2 23 24 carditis, pericarditis, arrhythmogenic right ventricular dys In some embodiments, the invention provides a label for plasia, hypertrophic cardiomyopathy, Williams syndrome, detecting a biological molecule comprising a binding partner heart valve diseases, endocarditis, bacterial, pulmonary atre for the biological molecule that is attached to a fluorescent sia, aortic valve Stenosis, Raynaud's disease, cholesterol moiety, wherein the fluorescent moiety is capable of emitting embolism, Wallenberg syndrome, Hippel-Lindau disease, at least about 200 photons when simulated by a laser emitting and telangiectasis. light at the excitation wavelength of the moiety, wherein the Inflammatory disease and autoimmune disease may be laser is focused on a spot not less than about 5 microns in included in other embodiments of the invention. Examples of diameter that contains the moiety, and wherein the total inflammatory disease and autoimmune disease include, but energy directed at the spot by the laser is no more than about are not limited to, rheumatoid arthritis, non-specific arthritis, 10 3 microJoules. In some embodiments, the moiety comprises a inflammatory disease of the larynx, inflammatory bowel dis plurality of fluorescent entities, e.g., about 2 to 4, 2 to 5, 2 to order, psoriasis, hypothyroidism (e.g., Hashimoto thyroid 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, or about 3 to 5, 3 to 6, 3 to 7, ism), colitis, Type 1 diabetes, pelvic inflammatory disease, 3 to 8, 3 to 9, or 3 to 10 fluorescententities. In some embodi inflammatory disease of the central nervous system, temporal ments, the moiety comprises about 2 to 4 fluorescent entities. arteritis, polymyalgia rheumatica, ankylosing spondylitis, 15 In some embodiments, the biological molecule is a protein or polyarteritis nodosa, Reiter's syndrome, Scleroderma, syste a small molecule. In some embodiments, the biological mol mis lupus and erythematosus. ecule is a protein. The fluorescent entities can be fluorescent The methods and compositions of the invention can also dye molecules. In some embodiments, the fluorescent dye provide laboratory information about markers of infectious molecules comprise at least one substituted indolium ring disease including markers of Adenovirus, Bordella pertussis, system in which the substituent on the 3-carbon of the indo Chlamydia pneumoiea, Chlamydia trachomatis, Cholera lium ring contains a chemically reactive group or a conju Toxin, Cholera Toxin B, Campylobacter jejuni, Cytomega gated Substance. In some embodiments, the dye molecules lovirus, Diptheria Toxin, Epstein-Barr NA, Epstein-Barr EA, are Alexa Fluor molecules selected from the group consisting Epstein-Barr VCA, Helicobacter Pylori, Hepatitis B virus of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 647, Alexa (HBV) Core, Hepatitis B virus (HBV) Envelope, Hepatitis B 25 Fluor 680 or Alexa Fluor 700. In some embodiments, the dye virus (HBV) Surface (Ay), Hepatitis C virus (HCV) Core, molecules are Alexa Fluor molecules selected from the group Hepatitis C virus (HCV) NS3, Hepatitis C virus (HCV) NS4, consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor Hepatitis C virus (HCV)NS5, Hepatitis A, Hepatitis D, Hepa 680 or Alexa Fluor 700. In some embodiments, the dye mol titis E virus (HEV) orf23 KD, Hepatitis E virus (HEV) orf2 ecules are Alexa Fluor 647 dye molecules. In some embodi 6 KD, Hepatitis E virus (HEV) orf33 KD, Human immuno 30 ments, the dye molecules comprise a first type and a second deficiency virus (HIV)-1 p24. Human immunodeficiency type of dye molecules, e.g., two different Alexa Fluor mol virus (HIV)-1 gp41, Human immunodeficiency virus ecules, e.g., where the first type and second type of dye (HIV)-1 gp120, Human papilloma virus (HPV), Herpes sim molecules have different emission spectra. The ratio of the plex virus HSV-1/2. Herpes simplex virus HSV-1 g). Herpes number of first type to second type of dye molecule can be, simplex virus HSV-2 gG, Human T-cell leukemia virus 35 e.g., 4 to 1, 3 to 1, 2 to 1, 1 to 1, 1 to 2, 1 to 3 or 1 to 4. The (HTLV)-1/2, Influenza A, Influenza A H3N2, Influenza B, binding partner can be, e.g., an antibody. Leishmania donovani, Lyme disease, Mumps, M. pneumo In some embodiments, the invention provides a label for niae, M. tuberculosis, Parainfluenza 1, Parainfluenza 2, the detection of a marker, wherein the label comprises a Parainfluenza 3, Polio Virus, Respiratory syncytial virus binding partner for the marker and a fluorescent moiety, (RSV), Rubella, Rubeola, Streptolysin O, Tetanus Toxin, T. 40 wherein the fluorescent moiety is capable of emitting at least pallidum 15 kd, T. pallidum p47. T. Cruzi, Toxoplasma, and about 200 photons when simulated by a laser emitting light at Varicella Zoster. the excitation wavelength of the moiety, wherein the laser is III. Labels focused on a spot not less than about 5 microns in diameter In some embodiments, the invention provides methods and that contains the moiety, and wherein the total energy directed compositions that include labels for the highly sensitive 45 at the spot by the laser is no more than about 3 microJoules. In detection and quantitation of molecules, e.g., markers. Some embodiments, the fluorescent moiety comprises a fluo One skilled in the art will recognize that many strategies rescent molecule. In some embodiments, the fluorescent moi can be used for labeling target molecules to enable their ety comprises a plurality of fluorescent molecules, e.g., about detection or discrimination in a mixture of particles. The 2 to 10, 2 to 8, 2 to 6, 2 to 4,3 to 10, 3 to 8, or 3 to 6 fluorescent labels may be attached by any known means, including meth 50 molecules. In some embodiments, the label comprises about ods that utilize non-specific or specific interactions of label 2 to 4 fluorescent molecules. In some embodiments, the fluo and target. Labels may provide a detectable signal or affect rescent dye molecules comprise at least one Substituted indo the mobility of the particle in an electric field. In addition, lium ring system in which the substituent on the 3-carbon of labeling can be accomplished directly or through binding the indolium ring contains a chemically reactive group or a partners. 55 conjugated Substance. In some embodiments, the fluorescent In some embodiments, the label comprises a binding part molecules are selected from the group consisting of Alexa ner to the molecule of interest, where the binding partner is Fluor 488, Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 680 attached to a fluorescent moiety. The compositions and meth or Alexa Fluor 700. In some embodiments, the fluorescent ods of the invention may utilize highly fluorescent moieties, molecules are selected from the group consisting of Alexa e.g., a moiety capable of emitting at least about 200 photons 60 Fluor 488, Alexa Fluor 532, Alexa Fluor 680 or Alexa Fluor when simulated by a laser emitting light at the excitation 700. In some embodiments, the fluorescent molecules are wavelength of the moiety, wherein the laser is focused on a Alexa Fluor 647 molecules. In some embodiments, the bind spot not less than about 5 microns in diameter that contains ing partner comprises an antibody. In some embodiments, the the moiety, and wherein the total energy directed at the spot antibody is a monoclonal antibody. In other embodiments, the by the laser is no more than about 3 microJoules. Moieties 65 antibody is a polyclonal antibody. suitable for the compositions and methods of the invention The antibody may be specific to any suitable marker. In are described in more detail below. Some embodiments, the antibody is specific to a marker that is US 8,450,069 B2 25 26 selected from the group consisting of cytokines, growth fac Proteoglycans Aggrecan, Mimecan, Agrin, NG2/MCSP, Big tors, oncology markers, markers of inflammation, endocrine lycan, Osteoadherin, Decorin, Podocan, DSPG3, delta markers, autoimmune markers, thyroid markers, cardiovas Sarcoglycan, Endocan, Syndecan-1/CD138, Endoglycan, cular markers, markers of diabetes, markers of infectious Syndecan-2, Endorepellin/Perlecan, Syndecan-3, Glypican disease, neurological markers, respiratory markers, gas 2, Syndecan-4, Glypican 3, Testican 1/SPOCK1, Glypican 5, trointestinal markers, musculoskeletal markers, dermatologi Testican2/SPOCK2, Glypican 6, Testican 3/SPOCK3, Lumi cal disorders, and metabolic markers. can, Versican, Proteoglycan Regulators, Arylsulfatase In some embodiments, the antibody is specific to a marker A/ARSA, Glucosamine (N-acetyl)-6-Sulfatase/GNS. Exos that is a cytokine. In some embodiments, the cytokine is tosin-like 2/EXTL2, HS6ST2, Exostosin-like 3/EXTL3, Idu selected from the group consisting of BDNF, CREB pS133, 10 ronate 2-Sulfatase/IDS, GalNAc4S-6ST: SCF, Flt-3 Ligand CREB Total, DR-5, EGFENA-78, Eotaxin, Fatty Acid Bind & M-CSF Flt-3, M-CSF R, Flt-3 Ligand, SCF, M-CSF, SCF ing Protein, FGF-basic, granulocyte colony-stimulating fac R/c-kit; TGF-beta Superfamily (same as listed for inflamma tor (G-CSF), GCP-2, Granulocyte-macrophage Colony tory markers); VEGF/PDGF Family Neuropilin-1, PIGF, stimulating Factor GM-CSF (GM-CSF), growth-related Neuropilin-2, PIGF-2, PDGF, VEGF, PDGF Ralpha, VEGF oncogene-keratinocytes (GRO-KC), HGF, ICAM-1, IFN-al 15 B, PDGF R beta, VEGF-C, PDGF-A, VEGF-D, PDGF-AB, pha, IFN-gamma, interleukins such as IL-10, IL-11, IL-12, VEGF R, PDGF-B, VEGF R1/Flt-1, PDGF-C, VEGF IL-12 p40, IL-12 p40/p70, IL-12 p70, IL-13, IL-15, IL-16, R2/KDR/Flk-1, PDGF-D, VEGFR3/Flt-4, Wnt-related Mol IL-17, IL-18, IL-1alpha, IL-1beta, IL-1 ra, IL-1 ra/IL-1F3, ecules Dickkopf Proteins & Wnt Inhibitors Dkk-1, Dkk-4, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, interferon Dkk-2, Soggy-1, Dkk-3, WIF-1 Frizzled & Related Proteins inducible protein (10 IP-10), JE/MCP-1, keratinocytes (KC). Frizzled-1, Frizzled-8, Frizzled-2, Frizzled-9, Frizzled-3, KC/GROa, LIF, Lymphotacin, M-CSF, monocyte chemoat sERP-1, Frizzled-4, shRP-2, Frizzled-5, shRP-3, Frizzled-6, tractant protein-1 (MCP-1), MCP-1 (MCAF), MCP-3, MCP sFRP-4, Frizzled-7, MFRP Wnt Ligands Wnt-1, Wnt-8a, 5, MDC, MIG, macrophage inflammatory (MIP-1 alpha), Wnt-2b, Wnt-8b, Wnt-3a, Wnt-9a, Wnt-4, Wnt-9b, Wnt-5a, MIP-1 beta, MIP-1 gamma, MIP-2, MIP-3 beta, OSM, Wnt-10a, Wnt-5b, Wnt-10b, Wnt-7a, Wnt-11, Wnt-7b; Other PDGF-BB, regulated upon activation, normal T cell. 25 Wnt-related Molecules APC, Kremen-2, Axin-1, LRP-1, expressed and secreted (RANTES), Rb (pT821), Rb (total), beta-Catenin, LRP-6, Dishevelled-1, Norrin, Dishevelled-3, Rb pSpT249/252, Tau (pS214), Tau (pS396), Tau (total), PKC beta 1, Glypican 3, Pygopus-1, Glypican 5, Pygopus-2. Tissue Factor, tumor necrosis factor-alpha (TNF-alpha), GSK-3 alpha/beta, R-Spondin 1, GSK-3 alpha, R-Spondin2, TNF-beta, TNF-RI, TNF-RII, VCAM-1, and VEGF. GSK-3beta, R-Spondin3, ICAT, RTK-like Orphan Receptor In some embodiments, the cytokine is selected from the 30 1/ROR1, Kremen-1, RTK-like Orphan Receptor 2/ROR, and group consisting of IL-12p70, IL-10, IL-1 alpha, IL-3, IL-12 Other Growth Factors CTGF/CCN2, beta-NGF, Cyró1/ p40, IL-1 ra, IL-12, IL-6, IL-4, IL-18, IL-10, IL-5, Eotaxin, CCN1, Norrin, DANCE, NOV/CCN3, EG-VEGF/PK1, IL-16, MIG, IL-8, IL-17, IL-7, IL-15, IL-13, IL-2R (soluble), Osteocrin, Hepassocin, PD-ECGF, HGF, Progranulin, IL-2, LIF/HILDA, IL-1 beta, Fas/CD95/Apo-1 and MCP-1. LECT2, Thrombopoietin, LEDGF, or WISP-1/CCN4. In some embodiments, the antibody is specific to a marker 35 In some embodiments, the antibody is specific to a marker that is a growth factor. In some embodiments, the antibody is that is a marker for cancer (oncology marker). In some specific to a marker that is a growth factor that is TGF-beta. In embodiments, the antibody is specific to a marker that is a Some embodiments, the growth factor is GF Ligands Such as marker for cancer that is EGF. In some embodiments, the Amphiregulin, LRIG3, Betacellulin, Neuregulin-1/NRG1. antibody is specific to a marker that is a marker for cancer that EGF. Neuregulin-3/NRG3, Epigen, TGF-alpha, Epiregulin, 40 is TNF-alpha. In some embodiments, the antibody is specific TMEFF1/Tomoregulin-1, HB-EGF, TMEFF2, LRIG1; EGF to a marker that is a marker for cancer that is PSA. In some R/ErbB Receptor Family such as EGF R, ErbB3, ErbB2, embodiments, the antibody is specific to a marker that is a ErbB4; FGF Family such as FGF Ligands, FGF acidic, FGF marker for cancer that is VEGF. In some embodiments, the 12, FGF basic, FGF-13, FGF-3, FGF-16, FGF-4, FGF-17, antibody is specific to a marker that is a marker for cancer that FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21, FGF-9, 45 is TGF-beta. In some embodiments, the antibody is specific to FGF-22, FGF-10, FGF-23, FGF-11, KGF/FGF-7, FGF a marker that is a marker for cancer that is FGFb. In some Receptors FGFR1-4, FGFR3, FGFR1, FGF R4, FGF R2, embodiments, the antibody is specific to a marker that is a FGF R5, FGF Regulators FGF-BP; the Hedgehog Family marker for cancer that is TRAIL. In some embodiments, the Desert Hedgehog, Sonic Hedgehog, Indian Hedgehog; antibody is specific to a marker that is a marker for cancer that Hedgehog Related Molecules & Regulators BOC, GLI-3, 50 is TNF-RI (p55). CDO, GSK-3alpha/beta, DISP1, GSK-3alpha, Gas1, GSK-3 In further embodiments, the antibody is specific to a beta, GLI-1, Hip, GLI-2; the IGF Family IGF Ligands IGF-I, marker for cancer that is alpha-Fetoprotein. In some embodi IGF-II, IGF-I Receptor (CD221)IGF-I R, and IGF Binding ments, the antibody is specific to a marker for cancer that is Protein (IGFBP) Family ALS, IGFBP-5, CTGF/CCN2, ER beta/NR3A2. In some embodiments, the antibody is spe IGFBP-6, Cyró1/CCN1, IGFBP-L1, Endocan, IGFBP-rp1/ 55 cific to a marker for cancer that is ErbB2. In some embodi IGFBP-7, IGFBP-1, IGFBP-rP10, IGFBP-2, NOV/CCN3, ments, the antibody is specific to a marker for cancer that is IGFBP-3, WISP-1/CCN4, IGFBP-4: Receptor Tyrosine Kallikrein 3/PSA. In some embodiments, the antibody is Kinases Ax1, FGFR4, C1q R1/CD93, FGFR5, DDRI, Flt-3, specific to a marker for cancer that is ER alpha/NR3A1. In DDR2, HGFR, Dtk, IGF-I R, EGF, RIGF-II R, Eph, INSRR, Some embodiments, the antibody is specific to a marker for EphA1, Insulin R/CD220, EphA2, M-CSF R, EphA3, Mer, 60 cancer that is Progesterone R/NR3C3. In some embodiments, EphA4, MSPR/Ron, EphA5, MuSK, EphA6, PDGFRalpha, the antibody is specific to a marker for cancer that is A33. In EphA7, PDGF R beta, EphA8, Ret, EphB 1, RTK-like Some embodiments, the antibody is specific to a marker for Orphan Receptor 1/ROR1, EphB2, RTK-like Orphan Recep cancer that is MIA. In some embodiments, the antibody is tor 2/ROR2, EphB3, SCF R/c-kit, EphB4, Tie-1, EphB6, specific to a marker for cancer that is Aurora A. In some Tie-2, ErbB2, TrkA, ErbB3, TrkB, ErbB4, TrkC, FGF, R1-4 65 embodiments, the antibody is specific to a marker for cancer VEGFR, FGFR1, VEGFR1/Flt-1, FGFR2, VEGFR2/KDR/ that is MMP-2. In some embodiments, the antibody is specific Flk-1, FGFR3, VEGFR3/Flt-4: Proteoglycans & Regulators to a marker for cancer that is Bcl-2. In some embodiments, the US 8,450,069 B2 27 28 antibody is specific to a marker for cancer that is MMP-3. In cancer that is Kallikrein 6/Neurosin. In some embodiments, Some embodiments, the antibody is specific to a marker for the antibody is specific to a marker for cancer that is TRAF-4. cancer that is Cadherin-13. In some embodiments, the anti In Some embodiments, the antibody is specific to a marker for body is specific to a marker for cancer that is MMP-9. In some cancer that is M-CSF. In some embodiments, the antibody is embodiments, the antibody is specific to a marker for cancer specific to a marker for cancer that is uPA. In some embodi that is E-Cadherin. In some embodiments, the antibody is ments, the antibody is specific to a marker for cancer that is specific to a marker for cancer that is NEK2. In some embodi Matriptase/ST14. In some embodiments, the antibody is spe ments, the antibody is specific to a marker for cancer that is cific to a marker for cancer that is uPAR. In some embodi Carbonic Anhydrase IX. In some embodiments, the antibody ments, the antibody is specific to a marker for cancer that is is specific to a marker for cancer that is Nestin. In some 10 Mesothelin. In some embodiments, the antibody is specific to embodiments, the antibody is specific to a marker for cancer a marker for cancer that is VCAM-1. In some embodiments, that is beta-Catenin. In some embodiments, the antibody is the antibody is specific to a marker for cancer that is Methion specific to a marker for cancer that is NG2/MCSP. In some ine Aminopeptidase. In some embodiments, the antibody is embodiments, the antibody is specific to a marker for cancer specific to a marker for cancer that is VEGF. In some embodi that is Cathepsin D. In some embodiments, the antibody is 15 ments, the antibody is specific to a marker for cancer that is specific to a marker for cancer that is Osteopontin. In some Methionine Aminopeptidase 2. embodiments, the antibody is specific to a marker for cancer In some embodiments, the antibody is specific to a marker that is CD44. In some embodiments, the antibody is specific that is a marker for inflammation. In some embodiments, the to a marker for cancer that is p21(CIP1/CDKN1A. In some antibody is specific to a marker that is a marker for inflam embodiments, the antibody is specific to a marker for cancer mation that is ICAM-1. In some embodiments, the antibody is that is CEACAM-6. In some embodiments, the antibody is specific to a marker that is a marker for inflammation that is specific to a marker for cancer that is p27/Kip 1. In some RANTES. In some embodiments, the antibody is specific to a embodiments, the antibody is specific to a marker for cancer marker that is a marker for inflammation that is MIP-2. In that is Cornulin. In some embodiments, the antibody is spe Some embodiments, the antibody is specific to a marker that is cific to a marker for cancer that is p53. In some embodiments, 25 a marker for inflammation that is MIP-1 beta. In some the antibody is specific to a marker for cancer that is DPPA4. embodiments, the antibody is specific to a marker that is a In Some embodiments, the antibody is specific to a marker for marker for inflammation that is MIP-1 alpha. In some cancer that is Prolactin. In some embodiments, the antibody is embodiments, the antibody is specific to a marker that is a specific to a marker for cancer that is ECM-1. In some marker for inflammation that is MMP-3. embodiments, the antibody is specific to a marker for cancer 30 In some embodiments, the antibody is specific to a marker that is PSP94. In some embodiments, the antibody is specific that is a marker for endocrine function. In some embodi to a marker for cancer that is EGF. In some embodiments, the ments, the antibody is specific to a marker that is a marker for antibody is specific to a marker for cancer that is S100B. In endocrine function that is 17 beta-estradiol (E2). In some Some embodiments, the antibody is specific to a marker for embodiments, the antibody is specific to a marker that is a cancer that is EGFR. In some embodiments, the antibody is 35 marker for endocrine function that is DHEA. In some specific to a marker for cancer that is S100P. In some embodi embodiments, the antibody is specific to a marker that is a ments, the antibody is specific to a marker for cancer that is marker for endocrine function that is ACTH. In some embodi EMMPRIN/CD147. In some embodiments, the antibody is ments, the antibody is specific to a marker that is a marker for specific to a marker for cancer that is SCF R/c-kit. In some endocrine function that is gastrin. In some embodiments, the embodiments, the antibody is specific to a marker for cancer 40 antibody is specific to a marker that is a marker for endocrine that is Fibroblast Activation Protein alpha/FAP. In some function that is growth hormone. embodiments, the antibody is specific to a marker for cancer In some embodiments, the antibody is specific to a marker that is Serpin E1/PAI-1. In some embodiments, the antibody that is a marker for autoimmune disease. In some embodi is specific to a marker for cancer that is FGF acidic. In some ments, the antibody is specific to a marker that is a marker for embodiments, the antibody is specific to a marker for cancer 45 autoimmune disease that is GM-CSF. In some embodiments, that is Serum Amyloid A4. In some embodiments, the anti the antibody is specific to a marker that is a marker for body is specific to a marker for cancer that is FGF basic. In autoimmune disease that is C-reactive protein (CRP). In some Some embodiments, the antibody is specific to a marker for embodiments, the antibody is specific to a marker that is a cancer that is Survivin. In some embodiments, the antibody is marker for autoimmune disease that is G-CSF. specific to a marker for cancer that is Galectin-3. In some 50 In some embodiments, the antibody is specific to a marker embodiments, the antibody is specific to a marker for cancer for thyroid function. In some embodiments, the antibody is that is TEM8. In some embodiments, the antibody is specific specific to a marker for thyroid function that is cyclic AMP. In to a marker for cancer that is Glypican 3. In some embodi Some embodiments, the antibody is specific to a marker for ments, the antibody is specific to a marker for cancer that is thyroid function. In some embodiments, the antibody is spe TIMP-1. In some embodiments, the antibody is specific to a 55 cific to a marker for thyroid function that is calcitonin. In marker for cancer that is HIN-1/Secretoglobulin 3A1. In Some embodiments, the antibody is specific to a marker for Some embodiments, the antibody is specific to a marker for thyroid function. In some embodiments, the antibody is spe cancer that is TIMP-2. In some embodiments, the antibody is cific to a marker for thyroid function that is parathyroid hor specific to a marker for cancer that is IGF-I. In some embodi O. ments, the antibody is specific to a marker for cancer that is 60 In some embodiments, the antibody is specific to a marker TIMP-3. In some embodiments, the antibody is specific to a for cardiovascular function. In some embodiments, the anti marker for cancer that is IGFBP-3. In some embodiments, the body is specific to a marker for cardiovascular function that is antibody is specific to a marker for cancer that is TIMP-4. In B-natriuretic peptide. In some embodiments, the antibody is Some embodiments, the antibody is specific to a marker for specific to a marker for cardiovascular function that is NT cancer that is IL-6. In some embodiments, the antibody is 65 proBNP. In some embodiments, the antibody is specific to a specific to a marker for cancer that is TNF-alpha/TNFSF1A. marker for cardiovascular function that is C-reactive protein, In Some embodiments, the antibody is specific to a marker for HS. In some embodiments, the antibody is specific to a US 8,450,069 B2 29 30 marker for cardiovascular function that is beta-thromboglo bombesin. In some embodiments, the antibody is specific to a bulin. In some embodiments, the antibody is specific to a marker that is CD14. In some embodiments, the antibody is marker for cardiovascular function that is a cardiac troponin. specific to a marker that is CD-26. In some embodiments, the In Some embodiments, the antibody is specific to a marker for antibody is specific to a marker that is CD-38. In some cardiovascular function that is cardiac troponin I. In some embodiments, the antibody is specific to a marker that is embodiments, the antibody is specific to a marker for cardio CD-40L. In some embodiments, the antibody is specific to a vascular function that is cardiac troponin T. marker that is CD-40s. In some embodiments, the antibody is In some embodiments, the antibody is specific to a marker specific to a marker that is CDK5. In some embodiments, the for diabetes. In some embodiments, the antibody is specific to antibody is specific to a marker that is Complement C3. In a marker for diabetes that is C-peptide. In some embodiments, 10 Some embodiments, the antibody is specific to a marker that is the antibody is specific to a marker for diabetes that is leptin. Complement C4. In some embodiments, the antibody is spe In some embodiments, the antibody is specific to a marker cific to a marker that is C-peptide. In some embodiments, the for infectious disease. In some embodiments, the antibody is antibody is specific to a marker that is CRP. In some embodi specific to a marker for infectious disease that is IFN gamma. ments, the antibody is specific to a marker that is EGF. In In Some embodiments, the antibody is specific to a marker for 15 Some embodiments, the antibody is specific to a marker that is infectious disease that is IFN alpha. In some embodiments, E-selectin. In some embodiments, the antibody is specific to the antibody is specific to a marker for infectious disease that a marker that is FAS. In some embodiments, the antibody is is TREM-1. specific to a marker that is FASLG. In some embodiments, the In some embodiments, the antibody is specific to a marker antibody is specific to a marker that is Fetuin A. In some for metabolism. In some embodiments, the antibody is spe embodiments, the antibody is specific to a marker that is cific to a marker for metabolism that is bio-intactPTH (1-84). fibrinogen. In some embodiments, the antibody is specific to In Some embodiments, the antibody is specific to a marker for a marker that is ghrelin. In some embodiments, the antibody metabolism that is PTH. is specific to a marker that is glucagon. In some embodiments, In some embodiments, the antibody is specific to a marker the antibody is specific to a marker that is growth hormone. In that is IL-1 beta. In some embodiments, the antibody is spe 25 Some embodiments, the antibody is specific to a marker that is cific to a marker that is TNF-alpha. In some embodiments, the haptoglobulin. In some embodiments, the antibody is specific antibody is specific to a marker that is IL-6. In some embodi to a marker that is hepatocyte growth factor. In some embodi ments, the antibody is specific to a marker that is Tn (cardiac ments, the antibody is specific to a marker that is HGF. In troponin I). In some embodiments, the antibody is specific to Some embodiments, the antibody is specific to a marker that is a marker that is IL-8. 30 ICAM1. In some embodiments, the antibody is specific to a In some embodiments, the antibody is specific to a marker marker that is IFNG. In some embodiments, the antibody is that is Abeta 40. In some embodiments, the antibody is spe specific to a marker that is IGF1. In some embodiments, the cific to a marker that is Abeta 42. In some embodiments, the antibody is specific to a marker that is IL-1RA. In some antibody is specific to a marker that is cAMP. In some embodiments, the antibody is specific to a marker that is embodiments, the antibody is specific to a marker that is FAS 35 Il-6Sr. In some embodiments, the antibody is specific to a Ligand. In some embodiments, the antibody is specific to a marker that is IL-8. In some embodiments, the antibody is marker that is FGF-basic. In some embodiments, the antibody specific to a marker that is IL-10. In some embodiments, the is specific to a marker that is GM-CSF. In some embodiments, antibody is specific to a marker that is IL-18. In some embodi the antibody is specific to a marker that is IFN-alpha. In some ments, the antibody is specific to a marker that is ILGFBP1. In embodiments, the antibody is specific to a marker that is 40 Some embodiments, the antibody is specific to a marker that is IFN-gamma. In some embodiments, the antibody is specific ILGFBP3. In some embodiments, the antibody is specific to a to a marker that is IL-1C. In some embodiments, the antibody marker that is insulin-like growth factor 1. In some embodi is specific to a marker that is IL-2. In some embodiments, the ments, the antibody is specific to a marker that is LEP. In some antibody is specific to a marker that is IL-4. In some embodi embodiments, the antibody is specific to a marker that is ments, the antibody is specific to a marker that is IL-5. In 45 M-CSF. In some embodiments, the antibody is specific to a Some embodiments, the antibody is specific to a marker that is marker that is MMP2. In some embodiments, the antibody is IL-7. In some embodiments, the antibody is specific to a specific to a marker that is MMP9. In some embodiments, the marker that is IL-12. In some embodiments, the antibody is antibody is specific to a marker that is NGF. In some embodi specific to a marker that is In some embodiments, the anti ments, the antibody is specific to a marker that is PAI-1. In body is specific to a marker that is IL-13. In some embodi 50 Some embodiments, the antibody is specific to a marker that is ments, the antibody is specific to a marker that is IL-17. In RAGE. In some embodiments, the antibody is specific to a Some embodiments, the antibody is specific to a marker that is marker that is RSP4. In some embodiments, the antibody is MCP-1. In some embodiments, the antibody is specific to a specific to a marker that is resistin. In some embodiments, the marker that is MIP-1a. In some embodiments, the antibody is antibody is specific to a marker that is sex hormone binding specific to a marker that is RANTES. In some embodiments, 55 globulin. In some embodiments, the antibody is specific to a the antibody is specific to a marker that is VEGF. marker that is SOCX3. In some embodiments, the antibody is In some embodiments, the antibody is specific to a marker specific to a marker that is TGFbeta. In some embodiments, that is ACE. In some embodiments, the antibody is specific to the antibody is specific to a marker that is thromboplastin. In a marker that is activin A. In some embodiments, the antibody Some embodiments, the antibody is specific to a marker that is is specific to a marker that is adiponectin. In some embodi 60 TNFR1. In some embodiments, the antibody is specific to a ments, the antibody is specific to a marker that is adipsin. In marker that is VCAM-1. In some embodiments, the antibody Some embodiments, the antibody is specific to a marker that is is specific to a marker that is VWF. In some embodiments, the AgRP. In some embodiments, the antibody is specific to a antibody is specific to a marker that is TSH. In some embodi marker that is AKT1. In some embodiments, the antibody is ments, the antibody is specific to a marker that is EPITOME. specific to a marker that is albumin. In some embodiments, 65 In some embodiments, the antibody is specific to a marker the antibody is specific to a marker that is betacellulin. In that is cardiac troponin I. In some embodiments, the antibody Some embodiments, the antibody is specific to a marker that is is specific to a marker that is TREM-1. In some embodiments, US 8,450,069 B2 31 32 the antibody is specific to a marker that is IL-6. In some In some embodiments, the antibody is a polyclonal anti embodiments, the antibody is specific to a marker that is IL-8. body. In other embodiments, the antibody is a monoclonal In some embodiments, the antibody is specific to a marker antibody. that is Leukotriene T4. In some embodiments, the antibody is Capture binding partners and detection binding partner specific to a marker that is Akt1. In some embodiments, the pairs, e.g., capture and detection antibody pairs, may be used antibody is specific to a marker that is TGF-beta. In some in embodiments of the invention. Thus, in some embodi embodiments, the antibody is specific to a marker that is Fas ments, a heterogeneous assay protocol is used in which, typi ligand. cally, two binding partners, e.g., two antibodies, are used. In some embodiments, the fluorescent moiety comprises a One binding partner is a capture partner, usually immobilized 10 on a solid Support, and the other binding partner is a detection fluorescent molecule. In some embodiments, the fluorescent binding partner, typically with a detectable label attached. moiety comprises a plurality of fluorescent molecules, e.g., Such antibody pairs are available from the sources described about 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 10, 3 to 8, or 3 to 6 above, e.g., Biospacific, Emeryville, Calif. Antibody pairs fluorescent molecules. In some embodiments, the label com can also be designed and prepared by methods well-known in prises about 2 to 4 fluorescent molecules. In some embodi 15 the art. Compositions of the invention include antibody pairs ments, the fluorescent molecule comprises a molecule that wherein one member of the antibody pair is a label as comprises at least one Substituted indolium ring system in described herein, and the other member is a capture antibody. which the substituent on the 3-carbon of the indolium ring In some embodiments it is useful to use an antibody that contains a chemically reactive group or a conjugated Sub cross-reacts with a variety of species, either as a capture stance group. In some embodiments, the fluorescent mol antibody, a detection antibody, or both. Such embodiments ecules are selected from the group consisting of Alexa Fluor include the measurement of drug toxicity by determining, 488, Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 680 or e.g., release of cardiac troponin into the blood as a marker of Alexa Fluor 700. In some embodiments, the fluorescent mol cardiac damage. A cross-reacting antibody allows studies of ecules are selected from the group consisting of Alexa Fluor toxicity to be done in one species, e.g., a non-human species, 488, Alexa Fluor 532, Alexa Fluor 680 or Alexa Fluor 700. In 25 and direct transfer of the results to studies or clinical obser Some embodiments, the fluorescent molecules are Alexa Vations of another species, e.g., humans, using the same anti Fluor 647 molecules. body or antibody pair in the reagents of the assays, thus A. Binding Partners decreasing variability between assays. Thus, in Some embodi Any Suitable binding partner with the requisite specificity ments, one or more of the antibodies for use as a binding 30 partner to the marker, e.g., cardiac troponin, Such as cardiac for the form of molecule, e.g., a marker, to be detected may be troponin I, may be across-reacting antibody. In some embodi used. If the molecule, e.g., a marker, has several different ments, the antibody cross-reacts with the marker, e.g., cardiac forms, various specificities of binding partners are possible. troponin, from at least two species selected from the group Suitable binding partners are known in the art and include consisting of human, monkey, dog, and mouse. In some antibodies, aptamers, lectins, and receptors. A useful and 35 embodiments the antibody cross-reacts with the marker e.g., Versatile type of binding partner is an antibody. cardiac troponin, from all of the group consisting of human, 1. Antibodies monkey, dog, and mouse. In some embodiments, the binding partner is an antibody B. Fluorescent Moieties specific for a molecule to be detected. The term “antibody, as In some embodiments of labels used in the invention, the used herein, is a broad term and is used in its ordinary sense, 40 binding partner, e.g., antibody, is attached to a fluorescent including, without limitation, to refer to naturally occurring moiety. The fluorescence of the moiety will be sufficient to antibodies as well as non-naturally occurring antibodies, allow detection in a single molecule detector, Such as the including, for example, single chain antibodies, chimeric, single molecule detectors described herein. bifunctional and humanized antibodies, as well as antigen A“fluorescent moiety.” as that term is used herein, includes binding fragments thereof. It will be appreciated that the 45 one or more fluorescent entities whose total fluorescence is choice of epitope or region of the molecule to which the Such that the moiety may be detected in the single molecule antibody is raised will determine its specificity, e.g., for vari detectors described herein. Thus, a fluorescent moiety may ous forms of the molecule, if present, or for total (e.g., all, or comprise a single entity (e.g., a Quantum Dot or fluorescent substantially all of the molecule). molecule) or a plurality of entities (e.g., a plurality of fluo Methods for producing antibodies are well-established. 50 rescent molecules). It will be appreciated that when "moiety.” One skilled in the art will recognize that many procedures are as that term is used herein, refers to a group of fluorescent available for the production of antibodies, for example, as entities, e.g., a plurality of fluorescent dye molecules, each described in Antibodies, A Laboratory Manual, Ed Harlow individual entity may be attached to the binding partner sepa and David Lane, Cold Spring Harbor Laboratory (1988), rately or the entities may be attached together, as long as the Cold Spring Harbor, N.Y. One skilled in the art will also 55 entities as a group provide Sufficient fluorescence to be appreciate that binding fragments or Fab fragments which detected. mimic antibodies can also be prepared from genetic informa Typically, the fluorescence of the moiety involves a com tion by various procedures (Antibody Engineering: A Practi bination of quantum efficiency and lack of photobleaching cal Approach (Borrebaeck, C., ed.), 1995, Oxford University sufficient that the moiety is detectable above background Press, Oxford; J. Immunol. 149,3914-3920 (1992)). Mono 60 levels in a single molecule detector, with the consistency clonal and polyclonal antibodies to molecules, e.g., proteins, necessary for the desired limit of detection, accuracy, and and markers also commercially available (R and D Systems, precision of the assay. For example, in Some embodiments, Minneapolis, Minn.; HyTest, HyTest Ltd., Turku Finland; the fluorescence of the fluorescent moiety is such that it Abcam Inc., Cambridge, Mass., USA, Life Diagnostics, Inc., allows detection and/or quantitation of a molecule, e.g., a West Chester, Pa., USA: Fitzgerald Industries International, 65 marker, at a limit of detection of less than about 10, 5, 4, 3, 2, Inc., Concord, Mass. 0.1742-3049 USA; BiosPacific, 1, 0.1, 0.01, 0.001, 0.00001, or 0.000001 pg/ml and with a Emeryville, Calif.). coefficient of variation of less than about 20, 15, 14, 13, 12, US 8,450,069 B2 33 34 11, 10,9,8,7,6, 5, 4, 3, 2, 1% or less, e.g., about 10% or less, capable of emitting an average of at least about 100 photons in the instruments described herein. In some embodiments, when simulated by a laser emitting light at the excitation the fluorescence of the fluorescent moiety is such that it wavelength of the moiety, where the laser is focused on a spot allows detection and/or quantitation of a molecule, e.g., a of not less than about 5 microns in diameter that contains the marker, at a limit of detection of less than about 5, 1, 0.5,0.1, moiety, and wherein the total energy directed at the spot by 0.05, 0.01, 0.005, 0.001 pg/ml and with a coefficient of varia the laser is no more than about 3 microJoules. In some tion of less than about 10%, in the instruments described embodiments, the invention utilizes a fluorescent dye moiety, herein. e.g., a single fluorescent dye molecule or a plurality of fluo “Limit of detection, or LoD, as those terms are used rescent dye molecules, that is capable of emitting an average herein, includes the lowest concentration at which one can 10 of at least about 150 photons when simulated by a laser identify a sample as containing a molecule of the Substance of emitting light at the excitation wavelength of the moiety, interest, e.g., the first non-zero value. It can be defined by the where the laser is focused on a spot of not less than about 5 variability of Zeros and the slope of the standard curve. For microns in diameter that contains the moiety, and wherein the example, the limit of detection of an assay may be determined total energy directed at the spot by the laser is no more than by running a standard curve, determining the standard curve 15 about 3 microJoules. In some embodiments, the invention Zero Value, and adding 2 standard deviations to that value. A utilizes a fluorescent dye moiety, e.g., a single fluorescent dye concentration of the Substance of interest that produces a molecule or a plurality of fluorescent dye molecules, that is signal equal to this value is the “lower limit of detection” capable of emitting an average of at least about 200 photons concentration. when simulated by a laser emitting light at the excitation Furthermore, the moiety has properties that are consistent wavelength of the moiety, where the laser is focused on a spot with its use in the assay of choice. In some embodiments, the of not less than about 5 microns in diameter that contains the assay is an immunoassay, where the fluorescent moiety is moiety, and wherein the total energy directed at the spot by attached to an antibody; the moiety must have properties Such the laser is no more than about 3 microJoules. In some that it does not aggregate with other antibodies or proteins, or embodiments, the invention utilizes a fluorescent dye moiety, experiences no more aggregation than is consistent with the 25 e.g., a single fluorescent dye molecule or a plurality of fluo required accuracy and precision of the assay. In some rescent dye molecules, that is capable of emitting an average embodiments, fluorescent moieties that are preferred are of at least about 300 photons when simulated by a laser fluorescent moieties, e.g., dye molecules that have a combi emitting light at the excitation wavelength of the moiety, nation of 1) high absorption coefficient; 2) high quantum where the laser is focused on a spot of not less than about 5 yield; 3) high photostability (low photobleaching); and 4) 30 microns in diameter that contains the moiety, and wherein the compatibility with labeling the molecule of interest (e.g., total energy directed at the spot by the laser is no more than protein) so that it may be analyzed using the analyzers and about 3 microJoules. In some embodiments, the invention systems of the invention (e.g., does not cause precipitation of utilizes a fluorescent dye moiety e.g., a single fluorescent dye the protein of interest, or precipitation of a protein to which molecule or a plurality of fluorescent dye molecules, that is the moiety has been attached). 35 capable of emitting an average of at least about 500 photons Fluorescent moieties, e.g., a single fluorescent dye mol when simulated by a laser emitting light at the excitation ecule or a plurality of fluorescent dye molecules, that are wavelength of the moiety, where the laser is focused on a spot useful in some embodiments of the invention may be defined of not less than about 5 microns in diameter that contains the in terms of their photon emission characteristics when stimu moiety, and wherein the total energy directed at the spot by lated by EM radiation. For example, in some embodiments, 40 the laser is no more than about 3 microJoules. the invention utilizes a fluorescent moiety, e.g., a moiety In some embodiments, the fluorescent moiety comprises comprising a single fluorescent dye molecule or a plurality of an average of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fluorescent dye molecules, that is capable of emitting an fluorescent entities, e.g., fluorescent molecules. In some average of at least about 10, 20,30, 40, 50, 75, 100, 125, 150, embodiments, the fluorescent moiety comprises an average of 175, 200, 225, 250, 275,300, 350, 400, 500, 600, 700, 800, 45 no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 fluorescent 900, or 1000, photons when simulated by a laser emitting entities, e.g., fluorescent molecules. In some embodiments, light at the excitation wavelength of the moiety, where the the fluorescent moiety comprises an average of about 1 to 11, laser is focused on a spot of not less than about 5 microns in or about 2 to 10, or about 2 to 8, or about 2 to 6, or about 2 to diameter that contains the moiety, and where the total energy 5, or about 2 to 4, or about 3 to 10, or about 3 to 8, or about 3 directed at the spot by the laser is no more than about 3 50 to 6, or about 3 to 5, or about 4 to 10, or about 4 to 8, or about microJoules. It will be appreciated that the total energy may 4 to 6, or about 2, 3, 4, 5, 6, or more than about 6 fluorescent beachieved by many different combinations of power output entities. In some embodiments, the fluorescent moiety com of the laser and length of time of exposure of the dye moiety. prises an average of about 2 to 8 fluorescent moieties are E.g., a laser of a power output of 1 mW may be used for 3 ms. attached. In some embodiments, the fluorescent moiety com 3 mW for 1 ms, 6 mW for 0.5ms, 12 mW for 0.25 ms, and so 55 prises an average of about 2 to 6 fluorescent entities. In some O. embodiments, the fluorescent moiety comprises an average of In some embodiments, the invention utilizes a fluorescent about 2 to 4 fluorescent entities. In some embodiments, the dye moiety, e.g., a single fluorescent dye molecule or a plu fluorescent moiety comprises an average of about 3 to 10 rality of fluorescent dye molecules, that is capable of emitting fluorescent entities. In some embodiments, the fluorescent an average of at least about 50 photons when simulated by a 60 moiety comprises an average of about 3 to 8 fluorescent laser emitting light at the excitation wavelength of the moiety, entities. In some embodiments, the fluorescent moiety com where the laser is focused on a spot of not less than about 5 prises an average of about 3 to 6 fluorescent entities. By microns in diameter that contains the moiety, and wherein the "average' it is meant that, in a given sample that is represen total energy directed at the spot by the laser is no more than tative of a group of labels of the invention, where the sample about 3 microJoules. In some embodiments, the invention 65 contains a plurality of the binding partner-fluorescent moiety utilizes a fluorescent dye moiety, e.g., a single fluorescent dye units, the molar ratio of the particular fluorescententity to the molecule or a plurality of fluorescent dye molecules, that is binding partner, as determined by standard analytical meth US 8,450,069 B2 35 36 ods, corresponds to the number or range of numbers speci moiety, and wherein the total energy directed at the spot by fied. For example, in embodiments wherein the label com the laser is no more than about 3 microJoules. In some prises a binding partner that is an antibody and a fluorescent embodiments, the invention utilizes a fluorescent dye moiety, moiety that comprises a plurality of fluorescent dye mol e.g., a single fluorescent dye molecule or a plurality of fluo ecules of a specific absorbance, a spectrophotometric assay rescent dye molecules, that is capable of emitting an average can be used in which a solution of the label is diluted to an of at least about 150 photons when simulated by a laser appropriate level and the absorbance at 280 nm is taken to emitting light at the excitation wavelength of the moiety, determine the molarity of the protein (antibody) and an absor where the laser is focused on a spot of not less than about 5 bance at, e.g., 650 nm (for Alexa Fluor 647), is taken to microns in diameter that contains the moiety, and wherein the determine the molarity of the fluorescent dye molecule. The 10 total energy directed at the spot by the laser is no more than ratio of the latter molarity to the former represents the average about 3 microJoules. In some embodiments, the invention number of fluorescent entities (dye molecules) in the fluores utilizes a fluorescent dye moiety, e.g., a single fluorescent dye cent moiety attached to each antibody. molecule or a plurality of fluorescent dye molecules, that is 1. Dyes capable of emitting an average of at least about 200 photons In some embodiments, the invention uses fluorescent moi 15 when simulated by a laser emitting light at the excitation eties that comprise fluorescent dye molecules. In some wavelength of the moiety, where the laser is focused on a spot embodiments, the invention utilizes a fluorescent dye mol of not less than about 5 microns in diameter that contains the ecule that is capable of emitting an average of at least about 50 moiety, and wherein the total energy directed at the spot by photons when simulated by a laser emitting light at the exci the laser is no more than about 3 microJoules. In some tation wavelength of the molecule, where the laser is focused embodiments, the invention utilizes a fluorescent dye moiety, on a spot of not less than about 5 microns in diameter that e.g., a single fluorescent dye molecule or a plurality of fluo contains the molecule, and wherein the total energy directed rescent dye molecules, that is capable of emitting an average at the spot by the laser is no more than about 3 microJoules. In of at least about 300 photons when simulated by a laser Some embodiments, the invention utilizes a fluorescent dye emitting light at the excitation wavelength of the moiety, molecule that is capable of emitting an average of at least 25 where the laser is focused on a spot of not less than about 5 about 75 photons when simulated by a laser emitting light at microns in diameter that contains the moiety, and wherein the the excitation wavelength of the molecule, where the laser is total energy directed at the spot by the laser is no more than focused on a spot of not less than about 5 microns in diameter about 3 microJoules. In some embodiments, the invention that contains the molecule, and wherein the total energy utilizes a fluorescent dye moiety, e.g., a single fluorescent dye directed at the spot by the laser is no more than about 3 30 molecule or a plurality of fluorescent dye molecules, that is microJoules. In some embodiments, the invention utilizes a capable of emitting an average of at least about 500 photons fluorescent dye molecule that is capable of emitting an aver when simulated by a laser emitting light at the excitation age of at least about 100 photons when simulated by a laser wavelength of the moiety, where the laser is focused on a spot emitting light at the excitation wavelength of the molecule, of not less than about 5 microns in diameter that contains the where the laser is focused on a spot of not less than about 5 35 moiety, and wherein the total energy directed at the spot by microns in diameter that contains the molecule, and wherein the laser is no more than about 3 microJoules. the total energy directed at the spot by the laser is no more A non-inclusive list of useful fluorescententities for use in than about 3 microJoules. In some embodiments, the inven the fluorescent moieties of the invention is given in Table 2, tion utilizes a fluorescent dye molecule that is capable of below. In some embodiments, the fluorescent dye is selected emitting an average of at least about 150 photons when simu 40 from the group consisting of Alexa Fluor 488, Alexa Fluor lated by a laser emitting light at the excitation wavelength of 532, Alexa Fluor 647, Alexa Fluor 700, Alexa Fluor 750, the molecule, where the laser is focused on a spot of not less Fluorescein, B-phycoerythrin, allophycocyanin, PBXL-3, than about 5 microns in diameter that contains the molecule, and Qdot 605. In some embodiments, the fluorescent dye is and wherein the total energy directed at the spot by the laser selected from the group consisting of Alexa Fluor 488, Alexa is no more than about 3 microJoules. In some embodiments, 45 the invention utilizes a fluorescent dye molecule that is Fluor 532, Alexa Fluor 700, Alexa Fluor 750, Fluorescein, capable of emitting an average of at least about 200 photons B-phycoerythrin, allophycocyanin, PBXL-3, and Qdot 605. when simulated by a laser emitting light at the excitation wavelength of the molecule, where the laser is focused on a TABLE 2 spot of not less than about 5 microns in diameter that contains 50 FLUORESCENTENTITIES the molecule, and wherein the total energy directed at the spot by the laser is no more than about 3 microJoules. Dye E EX (nm) E (M)-1 Em (nm) MMw In Some embodiments, the invention uses a fluorescent dye Bimane 380 5,700 458 282.31 moiety, e.g., a single fluorescent dye molecule or a plurality of Dapoxyl 373 22,000 551 362.83 fluorescent dye molecules, that is capable of emitting an 55 Dimethylamino 375 22,000 470 344.32 coumarin-4-acetic average of at least about 50 photons when simulated by a laser acid emitting light at the excitation wavelength of the moiety, Marina blue 365 19,000 460 367.26 where the laser is focused on a spot of not less than about 5 8-Anilino 372 480 naphthalene-1- microns in diameter that contains the moiety, and wherein the Sulfonic total energy directed at the spot by the laser is no more than 60 acid about 3 microJoules. In some embodiments, the invention Cascade blue 376 23,000 420 6O7.42 utilizes a fluorescent dye moiety, e.g., a single fluorescent dye Alexa Fluor 405 4O2 35,000 421 1028.26 molecule or a plurality of fluorescent dye molecules, that is Cascade blue 400 29,000 420 6O7.42 Cascade yellow 4O2 24,000 545 S63.54 capable of emitting an average of at least about 100 photons Pacific blue 410 46,000 455 339.21 when simulated by a laser emitting light at the excitation 65 PyMPO 415 26,000 570 582.41 wavelength of the moiety, where the laser is focused on a spot Alexa Fluor 430 433 15,000 539 701.75 of not less than about 5 microns in diameter that contains the US 8,450,069 B2 37 38 TABLE 2-continued TABLE 2-continued Atto-425 438 486 DY-730 734 18S.OOO 750 660.88 NBD 465 22,000 535 391.34 DY-731 736 22S.OOO 759 762.92 Alexa Fluor 488 495 73,000 519 643.41 DY-750 747 24OOOO 776 712.96 Fluorescein 494 79,000 S18 376.32 DY-751 751 22O.OOO 779 814.99 Oregon Green 488 496 76,000 524 SO9.38 DY-776 771 147.OOO 8O1 834.98 Atto 495 495 522 DY-78O-OH 770 7O.OOO 810 757.34 Cy2 489 150,000 SO6 713.78 DY-780-P 770 7O.OOO 810 957.55 DY-480-XL 500 40,000 630 514.60 DY-781 783 98.000 800 762.92 DY-485-XL 485 20,000 S60 502.59 DY-782 782 102.OOO 800 660.88 DY-490-XL 486 27,000 532 S36.58 10 EVOblue-10 651 101.440 664 389.88 DYSOO-XL 505 90,000 555 596.68 EVOblue-30 652 102.OOO 672 447.51 DY-520-XL 52O 40,000 664 514.60 Alexa Fluor 532 531 81,000 554 723.77 Quantum Dots: Qdot 525, QD 565, QD 585, QD 605, QD 655, QD 705, QD 800 BODIPY 530,550 534 77,000 554 S13.31 6-HEX 535 98,000 556 68O.O7 Suitable dyes for use in the invention include modified 6-JOE 522 75,000 550 6O2.34 15 Rhodamine 6G 525 108,000 555 555.59 carbocyanine dyes. On Such modification comprises modifi Atto-520 52O S42 cation of an indolium ring of the carbocyanine dye to permit Cy3B 558 130,000 572 658.00 a reactive group or conjugated Substance at the number three Alexa Fluor 610 612 138,000 628 position. The modification of the indolium ring provides dye Alexa Fluor 633 632 159,000 647 Cal. 1200 Alexa Fluor 647 6SO 250,000 668 Cal. 12SO conjugates that are uniformly and Substantially more fluores BODIPY 630,650 625 101,000 640 66O.SO cent on proteins, nucleic acids and other biopolymers, than Cy5 649 250,000 670 791.99 conjugates labeled with structurally similar carbocyanine Alexa Fluor 660 663 110,000 690 Alexa Fluor 680 679 184,000 702 dyes bound through the nitrogen atom at the number one Alexa Fluor 700 702 192,000 723 position. In addition to having more intense fluorescence Alexa Fluor 750 749 240,000 782 emission than structurally similar dyes at virtually identical B-phycoerythrin 546,565 2,410,000 575 240,000 25 R-phycoerythrin 480, 546,565 1960,000 578 240,000 wavelengths, and decreased artifacts in their absorption spec Allophycocyanin 6SO 700,000 660 700,000 tra upon conjugation to biopolymers, the modified carbocya PBXL-1 545 666 nine dyes have greater photostability and higher absorbance PBXL-3 614 662 (extinction coefficients) at the wavelengths of peak absor Atto-tec dyes 30 bance than the structurally similar dyes. Thus, the modified carbocyanine dyes result in greater sensitivity in assays using Name Ex (nm) Em (nm) QY T (ns) the modified dyes and their conjugates. Preferred modified Atto 425 436 486 O.9 3.5 dyes include compounds that have at least one Substituted Atto 495 495 522 O.45 2.4 indolium ring system in which the substituent on the 3-carbon Atto 520 52O S42 O.9 3.6 35 Atto S60 S61 585 O.92 3.4 of the indolium ring contains a chemically reactive group or a Atto 590 598 634 O.8 3.7 conjugated Substance. Other dye compounds include com Atto 610 60S 630 0.7 3.3 pounds that incorporate an aZabenzazolium ring moiety and Atto 655 665 690 O.3 1.9 at least one Sulfonate moiety. The modified carbocyanine Atto 680 68O 702 O.3 1.8 dyes that can be used to detect individual molecules in various Dyomics Fluors 40 embodiments of the invention are described in U.S. Pat. No. 6,977.305, which is herein incorporated by reference in its Molar molecular entirety. Thus, in some embodiments the labels of the inven absorbance weightii tion utilize a fluorescent dye that includes a substituted indo Label EX (nm) l mol-1 cm-1 Em (nm) g" mol-1 lium ring system in which the substituent on the 3-carbon of DY-495.5 495 70,000 520 489.47 45 the indolium ring contains a chemically reactive group or a DY-495, 6 495 70,000 520 489.47 conjugated Substance group. DY-495X5 495 70,000 520 525.95 DY-49SX6 495 70,000 520 525.95 In some embodiments, the label comprises a fluorescent DY-505/5 505 85,000 530 485.49 moiety that includes one or more Alexa Fluor dyes (Molecu DY-505/6 505 85,000 530 485.49 lar Probes, Eugene, Oreg.). The Alexa Fluor dyes are dis DY-505X5 505 85,000 530 523.97 50 closed in U.S. Pat. Nos. 6,977.305; 6,974,874; 6,130,101; and DY-505X6 505 85,000 530 523.97 DY-550 553 122,000 578 667.76 6,974,305 which are herein incorporated by reference in their DY-555 555 OO.OOO S8O 636.18 entirety. Some embodiments of the invention utilize a dye DY 610 609 81.000 629 667.75 chosen from the group consisting of Alexa Fluor 647, Alexa DY-615 621 2OO.OOO 641 578.73 Fluor 488, Alexa Fluor 532, Alexa Fluor 555, Alexa Fluor DY-630 636 2OO.OOO 657 634.84 55 DY-631 637 85.OOO 658 736.88 610, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750. DY-633 637 8O.OOO 657 751.92 Some embodiments of the invention utilize a dye chosen from DY-635 647 75.000 671 658.86 the group consisting of Alexa Fluor 488, Alexa Fluor 532, DY-636 645 90.OOO 671 760.91 Alexa Fluor 647, Alexa Fluor 700 and Alexa Fluor 750. Some DY-650 653 70.OOO 674 686.92 DY-651 653 6O.OOO 678 888.96 embodiments of the invention utilize a dye chosen from the DYQ-660 660 117,000 668.86 60 group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa DYQ-661 661 116,000 770.90 Fluor 555, Alexa Fluor 610, Alexa Fluor 680, Alexa Fluor DY-675 674 1.O.OOO 699 7O6.91 700, and Alexa Fluor 750. Some embodiments of the inven DY-676 674 45.OOO 699 807.95 DY-680 690 2S.OOO 709 634.84 tion utilize the Alexa Fluor 647 molecule, which has an DY-681 691 2S.OOO 708 736.88 absorption maximum between about 650 and 660 nm and an DY-700 702 96.OOO 723 668.86 65 emission maximum between about 660 and 670 nm. The DY.701 706 1S.OOO 731 770.90 Alexa Fluor 647 dye is used alone or in combination with other Alexa Fluor dyes. US 8,450,069 B2 39 40 Currently available organic fluors can be improved by ren single molecule analyzer is labeled with one quantum dot. In dering them less hydrophobic by adding hydrophilic groups some embodiments, the quantum dot is between 10 and 20 nm Such as polyethylene. Alternatively, currently sulfonated in diameter. In other embodiments, the quantum dot is organic fluors such as the Alexa Fluor 647 dye can be ren between 2 and 10 nm in diameter. In other embodiments, the dered less acidic by making them Zwitterionic. Particles such 5 quantum dot is about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 as antibodies that are labeled with the modified fluors are less nm, 9 mm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15V, 16 nm, likely to bind non-specifically to Surfaces and proteins in 17 nm, 18 nm, 19 nm or 20 nm in diameter. Useful Quantum immunoassays, and thus enable assays that have greater sen Dots comprise QD 605, QD 610, QD 655, and QD 705. A sitivity and lower backgrounds. Methods for modifying and preferred Quantum Dot is QD 605. improving the properties of fluorescent dyes for the purpose 10 C. Binding Partner-Fluorescent Moiety Compositions of increasing the sensitivity of a system that detects single The labels of the invention generally contain a binding molecules are known in the art. Preferably, the modification partner, e.g., antibody, bound to a fluorescent moiety to pro improves the Stokes shift while maintaining a high quantum vide the requisite fluorescence for detection and quantitation yield. in the instruments described herein. Any suitable combina 2. Quantum Dots 15 tion of binding partner and fluorescent moiety for detection in In some embodiments, the fluorescent label moiety that is the single molecule detectors described herein may be used as used to detect a molecule in a sample using the analyzer a label in the invention. In some embodiments, the invention systems of the invention is a quantum dot. Quantum dots provides a label for a marker of a biological state, where the (QDs), also known as semiconductor nanocrystals or artificial label includes an antibody to the marker and a fluorescent atoms, are semiconductor crystals that contain anywhere moiety. The marker may be any of the markers described between 100 to 1,000 electrons and range from 2-10 nm. above. The antibody may be any antibody as described above. Some QDs can be between 10-20 nm in diameter. QDs have A fluorescent moiety may be attached such that the label is high quantum yields, which makes them particularly useful capable of emitting an average of at least about 50, 75, 100, for optical applications. QDs are fluorophores that fluoresce 125, 150, 175, 200, 225, 250, 275,300, 350, 400, 500, 600, by forming excitons, which are similar to the excited State of 25 700, 800, 900, or 1000, photons when simulated by a laser traditional fluorophores, but have much longer lifetimes of up emitting light at the excitation wavelength of the moiety, to 200 nanoseconds. This property provides QDs with low where the laser is focused on a spot of not less than about 5 photobleaching. The energy level of QDs can be controlled by microns in diameter that contains the label, and wherein the changing the size and shape of the QD, and the depth of the total energy directed at the spot by the laser is no more than QDs' potential. One optical feature of small excitonic QDs is 30 about 3 microJoules. In some embodiments, the fluorescent coloration, which is determined by the size of the dot. The moiety may be a fluorescent moiety that is capable of emitting larger the dot, the redder, or more towards the red end of the an average of at least about 50, 100, 150, or 200 photons when spectrum the fluorescence. The smaller the dot, the bluer or simulated by a laser emitting light at the excitation wave more towards the blue end it is. The bandgap energy that length of the moiety, where the laser is focused on a spot of determines the energy and hence the color of the fluoresced 35 not less than about 5 microns in diameter that contains the light is inversely proportional to the square of the size of the moiety, and wherein the total energy directed at the spot by QD. Larger QDs have more energy levels which are more the laser is no more than about 3 microJoules. The fluorescent closely spaced, thus allowing the QD to absorb photons con moiety may be a fluorescent moiety that includes one or more taining less energy, i.e., those closer to the red end of the dye molecules with a structure that includes a substituted spectrum. Because the emission frequency of a dot is depen 40 indolium ring system in which the substituent on the 3-carbon dent on the bandgap, it is possible to control the output wave of the indolium ring contains a chemically reactive group or a length of a dot with extreme precision. In some embodiments conjugated Substance group. The label composition may the protein that is detected with the single molecule analyzer include a fluorescent moiety that includes one or more dye system is labeled with a QD. In some embodiments, the single molecules selected from the group consisting of Alexa Fluor molecule analyzer is used to detect a protein labeled with one 45 488, Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 700, or QD and using a filter to allow for the detection of different Alexa Fluor 750. The label composition may include a fluo proteins at different wavelengths. rescent moiety that includes one or more dye molecules QDS have broad excitation and narrow emission properties selected from the group consisting of Alexa Fluor 488, Alexa which, when used with color filtering, require only a single Fluor 532, Alexa Fluor 700, or Alexa Fluor 750. The label electromagnetic source to resolve individual signals during 50 composition may include a fluorescent moiety that includes multiplex analysis of multiple targets in a single sample. one or more dye molecules that are Alexa Fluor 488. The label Thus, in some embodiments, the analyzer system comprises composition may include a fluorescent moiety that includes one continuous wave laser and particles that are each labeled one or more dye molecules that are Alexa Fluor 555. The label with one QD. Colloidally prepared QDs are free floating and composition may include a fluorescent moiety that includes can be attached to a variety of molecules via metal coordinat 55 one or more dye molecules that are Alexa Fluor 610. The label ing functional groups. These groups include but are not lim composition may include a fluorescent moiety that includes ited to thiol, amine, nitrile, phosphine, phosphine oxide, one or more dye molecules that are Alexa Fluor 647. The label phosphonic acid, carboxylic acids or other ligands. By bond composition may include a fluorescent moiety that includes ing appropriate molecules to the Surface, the quantum dots one or more dye molecules that are Alexa Fluor 680. The label can be dispersed or dissolved in nearly any solvent or incor 60 composition may include a fluorescent moiety that includes porated into a variety of inorganic and organic films. Quan one or more dye molecules that are Alexa Fluor 700. The label tum dots (QDs) can be coupled to streptavidin directly composition may include a fluorescent moiety that includes through a maleimide ester coupling reaction or to antibodies one or more dye molecules that are Alexa Fluor 750. through a meleimide-thiol coupling reaction. This yields a In some embodiments the invention provides a composi material with a biomolecule covalently attached on the sur 65 tion for the detection of a marker of a biological state that face, which produces conjugates with high specific activity. includes an Alexa Fluor molecule, e.g., an Alexa Fluor mol In some embodiments, the protein that is detected with the ecule selected from the described groups. Such as an Alexa US 8,450,069 B2 41 42 Fluor 647 molecule attached to an antibody specific for the invention also encompasses binding partner pairs, e.g., anti marker. In some embodiments the composition includes an bodies, wherein both antibodies are specific to the same mol average of about 1 to 11, or about 2 to 10, or about 2 to 8, or ecule, e.g., the same marker, and wherein at least one member about 2 to 6, or about 2 to 5, or about 2 to 4, or about 3 to 10, of the pair is a label as described herein. Thus, for any label or about 3 to 8, or about 3 to 6, or about 3 to 5, or about 4 to that includes a binding-partner and a fluorescent moiety, the 10, or about 4 to 8, or about 4 to 6, or about 2,3,4, 5, 6, or more invention also encompasses a pair of binding partners than about 6 Alexa Fluor 647 molecules attached to an anti wherein the first binding partner, e.g., antibody, is part of the body for the marker. In some embodiments the invention label, and the second binding partner, e.g., antibody, is, typi provides a composition for the detection a marker of a bio cally, unlabeled and serves as a capture binding partner. In logical state that includes an average of about 1 to 11, or about 10 addition, binding partner pairs are frequently used in FRET 2 to 10, or about 2 to 8, or about 2 to 6, or about 2 to 5, or about assays. FRET assays useful in the invention are disclosed in 2 to 4, or about 3 to 10, or about 3 to 8, or about 3 to 6, or about U.S. patent application Ser. No. 11/048,660, incorporated by 3 to 5, or about 4 to 10, or about 4 to 8, or about 4 to 6, or about reference herein in its entirety, and the present invention also 2, 3, 4, 5, 6, or more than about 6 Alexa Fluor 647 molecules encompasses binding partner pairs, each of which includes a attached to an antibody specific to the marker. In some 15 FRET label. embodiments the invention provides a composition for the IV. Highly Sensitive Analysis of Molecules detection of a marker of a biological state that includes an In one aspect, the invention provides a method for deter average of about 2 to 10 Alexa Fluor 647 molecules attached mining the presence or absence of a single molecule, e.g., a to an antibody specific to the marker. In some embodiments molecule of a marker of a biological state, in a sample, by i) the invention provides a composition for the detection of a labeling the molecule if present, with a label; and ii) detecting marker of a biological state that includes an average of about the presence or absence of the label, where the detection of the 2 to 8 Alexa Fluor 647 molecules attached to an antibody presence of the label indicates the presence of the single specific to the marker. In some embodiments the invention molecule in the sample. In some embodiments, the method is provides a composition for the detection of a marker of a capable of detecting the moleculeata limit of detection of less biological state that includes an average of about 2 to 6 Alexa 25 than about 100, 80, 60, 50, 40, 30, 20, 15, 12, 10,9,8,7,6, 5, Fluor 647 molecules attached to an antibody specific to the 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6,0.5,0.4,0.3, 0.2,0.1, 0.05, 0.01, marker. In some embodiments the invention provides a com 0.005, or 0.001 femtomolar. In some embodiments, the position for the detection of a marker of a biological state that method is capable of detecting the molecule at a limit of includes an average of about 2 to 4 Alexa Fluor 647 molecules detection of less than about 100 femtomolar. In some embodi attached to an antibody specific to the marker. In some 30 ments, the method is capable of detecting the molecule at a embodiments the invention provides a composition for the limit of detection of less than about 10 femtomolar. In some detection of a marker of a biological state that includes an embodiments, the method is capable of detecting the mol average of about 3 to 8 Alexa Fluor 647 molecules attached to ecule at a limit of detection of less than about 1 femtomolar. an antibody specific to the marker. In some embodiments the In some embodiments, the method is capable of detecting the invention provides a composition for the detection of a 35 molecule at a limit of detection of less than about 0.1 femto marker of a biological state that includes an average of about molar. In some embodiments, the method is capable of detect 3 to 6 Alexa Fluor 647 molecules attached to an antibody ing the molecule at a limit of detection of less than about 0.01 specific to the marker. In some embodiments the invention femtomolar. In some embodiments, the method is capable of provides a composition for the detection of a marker of a detecting the molecule at a limit of detection of less than biological state that includes an average of about 4 to 8 Alexa 40 about 0.001 femtomolar. Detection limits may be determined Fluor 647 molecules attached to an antibody specific to the by use of an appropriate standard, e.g., National Institute of marker. Standards and Technology reference standard material. Attachment of the fluorescent moiety, or fluorescent enti The methods also provide methods of determining a con ties that make up the fluorescent moiety, to the binding part centration of a molecule, e.g., a marker indicative of a bio ner, e.g., antibody, may be by any suitable means; such meth 45 logical state, in a sample by detecting single molecules of the ods are well-known in the art and exemplary methods are molecule in the sample. The “detecting of a single molecule given in the Examples. In some embodiments, after attach includes detecting the molecule directly or indirectly. In the ment of the fluorescent moiety to the binding partner to form case of indirect detection, labels that correspond to single a label for use in the methods of the invention, and prior to the molecules, e.g., labels attached to the single molecules, can use of the label for labeling the protein of interest, it is useful 50 be detected. to perform a filtration step. E.g., an antibody-dye label may be In some embodiments, the invention provides a method for filtered prior to use, e.g., through a 0.2 micron filter, or any determining the presence or absence of a single molecule of a Suitable filter for removing aggregates. Other reagents for use protein in a biological sample, comprising labeling said mol in the assays of the invention may also be filtered, e.g., ecule with a label and detecting the presence or absence of through a 0.2 micron filter, or any suitable filter. Without 55 said label in a single molecule detector, wherein said label being bound by theory, it is thought that such filtration comprises a fluorescent moiety that is capable of emitting at removes a portion of the aggregates of the, e.g., antibody-dye least about 200 photons when simulated by a laser emitting labels. AS Such aggregates can bind as a unit to the protein of light at the excitation wavelength of the moiety, wherein the interest, but upon release in elution buffer are likely to disag laser is focused on a spot not less than about 5 microns in gregate, false positives may result; i.e., several labels will be 60 diameter that contains the moiety, and wherein the total detected from an aggregate that has bound to only a single energy directed at the spot by the laser is no more than about protein molecule of interest. Regardless of theory, filtration 3 microJoules. The single molecule detector may, in some has been found to reduce false positives in the Subsequent embodiments, comprise not more than one interrogation assay and to improve accuracy and precision. space. The limit of detection of the single molecule in the It will be appreciated that immunoassays often employ a 65 sample may be less than about 10, 1, 0.1, 0.01, or 0.001 sandwich format, in which binding partner pairs, e.g., anti femtomolar. In some embodiments, the limit of detection is bodies, to the same molecule, e.g., a marker, are used. The less than about 1 femtomolar. The detecting may comprise US 8,450,069 B2 43 44 detecting electromagnetic radiation emitted by said fluores the sample is a nasal Swab. In some embodiments, the sample cent moiety. The method may further comprise exposing said is a cell lysate. In some embodiments, the sample is a tissue fluorescent moiety to electromagnetic radiation, e.g., electro sample. magnetic radiation provided by a laser, Such as a laser with a B. Sample Preparation power output of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. In general, any method of sample preparation may be used 15, 16, 17, 18, 19, 20 mW. In some embodiments, the laser that produces a label corresponding to a molecule of interest, stimulus provides light to the interrogation space for between e.g., a marker of a biological state to be measured, where the about 10-1000 microseconds, or about 1000, 250, 100, 50, 25 label is detectable in the instruments described herein. As is or 10 microseconds. In some embodiments, the label further known in the art, sample preparation in which a label is added comprises a binding partner specific for binding said mol 10 to one or more molecules may be performed in a homoge neous or heterogeneous format. In some embodiments, the ecule. Such as an antibody. In some embodiments, the fluo sample preparation is formed in a homogenous format. In rescent moiety comprises a fluorescent dye molecule. Such as analyzer systems employing a homogenous format, unbound a dye molecule that comprises at least one Substituted indo label is not removed from the sample. See, e.g., U.S. patent lium ring system in which the substituent on the 3-carbon of 15 application Ser. No. 11/048,660. In some embodiments, the the indolium ring contains a chemically reactive group or a particle or particles of interest are labeled by addition of conjugated Substance. In some embodiments, the dye mol labeled antibody or antibodies that bind to the particle or ecule is an AlexFluor molecule selected from the group con particles of interest. sisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 647, In some embodiments, a heterogeneous assay format is Alexa Fluor 680 or Alexa Fluor 700. In some embodiments, used, where, typically, a step is employed for removing the dye molecule is an Alexa Fluor 647 dye molecule. In some unbound label. Such assay formats are well-known in the art. embodiments, the fluorescent moiety comprises a plurality of One particularly useful assay format is a sandwich assay, e.g., Alexa Fluor 647 molecules. In some embodiments, the plu a sandwich immunoassay. In this format, the molecule of rality of Alexa Fluor 647 molecules comprises about 2-4 interest, e.g., marker of a biological state, is captured, e.g., on Alexa Fluor 647 molecules, or about 3-6 Alexa Fluor 647 25 a Solid Support, using a capture binding partner. Unwanted molecules. In some embodiments, the fluorescent moiety is a molecules and other Substances may then optionally be quantum dot. The method may further comprise measuring washed away, followed by binding of a label comprising a the concentration of said protein in the sample. detection binding partner and a detectable label, e.g., fluores In some embodiments, detecting the presence or absence of cent moiety. Further washes remove unbound label, then the said label comprises: (i) passing a portion of said sample 30 detectable label is released, usually though not necessarily through an interrogation space; (ii) Subjecting said interroga still attached to the detection binding partner. In alternative tion space to exposure to electromagnetic radiation, said elec embodiments, sample and label are added to the capture tromagnetic radiation being Sufficient to stimulate said fluo binding partner without a wash in between, e.g., at the same rescent moiety to emit photons, if said label is present; and time. Other variations will be apparent to one of skill in the (iii) detecting photons emitted during said exposure of step 35 art. (ii). The method may further comprise determining a back In some embodiments, the method for detecting the mol ground photon level in said interrogation space, wherein said ecule of interest, e.g., marker of a biological state, uses a background level represents the average photon emission of sandwich assay with antibodies, e.g., monoclonal antibodies the interrogation space when it is subjected to electromag as capture binding partners. The method comprises binding netic radiation in the same manner as in step (ii), but without 40 molecules in a sample to a capture antibody that is immobi label in the interrogation space. The method may further lized on a binding Surface, and binding the label comprising a comprise comparing the amount of photons detected in step detection antibody to the molecule to form a “sandwich' (iii) to a threshold photon level, wherein said threshold pho complex. The label comprises the detection antibody and a ton level is a function of said background photon level. fluorescent moiety, as described herein, which is detected, wherein an amount of photons detected in Step (iii) greater 45 e.g., using the single molecule analyzers of the invention. that the threshold level indicates the presence of said label, Both the capture and detection antibodies specifically bind and an amount of photons detected in step (iii) equal to or less the molecule. Many examples of sandwich immunoassays are than the threshold level indicates the absence of said label. known, and some are described in U.S. Pat. No. 4,168,146 to A. Sample Grubb et al. and U.S. Pat. No. 4,366,241 to Tom et al., both of The sample may be any suitable sample. Typically, the 50 which are incorporated herein by reference. Further examples sample is a biological sample, e.g., a biological fluid. Such specific to specific markers are described in the Examples. fluids include, without limitation, bronchoalveolar lavage The capture binding partner may be attached to a solid fluid (BAL), blood, serum, plasma, urine, nasal Swab, cere Support, e.g., a microtiter plate or paramagnetic beads. In brospinal fluid, pleural fluid, synovial fluid, peritoneal fluid, Some embodiments, the invention provides a binding partner amniotic fluid, gastric fluid, lymph fluid, interstitial fluid, 55 for a molecule of interest, e.g., marker of a biological state, tissue homogenate, cell extracts, saliva, sputum, stool, physi attached to a paramagnetic bead. Any Suitable binding partner ological Secretions, tears, mucus, Sweat, milk, semen, semi that is specific for the molecule that it is wished to capture nal fluid, vaginal Secretions, fluid from ulcers and other Sur may be used. The binding partner may be an antibody, e.g., a face eruptions, blisters, and abscesses, and extracts of tissues monoclonal antibody. Production and sources of antibodies including biopsies of normal, malignant, and Suspect tissues 60 are described elsewhere herein. It will be appreciated that or any other constituents of the body which may contain the antibodies identified herein as useful as a capture antibody target particle of interest. Other similar specimens such as cell may also be useful as detection antibodies, and vice versa. or tissue culture or culture broth are also of interest. The attachment of the binding partner, e.g., antibody, to the In some embodiments, the sample is a blood sample. In Solid Support may be covalent or noncovalent. In some Some embodiments the sample is a plasma sample. In some 65 embodiments, the attachment is noncovalent. An example of embodiments the sample is a serum sample. In some embodi a noncovalent attachment well-known in the art is biotin ments, the sample is a urine sample. In some embodiments, avidin/streptavidin interactions. Thus, in Some embodiments, US 8,450,069 B2 45 46 a solid Support, e.g., a microtiterplate or aparamagnetic bead, of the molecule of interest to the capture binding partner, e.g., is attached to the capture binding partner, e.g., antibody, an antibody, is less than about 10 minutes. In some embodi through noncovalent attachment, e.g., biotin-avidin/strepta ments, the time allowed for binding of the molecule of interest vidin interactions. In some embodiments, the attachment is to the capture binding partner, e.g., an antibody, is less than covalent. Thus, in Some embodiments, a Solid Support, e.g., a about 5 minutes. microtiter plate or a paramagnetic bead, is attached to the In some embodiments, following the binding of particles of capture binding partner, e.g., antibody, through covalent the molecule of interest to the capture binding partner, e.g., a attachment. capture antibody, particles that bound nonspecifically, as well The capture antibody can be covalently attached in an as other unwanted Substances in the sample, are washed away orientation that optimizes the capture of the molecule of 10 leaving Substantially only specifically bound particles of the interest. For example, in some embodiments, a binding part molecule of interest. In other embodiments, no wash is used ner, e.g., an antibody, is attached in a orientated manner to a between additions of sample and label, which can reduce Solid Support, e.g., a microtiterplate or a paramagnetic micro sample preparation time. Thus, in Some embodiments, the particle. time allowed for both binding of the molecule of interest to An exemplary protocol for oriented attachment of an anti 15 the capture binding partner, e.g., an antibody, and binding of body to a solid support is as follows: IgG is dissolved in 0.1M the label to the molecule of interest, is less that about 12, 10, sodium acetate buffer, pH 5.5 to a final concentration of 1 8, 6, 4, 3, 2, or 1 hours, or less than about 60, 50, 40, 30, 25, mg/ml. An equal Volume of ice-cold 20 mMSodium periodate 20, 15, 10, or minutes. In some embodiments, the time in 0.1 M sodium acetate, pH 5.5 is added. The IgG is allowed allowed for both binding of the molecule of interest to the to oxidize for /2 hour on ice. Excess periodate reagent is capture binding partner, e.g., an antibody, and binding of the quenched by the addition of 0.15 volume of 1 M glycerol. label to the molecule of interest, is less that about 60 minutes. Low molecular weight byproducts of the oxidation reaction In some embodiments, the time allowed for both binding of are removed by ultrafiltration. The oxidized IgG fraction is the molecule of interest to the capture binding partner, e.g., an diluted to a suitable concentration (typically 0.5 micrograms antibody, and binding of the label to the molecule of interest, IgG per ml) and reacted with hydrazide-activated multiwell 25 is less that about 50 minutes. In some embodiments, the time plates for at least two hours at room temperature. Unbound allowed for both binding of the molecule of interest to the IgG is removed by washing the multiwell plate with borate capture binding partner, e.g., an antibody, and binding of the buffered saline or another suitable buffer. The plate may be label to the molecule of interest, is less than about 40 minutes. dried for storage, if desired. A similar protocol may be fol In some embodiments, the time allowed for both binding of lowed for microbeads if the material of the microbead is 30 the molecule of interest to the capture binding partner, e.g., an suitable for such attachment. antibody, and binding of the label to the molecule of interest, In some embodiments, the solid support is a microtiter is less than about 30 minutes. In some embodiments, the time plate. In some embodiments, the Solid Support is a paramag allowed for both binding of the molecule of interest to the netic bead. An exemplary paramagnetic bead is Streptavidin capture binding partner, e.g., an antibody, and binding of the C1 (Dynal, 650.01-03). Other suitable beads will be apparent 35 label to the molecule of interest, is less than about 20 minutes. to those of skill in the art. Methods for attachment of antibod In some embodiments, the time allowed for both binding of ies to paramagnetic beads are well-known in the art. One the molecule of interest to the capture binding partner, e.g., an example is given in Example 2. antibody, and binding of the label to the molecule of interest, The molecule of interest is contacted with the capture is less than about 15 minutes. In some embodiments, the time binding partner, e.g., capture antibody immobilized on a Solid 40 allowed for both binding of the molecule of interest to the Support. Some sample preparation may be used; e.g., prepa capture binding partner, e.g., an antibody, and binding of the ration of serum from blood samples or concentration proce label to the molecule of interest, is less than about 10 minutes. dures before the sample is contacted with the capture anti In some embodiments, the time allowed for both binding of body. Protocols for binding of proteins in immunoassays are the molecule of interest to the capture binding partner, e.g., an well-known in the art and are included in the Examples. 45 antibody, and binding of the label to the molecule of interest, The time allowed for binding will vary depending on the is less than about 5 minutes. conditions; it will be apparent that shorter binding times are Some immunoassay diagnostic reagents, including the desirable in some settings, especially in a clinical setting. The capture and signal antibodies used to measure the molecule of use of, e.g., paramagnetic beads can reduce the time required interest, can be derived from animal sera. Endogenous human for binding. In some embodiments, the time allowed for bind 50 heterophilic antibodies, or human anti-animal antibodies, ing of the molecule of interest to the capture binding partner, which have the ability to bind to immunoglobulins of other e.g., an antibody, is less that about 12, 10, 8, 6, 4, 3, 2, or 1 species, are present in the serum or plasma of more than 10% hours, or less than about 60, 50, 40, 30, 25, 20, 15, 10, or 5 of patients. These circulating heterophilic antibodies can minutes. In some embodiments, the time allowed for binding interfere with immunoassay measurements. In Sandwich of the molecule of interest to the capture binding partner, e.g., 55 immunoassays, these heterophilic antibodies can either an antibody, is less than about 60 minutes. In some embodi bridge the capture and detection (diagnostic) antibodies, ments, the time allowed for binding of the molecule of interest thereby producing a false-positive signal, or they can block to the capture binding partner, e.g., an antibody, is less than the binding of the diagnostic antibodies, thereby producing a about 40 minutes. In some embodiments, the time allowed for false-negative signal. In competitive immunoassays, the het binding of the molecule of interest to the capture binding 60 erophilic antibodies can bind to the analytic antibody and partner, e.g., an antibody, is less than about 30 minutes. In inhibit its binding to the molecule of interest. They can also some embodiments, the time allowed for binding of the mol either block or augment the separation of the antibody-mol ecule of interest to the capture binding partner, e.g., an anti ecule of interest complex from free molecule of interest, body, is less than about 20 minutes. In some embodiments, especially when antispecies antibodies are used in the sepa the time allowed for binding of the molecule of interest to the 65 ration systems. Therefore, the impact of these heterophilic capture binding partner, e.g., an antibody, is less than about 15 antibody interferences is difficult to predict and it can be minutes. In some embodiments, the time allowed for binding advantageous to block the binding of heterophilic antibodies. US 8,450,069 B2 47 48 In some embodiments of the invention, the immunoassay allowed for binding of the molecule of interest to the label, includes the step of depleting the sample of heterophilic anti e.g., an antibody-dye, is less than about 5 minutes. Excess bodies using one or more heterophilic antibody blockers. label is removed by washing. Methods for removing heterophilic antibodies from samples In some embodiments, the label is not eluted from the to be tested in immunoassays are known and include: heating 5 protein of interest. In other embodiments, the label is eluted the specimen in a sodium acetate buffer, pH 5.0, for 15 min from the protein of interest. Preferred elution buffers are utes at 90° C. and centrifuging at 1200 g for 10 minutes: effective in releasing the label without generating significant precipitating the heterophilic immunoglobulins using poly background. It is useful if the elution buffer is bacteriostatic. ethylene glycol (PEG); immunoextracting the interfering het Elution buffers used in the invention can comprise a chao erophilic immunoglobulins from the specimen using protein 10 trope, a buffer, an albuminto coat the surface of the microtiter plate, and a Surfactant selected so as to produce a relatively A or protein G, or adding nonimmune mouse IgG. Embodi low background. The chaotrope can comprise urea, a guani ments of the methods of the invention contemplate preparing dinium compound, or other useful chaotropes. The buffer can the sample prior to analysis with the single molecule detector. comprise borate buffered saline, or other useful buffers. The The appropriateness of the method of pretreatment can be 15 protein carrier can comprise, e.g., an albumin, Such as human, determined Biochemicals to minimize immunoassay interfer bovine, or fish albumin, an IgG, or other useful carriers. The ence caused by heterophilic antibodies are commercially Surfactant can comprise anionic or nonionic detergent includ available. For example, a product called MAK33, which is an ing Tween 20, Triton X-100, sodium dodecyl sulfate (SDS), IgG1 monoclonal antibody to h-CK-MM, can be obtained and others. from Boehringer Mannheim. The MAK33 plus product con In another embodiment, the Solid phase binding assay can tains a combination of IgG1 and IgG1-Fab. polyMAK33 be a competitive binding assay. One such method is as fol contains IgG1-Fab polymerized with IgG1, and the poly lows. First, a capture antibody immobilized on a binding MAC 2b/2a contains IgG2a-Fab polymerized with IgG2b. surface is competitively bound by i) a molecule of interest, Bioreclamation Inc., East Meadow, N.Y., markets a second e.g., marker of a biological state, in a sample, and ii) a labeled commercial source of biochemicals to neutralize heterophilic 25 analog of the molecule comprising a detectable label (the antibodies known as Immunoglobulin Inhibiting Reagent. detection reagent). Second, the amount of the label using a This product is a preparation of immunoglobulins (IgG and single molecule analyzer is measured. Another Such method IgM) from multiple species, mainly murine IgG2a, IgG2b. is as follows. First, an antibody having a detectable label (the and IgG3 from Balb/c mice. In some embodiments the het detection reagent) is competitively bound to i) a molecule of erophilic antibody can be immunoextracted from the sample 30 interest, e.g., marker of a biological state in a sample, and ii) using methods known in the art, e.g., depleting the sample of an analog of the molecule that is immobilized on a binding the heterophilic antibody by binding the interfering antibody surface (the capture reagent). Second, the amount of the label to protein A or protein G. In some embodiments, the hetero using a single molecule analyzer is measured. An “analog of philic antibody can be neutralized using one or more hetero a molecule' refers, herein, to a species that competes with a philic antibody blockers. Heterophilic blockers can be 35 molecule for binding to a capture antibody. Examples of selected from the group consisting of anti-isotype hetero competitive immunoassays are disclosed in U.S. Pat. No. philic antibody blockers, anti-idiotype heterophilic antibody 4,235,601 to Deutsch et al., U.S. Pat. No. 4,442,204 to Liotta, blockers, and anti-anti-idiotype heterophilic antibody block and U.S. Pat. No. 5,208,535 to Buechler et al., all of which are ers. In some embodiments, a combination of heterophilic incorporated herein by reference. antibody blockers can be used. 40 C. Detection of Molecule of Interest and Determination of Label is added either with or following the addition of Concentration sample and washing. Protocols for binding antibodies and Following elution, the label is run through a single mol other immunolabels to proteins and other molecules are well ecule detectorin, e.g., the elution buffer. A processing sample known in the art. If the label binding step is separate from that may contain no label, a single label, or a plurality of labels. of capture binding, the time allowed for label binding can be 45 The number of labels corresponds or is proportional to (if important, e.g., inclinical applications or other time sensitive dilutions or fractions of samples are used) the number of settings. In some embodiments, the time allowed for binding molecules of the molecule of interest, e.g., marker of a bio of the molecule of interest to the label, e.g., an antibody-dye, logical state captured during the capture step. is less than about 12, 10, 8, 6, 4, 3, 2, or 1 hours, or less than Any Suitable single molecule detector capable of detecting about 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes. In some 50 the label used with the molecule of interest may be used. embodiments, the time allowed for binding of the molecule of Suitable single molecule detectors are described herein. Typi interest to the label, e.g., an antibody-dye, is less than about cally the detector will be part of a system that includes an 60 minutes. In some embodiments, the time allowed for bind automatic sampler for sampling prepared samples, and, ing of the molecule of interest to the label, e.g., an antibody optionally, a recovery system to recover samples. dye, is less than about 50 minutes. In some embodiments, the 55 In some embodiments, the processing sample is analyzed time allowed for binding of the molecule of interest to the in a single molecule analyzer that utilizes a capillary flow label, e.g., an antibody-dye, is less than about 40 minutes. In system, and that includes a capillary flow cell, a laser to some embodiments, the time allowed for binding of the mol illuminate an interrogation space in the capillary through ecule of interest to the label, e.g., an antibody-dye, is less than which processing sample is passed, a detector to detect radia about 30 minutes. In some embodiments, the time allowed for 60 tion emitted from the interrogation space, and a source of binding of the molecule of interest to the label, e.g., an anti motive force to move a processing sample through the inter body-dye, is less than about 20 minutes. In some embodi rogation space. In some embodiments, the single molecule ments, the time allowed for binding of the molecule of interest analyzer further comprises a microscope objective lens that to the label, e.g., an antibody-dye, is less than about 15 min collects light emitted from processing sample as it passes utes. In some embodiments, the time allowed for binding of 65 through the interrogation space, e.g., a high numerical aper the molecule of interest to the label, e.g., an antibody-dye, is ture microscope objective. In some embodiments, the laser less than about 10 minutes. In some embodiments, the time and detector are in a confocal arrangement. In some embodi US 8,450,069 B2 49 50 ments, the laser is a continuous wave laser. In some embodi than about 10um, less than about 5um, less than about 4 ments, the detector is an avalanche photodiode detector. In um, less than about 3 um, less than about 2.um, or less than Some embodiments, the source of motive force is a pump to about 1 um. In some embodiments, the volume of the inter provide pressure. In some embodiments, the invention pro rogation space is between about 1 um and about 10000 um. vides an analyzer system that includes a sampling system In some embodiments, the interrogation space is between capable of automatically sampling a plurality of Samples about 1 um and about 1000 um. In some embodiments, the providing a fluid communication between a sample container interrogation space is between about 1 um and about 100 and the interrogation space. In some embodiments, the inter um. In some embodiments, the interrogation space is rogation space has a volume of between about 0.001 and 500 between about 1 um and about 50 um. In some embodi pI, or between about 0.01 pland 100 p., or between about 10 ments, the interrogation space is between about 1 Lum and 0.01 pl. and 10 pl. or between about 0.01 pl. and 5 p., or about 10um. In some embodiments, the interrogation space between about 0.01 pl. and 0.5 pL, or between about 0.02 pl. is between about 2 um and about 10 um. In some embodi and about 300 pl. or between about 0.02 pl. and about 50 pl. ments, the interrogation space is between about 3 um and or between about 0.02 pl. and about 5 pl. or between about about 7 m. 0.02 pl. and about 0.5 pL or between about 0.02 pl. and about 15 In Some embodiments, the single molecule detector used in 2 pl. or between about 0.05 pl. and about 50 p., or between the methods of the invention utilizes a capillary flow system, about 0.05 pl. and about 5p, or between about 0.05 pl. and and includes a capillary flow cell, a continuous wave laser to about 0.5 pL, or between about 0.05 pl. and about 0.2 pl. or illuminate an interrogation space in the capillary through between about 0.1 pI and about 25 pl. In some embodi which processing sample is passed, a high numerical aperture ments, the interrogation space has a volume between about microscope objective lens that collects light emitted from 0.004 pland 100 pl. In some embodiments, the interrogation processing sample as it passes through the interrogation space has a volume between about 0.02 pland 50 pI. In some space, an avalanche photodiode detector to detect radiation embodiments, the interrogation space has a volume between emitted from the interrogation space, and a pump to provide about 0.001 pl. and 10 pl. In some embodiments, the inter pressure to move a processing sample through the interroga rogation space has a volume between about 0.001 pl. and 10 25 tion space, where the interrogation space is between about pL. In some embodiments, the interrogation space has a Vol 0.02 pl. and about 50 pl. In some embodiments, the single ume between about 0.01 pl. and 5 pl. In some embodiments, molecule detector used in the methods of the invention uti the interrogation space has a volume between about 0.02 pl. lizes a capillary flow system, and includes a capillary flow and about 5 p. In some embodiments, the interrogation cell, a continuous wave laser to illuminate an interrogation space has a volume between about 0.05 pl. and 5 pl. In some 30 space in the capillary through which processing sample is embodiments, the interrogation space has a volume between passed, a high numerical aperture microscope objective lens about 0.05 p, and 10 p. In some embodiments, the interro that collects light emitted from processing sample as it passes gation space has a volume between about 0.5 pL and about 5 through the interrogation space wherein the lens has a pL. In some embodiments, the interrogation space has a Vol numerical aperture of at least about 0.8, an avalanche photo ume between about 0.02 pl. and about 0.5 pI. 35 diode detector to detect radiation emitted from the interroga In some embodiments, the interrogation space has a Vol tion space, and a pump to provide pressure to move a pro ume of more than about 1 Lim, more than about 2 um, more cessing sample through the interrogation space, where the than about 3 um, more than about 4 um, more than about 5 interrogation space is between about 0.004 pl. and about 100 um, more than about 10um, more than about 15um, more pL. In some embodiments, the single molecule detector used than about 30 um, more than about 50 um, more than about 40 in the methods of the invention utilizes a capillary flow sys 75um, more than about 100 um, more than about 150 um, tem, and includes a capillary flow cell, a continuous wave more than about 200 um, more than about 250 um, more laser to illuminate an interrogation space in the capillary than about 300 um, more than about 400 um, more than through which processing sample is passed, a high numerical about 500 um, more than about 550 um, more than about aperture microscope objective lens that collects light emitted 600 um, more than about 750 um, more than about 1000 45 from processing sample as it passes through the interrogation um, more than about 2000 um, more than about 4000 um, space wherein the lens has a numerical aperture of at least more than about 6000 um, more than about 8000 um, more about 0.8, an avalanche photodiode detector to detect radia than about 10000 um, more than about 12000 um, more tion emitted from the interrogation space, and a pump to than about 13000 um, more than about 14000 um, more provide pressure to move a processing sample through the than about 15000 um, more than about 20000 um, more 50 interrogation space, where the interrogation space is between than about 30000 um, more than about 40000 um, or more about 0.05 pl. and about 10 pl. In some embodiments, the than about 50000 um. In some embodiments, the interroga single molecule detector used in the methods of the invention tion space is of a volumeless than about 50000 um, less than utilizes a capillary flow system, and includes a capillary flow about 40000 um, less than about 30000 um, less than about cell, a continuous wave laser to illuminate an interrogation 20000 um, less than about 15000 um, less than about 14000 55 space in the capillary through which processing sample is um, less than about 13000 um, less than about 12000 um, passed, a high numerical aperture microscope objective lens less than about 11000 um, less than about 9500 um, less that collects light emitted from processing sample as it passes than about 8000 um, less than about 6500 um, less than through the interrogation space wherein the lens has a about 6000 um, less than about 5000 um, less than about numerical aperture of at least about 0.8, an avalanche photo 4000 um, less than about 3000 um, less than about 2500 60 diode detector to detect radiation emitted from the interroga um, less than about 2000 um, less than about 1500 um, less tion space, and a pump to provide pressure to move a pro than about 1000um, less than about 800 um, less than about cessing sample through the interrogation space, where the 600 um, less than about 400 um, less than about 200 um, interrogation space is between about 0.05 pl. and about 5 pl. less than about 100 um, less than about 75 um, less than In some embodiments, the single molecule detector used in about 50 um, less than about 25um, less than about 20 um, 65 the methods of the invention utilizes a capillary flow system, less than about 15um, less than about 14 um, less than about and includes a capillary flow cell, a continuous wave laser to 13 um, less than about 12 um, less than about 11 um, less illuminate an interrogation space in the capillary through US 8,450,069 B2 51 52 which processing sample is passed, a high numerical aperture from the interrogation space, and a scan motor for translating microscope objective lens that collects light emitted from the interrogation space through the sample, wherein the inter processing sample as it passes through the interrogation space rogation space is between about 2 um and about 10 Lum. In wherein the lens has a numerical aperture of at least about 0.8. Some embodiments, the single molecule detector used in the an avalanche photodiode detector to detect radiation emitted methods of the invention uses a sample plate, a continuous from the interrogation space, and a pump to provide pressure wave laser directed toward an interrogation space located to move a processing sample through the interrogation space, within the sample, a high numerical aperture microscope where the interrogation space is between about 0.5 pI, and objective lens that collects light emitted from the sample as about 5 p. In any of these embodiments the analyzer may the interrogation space is translated through the sample, contain not more than one interrogation space. 10 wherein the lens has a numerical aperture of at least about 0.8. In some embodiments, the single molecule detector com an avalanche photodiode detector to detect radiation emitted prises a scanning analyzer system, as disclosed in U.S. patent from the interrogation space, and a scan motor for translating application Ser. No. 12/338,955, filed Dec. 18, 2008 and the interrogation space through the sample, wherein the inter entitled “Scanning Analyzer for Single Molecule Detection rogation space is between about 2 um and about 8 Lum. In and Methods of Use.” In some embodiments, the single mol 15 Some embodiments, the single molecule detector used in the ecule detector used in the methods of the invention uses a methods of the invention uses a sample plate, a continuous sample plate, a continuous wave laser directed toward a wave laser directed toward an interrogation space located sample plate in which the sample is contained, a high numeri within the sample, a high numerical aperture microscope cal aperture microscope objective lens that collects light emit objective lens that collects light emitted from the sample as ted from the sample as interrogation space is translated the interrogation space is translated through the sample, through the sample, wherein the lens has a numerical aperture wherein the lens has a numerical aperture of at least about 0.8. of at least about 0.8, an avalanche photodiode detector to an avalanche photodiode detector to detect radiation emitted detect radiation emitted from the interrogation space, and a from the interrogation space, and a scan motor for translating scan motor with a moveable mirror to translate the interroga the interrogation space through the sample, wherein the inter tion space through the sample wherein the interrogation space 25 rogation space is between about 3 um and about 7 um. In is between about 1 um and about 10000 um. In some any of these embodiments, the analyzer can contain only one embodiments, the single molecule detector used in the meth interrogation space. ods of the invention uses a sample plate, a continuous wave In other embodiments, the single molecule detector used in laser directed toward an interrogation space located within the methods of the invention uses a sample plate, a continuous the sample, a high numerical aperture microscope objective 30 wave laser directed toward a sample plate in which the sample lens that collects light emitted from the sample as the inter is contained, a high numerical aperture microscope objective rogation space is translated through the sample, wherein the lens that collects light emitted from the sample as interroga lens has a numerical aperture of at least about 0.8, an ava tion space is translated through the sample, an avalanche lanche photodiode detector to detect radiation emitted from photodiode detector to detect radiation emitted from the inter the interrogation space, and a scan motor for translating the 35 rogation space, and a scan motor with a moveable mirror to interrogation space through the sample, wherein the interro translate the interrogation space through the sample wherein gation space is between about 1 um and about 1000 um. In the interrogation space is between about 1 Lum and about Some embodiments, the single molecule detector used in the 10000um. In some embodiments, the single molecule detec methods of the invention uses a sample plate, a continuous tor used in the methods of the invention uses a sample plate, wave laser directed toward an interrogation space located 40 a continuous wave laser directed toward an interrogation within the sample, a high numerical aperture microscope space located within the sample, a high numerical aperture objective lens that collects light emitted from the sample as microscope objective lens that collects light emitted from the the interrogation space is translated through the sample, sample as the interrogation space is translated through the wherein the lens has a numerical aperture of at least about 0.8. sample, an avalanche photodiode detector to detect radiation an avalanche photodiode detector to detect radiation emitted 45 emitted from the interrogation space, and a scan motor for from the interrogation space, and a scan motor for translating translating the interrogation space through the sample, the interrogation space through the sample, wherein the inter wherein the interrogation space is between about 1 Lum and rogation space is between about 1 um and about 100 um. In about 1000 um. In some embodiments, the single molecule Some embodiments, the single molecule detector used in the detector used in the methods of the invention uses a sample methods of the invention uses a sample plate, a continuous 50 plate, a continuous wave laser directed toward an interroga wave laser directed toward an interrogation space located tion space located within the sample, a high numerical aper within the sample, a high numerical aperture microscope ture microscope objective lens that collects light emitted from objective lens that collects light emitted from the sample as the sample as the interrogation space is translated through the the interrogation space is translated through the sample, sample, an avalanche photodiode detector to detect radiation wherein the lens has a numerical aperture of at least about 0.8. 55 emitted from the interrogation space, and a scan motor for an avalanche photodiode detector to detect radiation emitted translating the interrogation space through the sample, from the interrogation space, and a scan motor for translating wherein the interrogation space is between about 1 um and the interrogation space through the sample, wherein the inter about 100 um. In some embodiments, the single molecule rogation space is between about 1 um and about 10 um. In detector used in the methods of the invention uses a sample Some embodiments, the single molecule detector used in the 60 plate, a continuous wave laser directed toward an interroga methods of the invention uses a sample plate, a continuous tion space located within the sample, a high numerical aper wave laser directed toward an interrogation space located ture microscope objective lens that collects light emitted from within the sample, a high numerical aperture microscope the sample as the interrogation space is translated through the objective lens that collects light emitted from the sample as sample, an avalanche photodiode detector to detect radiation the interrogation space is translated through the sample, 65 emitted from the interrogation space, and a scan motor for wherein the lens has a numerical aperture of at least about 0.8. translating the interrogation space through the sample, an avalanche photodiode detector to detect radiation emitted wherein the interrogation space is between about 1 um and US 8,450,069 B2 53 54 about 10 Lum. In some embodiments, the single molecule detector, where the volume of the first sample and said second detector used in the methods of the invention uses a sample sample introduced into the analyzer is less than about 20 ul, plate, a continuous wave laser directed toward an interroga and wherein the analyte is present at a concentration of less tion space located within the sample, a high numerical aper than about 20 femtomolar. In some embodiments, the meth ture microscope objective lens that collects light emitted from ods of the invention utilize a single molecule detector capable the sample as the interrogation space is translated through the detecting a difference of less than about 20% in concentration sample, an avalanche photodiode detector to detect radiation of an analyte between a first sample and a second sample that emitted from the interrogation space, and a scan motor for are introduced into the detector, where the volume of the first translating the interrogation space through the sample, sample and said second sample introduced into the analyzeris wherein the interrogation space is between about 2 Lum and 10 about 10 Lum. In some embodiments, the single molecule less than about 10 ul, and wherein the analyte is present at a detector used in the methods of the invention uses a sample concentration of less than about 10 femtomolar. In some plate, a continuous wave laser directed toward an interroga embodiments, the methods of the invention utilize a single tion space located within the sample, a high numerical aper molecule detector capable detecting a difference of less than ture microscope objective lens that collects light emitted from 15 about 20% in concentration of an analyte between a first the sample as the interrogation space is translated through the sample and a second sample that are introduced into the sample, an avalanche photodiode detector to detect radiation detector, where the volume of the first sample and said second emitted from the interrogation space, and a scan motor for sample introduced into the analyzer is less than about 5 ul, translating the interrogation space through the sample, and wherein the analyte is present at a concentration of less wherein the interrogation space is between about 2 Lum and than about 5 femtomolar. In some embodiments, the methods about 8 Lum. In some embodiments, the single molecule of the invention utilize a single molecule detector capable detector used in the methods of the invention uses a sample detecting a difference of less than about 20% in concentration plate, a continuous wave laser directed toward an interroga of an analyte between a first sample and a second sample that tion space located within the sample, a high numerical aper are introduced into the detector, where the volume of the first ture microscope objective lens that collects light emitted from 25 sample and said second sample introduced into the analyzeris the sample as the interrogation space is translated through the less than about 5 ul, and wherein the analyte is present at a sample, an avalanche photodiode detector to detect radiation concentration of less than about 50 femtomolar. emitted from the interrogation space, and a scan motor for The single molecule detector and systems are described in translating the interrogation space through the sample, more detail below. Further embodiments of single molecule wherein the interrogation space is between about 3 Lum and 30 analyzers useful in the methods of the invention, such as about 7um. In any of these embodiments, the analyzer can detectors with more than one interrogation window, detectors contain only one interrogation space. utilize electrokinetic or electrophoretic flow, and the like, In some embodiments, the single molecule detector is may be found in U.S. patent application Ser. No. 11/048,660, capable of determining a concentration for a molecule of incorporated by reference herein in its entirety. interest in a sample where sample may range in concentration 35 Between runs the instrument may be washed. A wash over a range of at least about 100-fold, or 1000-fold, or buffer that maintains the salt and Surfactant concentrations of 10,000-fold, or 100,000-fold, or 300,00-fold, or 1,000,000 the sample may be used in Some embodiments to maintain the fold, or 10,000,000-fold, or 30,000,000-fold. conditioning of the capillary; i.e., to keep the capillary Surface In some embodiments, the methods of the invention utilize relatively constant between samples to reduce variability. a single molecule detector capable detecting a difference of 40 A feature that contributes to the extremely high sensitivity less than about 50%, 40%, 30%, 20%, 15%, or 10% in con of the instruments and methods of the invention is the method centration of an analyte between a first sample and a second of detecting and counting labels, which, in some embodi sample that are introduced into the detector, where the Vol ments, are attached to single molecules to be detected or, ume of the first sample and said second sample introduced more typically, correspond to a single molecule to be into the analyzer is less than about 100,90, 80, 70, 60, 50, 40, 45 detected. Briefly, the processing sample flowing through the 30, 20, 15, 10, 5, 4, 3, 2, or 1 ul, and wherein the analyte is capillary or contained on a sample plate is effectively divided present at a concentration of less than about 100, 90, 80, 70. into a series of detection events, by Subjecting a given inter 60, 50, 40, 30, 20, 15, 10, 5, 4, 3, 2, or 1 femtomolar. In some rogation space of the capillary to EM radiation from a laser embodiments, the methods of the invention utilize a single that emits light at an appropriate excitation wavelength for the molecule detector capable detecting a difference of less than 50 fluorescent moiety used in the label for a predetermined about 50% in concentration of an analyte between a first period of time, and detecting photons emitted during that sample and a second sample that are introduced into the time. Each predetermined period of time is a “bin.” If the total detector, where the volume of the first sample and said second number of photons detected in a given bin exceeds a prede sample introduced into the analyzer is less than about 100 ul, termined threshold level, a detection event is registered for and wherein the analyte is present at a concentration of less 55 that bin, i.e., a label has been detected. If the total number of than about 100 femtomolar. In some embodiments, the meth photons is not at the predetermined threshold level, no detec ods of the invention utilize a single molecule detector capable tion event is registered. In some embodiments, processing detecting a difference of less than about 40% in concentration sample concentration is dilute enough that, for a large per of an analyte between a first sample and a second sample that centage of detection events, the detection event represents are introduced into the detector, where the volume of the first 60 only one label passing through the window, which corre sample and said second sample introduced into the analyzeris sponds to a single molecule of interest in the original sample, less than about 50 ul, and wherein the analyte is present at a that is, few detection events represent more than one label in concentration of less than about 50 femtomolar. In some a single bin. In some embodiments, further refinements are embodiments, the methods of the invention utilize a single applied to allow greater concentrations of label in the pro molecule detector capable detecting a difference of less than 65 cessing sample to be detected accurately, i.e., concentrations about 20% in concentration of an analyte between a first at which the probability of two or more labels being detected sample and a second sample that are introduced into the as a single detection event is no longer insignificant. US 8,450,069 B2 55 56 Although other bin times can be used without departing ments, the bin time is about 750 microseconds. In some from the scope of the present invention, in Some embodiments embodiments, the bin time is about 1000 microseconds. the bintimes are selected in the range of about 1 microsecond In some embodiments, determining the concentration of a to about 5 ms. In some embodiments, the bin time is more particle-label complex in a sample comprises determining the than about 1,2,3,4,5,6,7,8,9, 10, 15, 20, 30, 40, 50, 60, 70, background noise level. In some embodiments, the back 80,90, 100, 200, 250,300, 400, 500, 600, 700, 750, 800, 900, ground noise level is determined from the mean noise level, or 1000, 2000, 3000, 4000, or 5000 microseconds. In some the root-mean-square noise. In other cases, a typical noise embodiments, the bintime is less than about 2, 3, 4, 5, 6, 7, 8, value or a statistical value is chosen. In most cases, the noise 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, is expected to follow a Poisson distribution. 400, 500, 600, 700, 750, 800,900, 1000, 2000, 3000, 4000, or 10 Thus, as a label is encountered in the interrogation space, it 5000 microseconds. In some embodiments, the bin time is is irradiated by the laser beam to generate a burst of photons. about 1 to 1000 microseconds. In some embodiments, the bin The photons emitted by the label are discriminated from time is about 1 to 750 microseconds. In some embodiments, background light or background noise emission by consider the bintime is about 1 to 500 microseconds. In some embodi ing only the bursts of photons that have energy above a pre ments, the bin time is about 1 to 250 microseconds. In some 15 determined threshold energy level which accounts for the embodiments, the bintime is about 1 to 100 microseconds. In amount of background noise that is present in the sample. some embodiments, the bin time is about 1 to 50 microsec Background noise typically comprises low frequency emis onds. In some embodiments, the bin time is about 1 to 40 sion produced, for example, by the intrinsic fluorescence of microseconds. In some embodiments, the bin time is about 1 non-labeled particles that are present in the sample, the buffer to 30 microseconds. In some embodiments, the bin time is or diluent used in preparing the sample for analysis, Raman about 1 to 25 microseconds. In some embodiments, the bin scattering and electronic noise. In some embodiments, the time is about 1 to 20 microseconds. In some embodiments, the value assigned to the background noise is calculated as the bin time is about 1 to 10 microseconds. In some embodi average background signal noise detected in a plurality of ments, the bin time is about 1 to 7.5 microseconds. In some bins, which are measurements of photon signals that are embodiments, the bin time is about 1 to 5 microseconds. In 25 detected in an interrogation space during a predetermined some embodiments, the bin time is about 5 to 500 microsec length of time. Thus in some embodiments, background noise onds. In some embodiments, the bin time is about 5 to 250 is calculated for each sample as a number specific to that microseconds. In some embodiments, the bin time is about 5 sample. to 100 microseconds. In some embodiments, the bin time is Given the value for the background noise, the threshold about 5 to 50 microseconds. In some embodiments, the bin 30 energy level can be assigned. As discussed above, the thresh time is about 5 to 20 microseconds. In some embodiments, the old value is determined to discriminate true signals (due to bin time is about 5 to 10 microseconds. In some embodi fluorescence of a label) from the background noise. Care must ments, the bin time is about 10 to 500 microseconds. In some be taken in choosing a threshold value such that the number of embodiments, the bin time is about 10 to 250 microseconds. false positive signals from random noise is minimized while In some embodiments, the bin time is about 10 to 100 micro 35 the number of true signals which are rejected is also mini seconds. In some embodiments, the bin time is about 10 to 50 mized Methods for choosing a threshold value include deter microseconds. In some embodiments, the bintime is about 10 mining a fixed value above the noise level and calculating a to 30 microseconds. In some embodiments, the bin time is threshold value based on the distribution of the noise signal. about 10 to 20 microseconds. In some embodiments, the bin In one embodiment, the threshold is set at a fixed number of time is about 1 microsecond. In some embodiments, the bin 40 standard deviations above the background level. Assuming a time is about 2 microseconds. In some embodiments, the bin Poisson distribution of the noise, using this method one can time is about 3 microseconds. In some embodiments, the bin estimate the number of false positive signals over the time time is about 4 microseconds. In some embodiments, the bin course of the experiment. In some embodiments, the thresh time is about 5 microseconds. In some embodiments, the bin old level is calculated as a value of 4 sigma above the back time is about 6 microseconds. In some embodiments, the bin 45 ground noise. For example, given an average background time is about 7 microseconds. In some embodiments, the bin noise level of 200 photons, the analyzer system establishes a time is about 8 microseconds. In some embodiments, the bin threshold level of 4V200 above the average background/noise time is about 9 microseconds. In some embodiments, the bin level of 200 photons to be 256 photons. Thus, in some time is about 10 microseconds. In some embodiments, the bin embodiments, determining the concentration of a label in a time is about 11 microseconds. In some embodiments, the bin 50 sample includes establishing the threshold level above which time is about 12 microseconds. In some embodiments, the bin photon signals represent the presence of a label. Conversely, time is about 13 microseconds. In some embodiments, the bin photon signals that have an energy level that is not greater time is about 14 microseconds. In some embodiments, the bin than that of the threshold level indicate the absence of a label. time is about 5 microseconds. In some embodiments, the bin Many bin measurements are taken to determine the con time is about 15 microseconds. In some embodiments, the bin 55 centration of a sample, and the absence or presence of a label time is about 16 microseconds. In some embodiments, the bin is ascertained for each bin measurement. Typically, 60,000 time is about 17 microseconds. In some embodiments, the bin measurements or more can made in one minute (e.g., in time is about 18 microseconds. In some embodiments, the bin embodiments in which the bin size is 1 ms—for smaller bin time is about 19 microseconds. In some embodiments, the bin sizes the number of measurements is correspondingly larger, time is about 20 microseconds. In some embodiments, the bin 60 e.g., 6,000,000 measurements per minute for a bin size of 10 time is about 25 microseconds. In some embodiments, the bin microseconds). Thus, no single measurement is crucial and time is about 30 microseconds. In some embodiments, the bin the method provides for a high margin of error. The bins that time is about 40 microseconds. In some embodiments, the bin are determined not to contain a label (“no bins) are dis time is about 50 microseconds. In some embodiments, the bin counted and only the measurements made in the bins that are time is about 100 microseconds. In some embodiments, the 65 determined to contain label (“yes” bins) are accounted in bin time is about 250 microseconds. In some embodiments, determining the concentration of the label in the processing the bin time is about 500 microseconds. In some embodi sample. Discounting measurements made in the “no bins or US 8,450,069 B2 57 58 bins that are devoid of label increases the signal to noise ratio As discussed above, when the interrogation space encom and the accuracy of the measurements. Thus, in some passes the entire cross-section of the sample stream, only the embodiments, determining the concentration of a label in a number of labels counted passing through a cross-section of sample comprises detecting the bin measurements that reflect the sample stream in a set length of time (bin) and the Volume the presence of a label. of sample that was interrogated in the bin are needed to The signal to noise ratio or the sensitivity of the analyzer calculate the concentration the sample. The total number of system can be increased by minimizing the time that back labels contained in the “yes” bins is determined and related to ground noise is detected during a bin measurement in which the sample volume represented by the total number of bins a particle-label complex is detected. For example, in a bin used in the analysis to determine the concentration of labels in 10 the processing sample. Thus, in one embodiment, determin measurement lasting 1 millisecond during which one par ing the concentration of a label in a processing sample com ticle-label complex is detected when passing across an inter prises determining the total number of labels detected “yes” rogation space within 250 microseconds, 750 microseconds bins and relating the total number of detected labels to the of the 1 millisecond are spent detecting background noise total sample Volume that was analyzed. The total sample emission. The signal to noise ratio can be improved by 15 Volume that is analyzed is the sample Volume that is passed decreasing the bintime. In some embodiments, the bintime is through the capillary flow cell and across the interrogation 1 millisecond. In other embodiments, the bintime is 750, 500, space in a specified time interval. Alternatively, the concen 250 microseconds, 100 microseconds, 50 microseconds, 25 tration of the label complex in a sample is determined by microseconds or 10 microseconds. Other bin times are as interpolation of the signal emitted by the label in a number of described herein. bins from a standard curve that is generated by determining Other factors that affect measurements are the brightness the signal emitted by labels in the same number of bins by or dimness of the fluorescent moiety, the flow rate, and the standard samples containing known concentrations of the power of the laser. Various combinations of the relevant fac label. tors that allow for detection of label will be apparent to those In some embodiments, the number of individual labels that of skill in the art. In some embodiments, the bin time is 25 are detected in a bin is related to the relative concentration of adjusted without changing the flow rate. It will be appreciated the particle in the processing sample. At relatively low con by those of skill in the art that as bin time decreases, laser centrations, for example at concentrations below about 10' power output directed at the interrogation space must increase M the number of labels is proportional to the photon signal to maintain a constant total energy applied to the interrogation that is detected in a bin. Thus, at low concentrations of label space during the bin time. For example, if bin time is 30 the photon signal is provided as a digital signal. At relatively decreased from 1000 microseconds to 250 microseconds, as a higher concentrations, for example at concentrations greater first approximation, laser power output must be increased than about 10' M, the proportionality of photon signal to a approximately four-fold. These settings allow for the detec label is lost as the likelihood of two or more labels crossing tion of the same number of photons in a 250 us as the number the interrogation space at about the same time and being of photons counted during the 1000 us given the previous 35 counted as one becomes significant. Thus, in some embodi settings, and allow for faster analysis of sample with lower ments, individual particles in a sample of a concentration backgrounds and thus greater sensitivity. In addition, flow greater than about 10' M are resolved by decreasing the rates may be adjusted in order to speed processing of sample. length of time of the bin measurement. These numbers are merely exemplary, and the skilled practi Alternatively, in other embodiments, the total photon sig tioner can adjust the parameters as necessary to achieve the 40 nal that is emitted by a plurality of particles that are present in desired result. any one bin is detected. These embodiments allow for single In some embodiments, the interrogation space encom molecule detectors of the invention wherein the dynamic passes the entire cross-section of the sample stream. When range is at least 3, 3.5, 4, 4.5, 5.5, 6, 6.5, 7, 7.5, 8, or more than the interrogation space encompasses the entire cross-section 8 logs. of the sample stream, only the number of labels counted and 45 “Dynamic range.” as that term is used herein, refers to the the Volume passing through a cross-section of the sample range of sample concentrations that may be quantitated by the stream in a set length of time are needed to calculate the instrument without need for dilution or other treatment to concentration of the label in the processing sample. In some alter the concentration of Successive samples of differing embodiments, the interrogation space can be defined to be concentrations, where concentrations are determined with an Smaller than the cross-sectional area of sample stream by, for 50 accuracy appropriate for the intended use. For example, if a example, the interrogation space is defined by the size of the microtiter plate contains a sample of 1 femtomolar concen spot illuminated by the laser beam. In some embodiments, the tration for an analyte of interest in one well, a sample of interrogation space can be defined by adjusting the apertures 10,000 femtomolar concentration for an analyte of interest in 306 (FIG. 1A) or 358 and 359 (FIG. 1B) of the analyzer and another well, and a sample of 100 femtomolar concentration reducing the illuminated Volume that is imaged by the objec 55 for the analyte in a third well, an instrument with a dynamic tive lens to the detector. In the embodiments when the inter range of at least 4 logs and a lower limit of quantitation of 1 rogation space is defined to be smaller than the cross-sec femtomolar is able to accurately quantitate the concentration tional area of sample stream, the concentration of the label of all the samples without the need for further treatment to can be determined by interpolation of the signal emitted by adjust concentration, e.g., dilution. Accuracy may be deter the complex from a standard curve that is generated using one 60 mined by standard methods, e.g., using a series of standards or more samples of known standard concentrations. In yet of concentrations that span the dynamic range and construct other embodiments, the concentration of the label can be ing a standard curve. Standard measures of fit of the resulting determined by comparing the measured particles to an inter standard curve may be used as a measure of accuracy, e.g., an nal label standard. In embodiments when a diluted sample is r greater than about 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, analyzed, the dilution factor is accounted in calculating the 65 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99. concentration of the molecule of interest in the starting Increased dynamic range is achieved by altering the man sample. ner in which data from the detector is analyzed, and/or by the US 8,450,069 B2 59 60 use of an attenuator between the detector and the interroga In some embodiments, an analyzer or analyzer system of tion space. At the low end of the range, where processing the invention is capable of detecting an analyte, e.g., a biom sample is sufficiently dilute that each detection event, i.e., arker, at a limit of detection of less than 1 nanomolar, or 1 each burst of photons above a threshold level in a bin (the picomolar, or 1 femtomolar, or 1 attomolar, or 1 Zeptomolar. “event photons'), likely represents only one label, the data is 5 In some embodiments, the analyzer or analyzer system is analyzed to count detection events as single molecules. capable of detecting a change in concentration of the analyte, Thereby each bin is analyzed as a simple “yes” or 'no' for the or of multiple analytes, e.g., a biomarker or biomarkers, from presence of label, as described above. For a more concen one sample to another sample of less than about 0.1%, 1%, trated processing sample, where the likelihood of two or more 2%. 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% when labels occupying a single bin becomes significant, the number 10 the biomarker is present at a concentration of less than 1 of event photons in a significant number of bins is found to be nanomolar, or 1 picomolar, or 1 femtomolar, or 1 attomolar, or Substantially greater than the number expected for a single 1 Zeptomolar, in the samples, and when the size of each of the label, e.g., the number of event photons in a significant num sample is less than about 100, 50, 40, 30, 20, 10, 5, 2, 1, 0.1, ber of bins corresponds to two-fold, three-fold, or more, than 0.01, 0.001, or 0.0001 ul. In some embodiments, the analyzer the number of event photons expected for a single label. For 15 or analyzer system is capable of detecting a change in con these samples, the instrument changes its method of data centration of the analyte from a first sample to a second analysis to one of integrating the total number of event pho sample of less than about 20%, when the analyte is present at tons for the bins of the processing sample. This total will be a concentration of less than about 1 picomolar, and when the proportional to the total number of labels that were in all the size of each of the samples is less than about 50 ul. In some bins. For an even more concentrated processing sample, 20 embodiments, the analyzer or analyzer system is capable of where many labels are present in most bins, background noise detecting a change in concentration of the analyte from a first becomes an insignificant portion of the total signal from each sample to a second sample of less than about 20%, when the bin, and the instrument changes its method of data analysis to analyte is present at a concentration of less than about 100 one of counting total photons per bin (including background). femtomolar, and when the size of each of the samples is less An even further increase in dynamic range can beachieved by 25 than about 50 ul. In some embodiments, the analyzer or the use of an attenuator between the flow cell and the detector, analyzer system is capable of detecting a change in concen when concentrations are such that the intensity of light reach tration of the analyte from a first sample to a second sample of ing the detector would otherwise exceed the capacity of the less than about 20%, when the analyte is present at a concen detector for accurately counting photons, i.e., Saturate the tration of less than about 50 femtomolar, and when the size of detector. 30 each of the samples is less than about 50 ul. In some embodi The instrument may include a data analysis system that ments, the analyZeror analyzer system is capable of detecting receives input from the detector and determines the appropri a change in concentration of the analyte from a first sample to ate analysis method for the sample being run, and outputs a second sample of less than about 20%, when the analyte is values based on Such analysis. The data analysis system may present at a concentration of less than about 5 femtomolar, further output instructions to use or not use an attenuator, if an 35 and when the size of each of the samples is less than about 50 attenuator is included in the instrument. ul. In some embodiments, the analyzer or analyzer system is By utilizing Such methods, the dynamic range of the instru capable of detecting a change in concentration of the analyte ment can be dramatically increased. Thus, in some embodi from a first sample to a second sample of less than about 20%, ments, the instrument is capable of measuring concentrations when the analyte is present at a concentration of less than of samples over a dynamic range of more than about 1000 (3 40 about 5 femtomolar, and when the size of each of the samples log), 10,000 (4 log), 100,000 (5 log),350,000 (5.5 log), 1,000, is less than about 5ul. In some embodiments, the analyzer or 000 (6 log), 3,500,000 (6.5 log), 10,000,000 (7 log), 35,000, analyzer system is capable of detecting a change in concen 000 (7.5 log), or 100,000,000 (8 log). In some embodiments, tration of the analyte from a first sample to a second sample of the instrument is capable of measuring concentrations of less than about 20%, when the analyte is present at a concen samples over a dynamic range of more than about 100,000 (5 45 tration of less than about 1 femtomolar, and when the size of log). In some embodiments, the instrument is capable of each of the samples is less than about 5 LA measuring concentrations of samples over a dynamic range of The single molecule detectors of the present invention are more than about 1,000,000 (6 log). In some embodiments, the capable of detecting molecules of interestina highly sensitive instrument is capable of measuring concentrations of samples manner with a very low coefficient of variation (CV). In some over a dynamic range of more than about 10,000,000 (7 log). 50 embodiments, the CV is less than about 50%, 40%, 30%, In some embodiments, the instrument is capable of measuring 25%, 20%, 15%, 10%, 5%, or less than about 1%. In some the concentrations of samples over a dynamic range of from embodiments, the CV is less than about 50%. In some about 1-10 femtomolar to at least about 1000; 10,000; 100, embodiments, the CV is less than about 40%. In some 000; 350,000; 1,000,000; 3,500,000; 10,000,000; or 35,000, embodiments, the CV is less than about 30%. In some 000 femtomolar. In some embodiments, the instrument is 55 embodiments, the CV is less than about 25%. In some capable of measuring the concentrations of samples over a embodiments, the CV is less than about 20%. In some dynamic range of from about 1-10 femtomolar to at least embodiments, the CV is less than about 15%. In some about 10,000 femtomolar. In some embodiments, the instru embodiments, the CV is less than about 10%. In some ment is capable of measuring the concentrations of samples embodiments, the CV is less than about 5%. In some embodi over a dynamic range of from about 1-10 femtomolar to at 60 ments, the CV is less than about 1%. In some embodiments, least about 100,000 femtomolar. In some embodiments, the the limit of detection (LOD) is less than about 100 pg/ml and instrument is capable of measuring the concentrations of the CV is less than about 10%. In some embodiments, the samples overa dynamic range of from about 1-10 femtomolar limit of detection (LOD) is less than about 50 pg/ml and the to at least about 1,000,000 femtomolar. In some embodi CV is less than about 10%. In some embodiments, the limit of ments, the instrument is capable of measuring the concentra- 65 detection (LOD) is less than about 40 pg/ml and the CV is less tions of Samples over a dynamic range of from about 1-10 than about 10%. In some embodiments, the limit of detection femtomolar to at least about 10,000,000. (LOD) is less than about 30 pg/ml and the CV is less than US 8,450,069 B2 61 62 about 10%. In some embodiments, the limit of detection ting at least about 200 photons when simulated by a laser (LOD) is less than about 20 pg/ml and the CV is less than emitting light at the excitation wavelength of the moiety, about 10%. In some embodiments, the limit of detection where the laser is focused on a spot not less than about 5 (LOD) is less than about 15 pg/ml and the CV is less than microns in diameter that contains the moiety, and where the about 10%. In some embodiments, the limit of detection total energy directed at the spot by the laser is no more than (LOD) is less than about 10 pg/ml and the CV is less than about 3 microJoules. about 10%. In some embodiments, the limit of detection One embodiment of an analyzer kit of the invention is (LOD) is less than about 5 pg/ml and the CV is less than about depicted in FIG. 15. The kit includes a label for a protein 10%. In some embodiments, the limit of detection (LOD) is molecule that includes a binding partner for a protein mol less than about 1 pg/ml and the CV is less than about 10%. In 10 ecule and a fluorescent moiety. The kit further includes an some embodiments, the limit of detection (LOD) is less than analyzer system for detecting a single protein molecule (300) about 0.05 pg/ml and the CV is less than about 10%. In some that includes an electromagnetic radiation source 301 for embodiments, the limit of detection (LOD) is less than about stimulating the fluorescent moiety, a capillary flow cell 313 0.01 pg/ml and the CV is less than about 10%. In some for passing the label; a source of motive force for moving the embodiments, the limit of detection (LOD) is less than about 15 label in the capillary flow cell (not shown); an interrogation 10 pg/ml and the CV is less than about 50%. In some embodi space defined within the capillary flow cell for receiving ments, the limit of detection (LOD) is less than about 10 electromagnetic radiation emitted from the electromagnetic pg/ml and the CV is less than about 25%. In some embodi source 314 (FIG. 2A); and an electromagnetic radiation ments, the limit of detection (LOD) is less than about 10 detector 309 operably connected to the interrogation space pg/ml and the CV is less than about 10%. In some embodi for measuring an electromagnetic characteristic of the stimu ments, the limit of detection (LOD) is less than about 10 lated fluorescent moiety, where the fluorescent moiety is pg/ml and the CV is less than about 5%. In some embodi capable of emitting at least about 200 photons when simu ments, the limit of detection (LOD) is less than about 10 lated by a laser emitting light at the excitation wavelength of pg/ml and the CV is less than about 1%. In some embodi the moiety, where the laser is focused on a spot not less than ments, the limit of detection (LOD) is less than about 5 pg/ml 25 about 5 microns in diameter that contains the moiety, and and the CV is less than about 100%. In some embodiments, where the total energy directed at the spot by the laser is no the limit of detection (LOD) is less than about 5 pg/ml and the more than about 3 microJoules. In some embodiments, the CV is less than about 50%. In some embodiments, the limit of beam 311 from an electromagnetic radiation source 301 is detection (LOD) is less than about 5 pg/ml and the CV is less focused by the microscope objective 315 to form one inter than about 25%. In some embodiments, the limit of detection 30 rogation space 314 (FIG. 2A) within the capillary flow cell (LOD) is less than about 5 pg/ml and the CV is less than about 313. The microscope objective may have a numerical aper 10%. In some embodiments, the limit of detection (LOD) is ture of equal to or greater than 0.7, 0.8, 0.9, or 1.0 in some less than about 5 pg/ml and the CV is less than about 5%. In embodiments. some embodiments, the limit of detection (LOD) is less than In some embodiments of the analyzer system kit, the ana about 5 pg/ml and the CV is less than about 1%. In some 35 lyZer comprises not more than one interrogation space. In embodiments, the limit of detection (LOD) is less than about Some embodiments, the electromagnetic radiation source is a 1 pg/ml and the CV is less than about 100%. In some embodi laser that has a power output of at least about 3, 5, 10, or 20 ments, the limit of detection (LOD) is less than about 1 pg/ml mW. In some embodiments, the fluorescent moiety comprises and the CV is less than about 50%. In some embodiments, the a fluorescent molecule. In some embodiments, the fluorescent limit of detection (LOD) is less than about 1 pg/ml and the CV 40 molecule is a dye molecule. Such as a dye molecule that is less than about 25%. In some embodiments, the limit of comprises at least one Substituted indolium ring system in detection (LOD) is less than about 1 pg/ml and the CV is less which the substituent on the 3-carbon of the indolium ring than about 10%. In some embodiments, the limit of detection contains a chemically reactive group or a conjugated Sub (LOD) is less than about 1 pg/ml and the CV is less than about stance. In some embodiments, the fluorescent moiety is a 5%. In some embodiments, the limit of detection (LOD) is 45 quantum dot. In some embodiments, the electromagnetic less than about 1 pg/ml and the CV is less than about 1%. radiation source is a continuous wave electromagnetic radia V. Instruments and Systems Suitable for Highly Sensitive tion source. Such as a light-emitting diode or a continuous Analysis of Molecules wave laser. In some embodiments, the motive force is pres The methods of the invention utilize analytical instruments Sure. In some embodiments, the detector is an avalanche of high sensitivity, e.g., single molecule detectors. Such 50 photodiode detector. In some embodiments, the analyZeruti single molecule detectors include embodiments as hereinaf lizes a confocal optical arrangement for deflecting a laser ter described. beam onto said interrogation space and for imaging said In some embodiments, the invention provides an analyzer stimulated dye molecule (shown in FIGS. 1,3), wherein said system kit for detecting a single protein molecule in a sample, confocal optical arrangement comprises an objective lens said system includes an analyzer System for detecting a single 55 having a numerical aperture of at least about 0.8. In some protein molecule in a sample and least one label that includes embodiments, the analyzer further comprises a sampling sys a fluorescent moiety and a binding partner for the protein tem capable of automatically sampling a plurality of samples molecule, where the analyzer includes an electromagnetic and providing a fluid communication between a sample con radiation source for stimulating the fluorescent moiety; a tainer and said interrogation space. In some embodiments, the capillary flow cell for passing the label; a source of motive 60 analyzer system further comprises a sample recovery system force for moving the label in the capillary flow cell; an inter in fluid communication with said interrogation space, rogation space defined within the capillary flow cell for wherein said recovery system is capable of recovering Sub receiving electromagnetic radiation emitted from the electro stantially all of said sample. In some embodiments, the kit magnetic source; and an electromagnetic radiation detector further includes instructions for use of the system. operably connected to the interrogation space for measuring 65 A. Apparatus/System an electromagnetic characteristic of the stimulated fluores In one aspect, the methods described herein utilize an cent moiety, where the fluorescent moiety is capable of emit analyzer system capable of detecting a single molecule in a US 8,450,069 B2 63 64 sample. In one embodiment, the analyzer system is capable of an electromagnetic radiation source 301, a mirror 302, a lens single molecule detection of a fluorescently labeled particle 303, a capillary flow cell 313, a microscopic objective lens wherein the analyzer system detects energy emitted by an 305, an aperture 306, a detector lens 307, a detector filter 308, excited fluorescent label in response to exposure by an elec a single photon detector 309, and a processor 310 operatively tromagnetic radiation source when the single particle is connected to the detector. present in an interrogation space defined within a capillary In operation the electromagnetic radiation source 301 is flow cell fluidly connected to the sampling system of the aligned so that its output 311 is reflected off of a front surface analyzer system. In a further embodiment of the analyzer 312 of mirror 302. The lens 303 focuses the beam 311 onto a system, the single particle moves through the interrogation single interrogation space (an illustrative example of an inter space of the capillary flow cell by means of a motive force. In 10 rogation space 314 is shown in FIG. 2A) in the capillary flow another embodiment of the analyzer System, an automatic cell 313. The microscope objective lens 305 collects light sampling system may be included in the analyzer system for from sample particles and forms images of the beam onto the introducing the sample into the analyzer system. In another aperture 306. The aperture 306 affects the fraction of light embodiment of the analyzer system, a sample preparation emitted by the specimen in the interrogation space of the system may be included in the analyzer System for preparing 15 capillary flow cell 313 that can be collected. The detector lens a sample. In a further embodiment, the analyzer system may 307 collects the light passing through the aperture 306 and contain a sample recovery system for recovering at least a focuses the light onto an active area of the detector 309 after portion of the sample after analysis is complete. it passes through the detector filters 308. The detector filters In one aspect, the analyzer System consists of an electro 308 minimize aberrant noise signals due to light scatter or magnetic radiation Source for exciting a single particle ambient light while maximizing the signal emitted by the labeled with a fluorescent label. In one embodiment, the excited fluorescent moiety bound to the particle. The proces electromagnetic radiation source of the analyzer system is a Sor 310 processes the light signal from the particle according laser. In a further embodiment, the electromagnetic radiation to the methods described herein. Source is a continuous wave laser. In one embodiment, the microscope objective lens 305 is a In a typical embodiment, the electromagnetic radiation 25 high numerical aperture microscope objective. As used Source excites a fluorescent moiety attached to a label as the herein, “high numerical aperture lens' include a lens with a label passes through the interrogation space of the capillary numerical aperture of equal to or greater than 0.6. The flow cell. In some embodiments, the fluorescent label moiety numerical aperture is a measure of the number of highly includes one or more fluorescent dye molecules. In some diffracted image-forming light rays captured by the objective. embodiments, the fluorescent label moiety is a quantum dot. 30 A higher numerical aperture allows increasingly oblique rays Any fluorescent moiety as described herein may be used in to enter the objective lens and thereby produce a more highly the label. resolved image. Additionally, the brightness of an image A label is exposed to electromagnetic radiation when the increases with a higher numerical aperture. High numerical label passes through an interrogation space located within the aperture lenses are commercially available from a variety of capillary flow cell. The interrogation space is typically fluidly 35 Vendors, and any one lens having a numerical aperture of connected to a sampling system. In some embodiments the equal to or greater than approximately 0.6 may be used in the label passes through the interrogation space of the capillary analyzer system. In some embodiments, the lens has a flow cell due to a motive force to advance the label through the numerical aperture of about 0.6 to about 1.3. In some embodi analyzer system. The interrogation space is positioned Such ments, the lens has a numerical aperture of about 0.6 to about that it receives electromagnetic radiation emitted from the 40 1.0. In some embodiments, the lens has a numerical aperture radiation source. In some embodiments, the sampling system of about 0.7 to about 1.2. In some embodiments, the lens has is an automated sampling system capable of sampling a plu a numerical aperture of about 0.7 to about 1.0. In some rality of samples without intervention from a human operator. embodiments, the lens has a numerical aperture of about 0.7 The label passes through the interrogation space and emits to about 0.9. In some embodiments, the lens has a numerical a detectable amount of energy when excited by the electro 45 aperture of about 0.8 to about 1.3. In some embodiments, the magnetic radiation source. In one embodiment, an electro lens has a numerical aperture of about 0.8 to about 1.2. In magnetic radiation detectoris operably connected to the inter Some embodiments, the lens has a numerical aperture of rogation space. The electromagnetic radiation detector is about 0.8 to about 1.0. In some embodiments, the lens has a capable of detecting the energy emitted by the label, e.g., by numerical aperture of at least about 0.6. In some embodi the fluorescent moiety of the label. 50 ments, the lens has a numerical aperture of at least about 0.7. In a further embodiment of the analyzer system, the system In some embodiments, the lens has a numerical aperture of at further includes a sample preparation mechanism where a least about 0.8. In some embodiments, the lens has a numeri sample may be partially or completely prepared for analysis cal aperture of at least about 0.9. In some embodiments, the by the analyzer system. In some embodiments of the analyzer lens has a numerical aperture of at least about 1.0. In some system, the sample is discarded after it is analyzed by the 55 embodiments, the aperture of the microscope objective lens system. In other embodiments, the analyzer system further 305 is approximately 1.25. In an embodiment where a micro includes a sample recovery mechanism whereby at least a scope objective lens 305 of 0.8 is used, a Nikon 60x/0.8 NA portion, or alternatively all or substantially all, of the sample Achromat lens (Nikon, Inc., USA) can be used. may be recovered after analysis. In Such an embodiment, the In some embodiments, the electromagnetic radiation sample can be returned to the origin of the sample. In some 60 source 301 is a laser that emits light in the visible spectrum. In embodiments, the sample can be returned to microtiter wells all embodiments, the electromagnetic radiation source is set on a sample microtiter plate. The analyzer system typically such that wavelength of the laser is set such that it is of a further consists of a data acquisition system for collecting and sufficient wavelength to excite the fluorescent label attached reporting the detected signal. to the particle. In some embodiments, the laser is a continuous B. Single Particle Analyzer 65 wave laser with a wavelength of 639 nm. In other embodi As shown in FIG. 1A, described herein is one embodiment ments, the laser is a continuous wave laser with a wavelength of an analyzer system 300. The analyzer system 300 includes of 532 nm. In other embodiments, the laser is a continuous US 8,450,069 B2 65 66 wave laser with a wavelength of 422 nm. In other embodi focused spot of the beam is about 5 microns. In some embodi ments, the laser is a continuous wave laser with a wavelength ments, the diameter of the focused spot of the beam is about of 405 nm. Any continuous wave laser with a wavelength 10 microns. In some embodiments, the diameter of the Suitable for exciting a fluorescent moiety as used in the meth focused spot of the beam is about 12 microns. In some ods and compositions of the invention may be used without embodiments, the diameter of the focused spot of the beam is departing from the scope of the invention. about 13 microns. In some embodiments, the diameter of the In a single particle analyzer system 300, as each particle focused spot of the beam is about 14 microns. In some passes through the beam 311 of the electromagnetic radiation embodiments, the diameter of the focused spot of the beam is source, the particle enters into an excited state. When the about 15 microns. In some embodiments, the diameter of the particle relaxes from its excited state, a detectable burst of 10 focused spot of the beam is about 16 microns. In some light is emitted. The excitation-emission cycle is repeated embodiments, the diameter of the focused spot of the beam is many times by each particle in the length of time it takes for about 17 microns. In some embodiments, the diameter of the it to pass through the beam allowing the analyzer system 300 focused spot of the beam is about 18 microns. In some to detect tens to thousands of photons for each particle as it embodiments, the diameter of the focused spot of the beam is passes through an interrogation space 314. Photons emitted 15 about 19 microns. In some embodiments, the diameter of the by fluorescent particles are registered by the detector 309 focused spot of the beam is about 20 microns. (FIG. 1A) with a time delay indicative of the time for the In an alternate embodiment of the single particle analyzer particle label complex to pass through the interrogation system, more than one electromagnetic radiation Source can space. The photon intensity is recorded by the detector 309 be used to excite particles labeled with fluorescent labels of and sampling time is divided into bins, which are uniform, different wavelengths. In another alternate embodiment, arbitrary, time segments with freely selectable time channel more than one interrogation space in the capillary flow cell widths. The number of signals contained in each bin evalu can be used. In another alternate embodiment, multiple detec ated. One or a combination of several statistical analytical tors can be employed to detect different emission wave methods are employed in order to determine when a particle lengths from the fluorescent labels. An illustration incorpo is present. Such methods include determining the baseline 25 rating each of these alternative embodiments of an analyzer noise of the analyzer System and setting a signal strength for system is shown in FIG. 1B. These embodiments are incor the fluorescent label at a statistical level above baseline noise porated by reference from previous U.S. patent application to eliminate false positive signals from the detector. Ser. No. 11/048,660. The electromagnetic radiation source 301 is focused onto a In some embodiments of the analyzer system 300, a motive capillary flow cell 313 of the analyzer system 300 where the 30 force is required to move a particle through the capillary flow capillary flow cell 313 is fluidly connected to the sample cell 313 of the analyzer system 300. In one embodiment, the system. An interrogation space 314 is shown in FIG. 2A. The motive force can be a form of pressure. The pressure used to beam 311 from the continuous wave electromagnetic radia move a particle through the capillary flow cell can be gener tion source 301 of FIG. 1A is optically focused to a specified ated by a pump. In some embodiments, a Scivex, Inc. HPLC depth within the capillary flow cell 313. The beam 311 is 35 pump can be used. In some embodiments where a pump is directed toward the sample-filled capillary flow cell 313 at an used as a motive force, the sample can pass through the angle perpendicular to the capillary flow cell 313. The beam capillary flow cell at a rate of 1 uL/minto about 20 uL/min, or 311 is operated at a predetermined wavelength that is selected about 5 LL/min to about 20 Jul/min. In some embodiments, to excite a particular fluorescent label used to label the par the sample can pass through the capillary flow cell at a rate of ticle of interest. The size or volume of the interrogation space 40 about 5 LL/min. In some embodiments, the sample can pass 314 is determined by the diameter of the beam 311 together through the capillary flow cell at a rate of about 10 uL/min. In with the depth at which the beam 311 is focused. Alterna Some embodiments, the sample can pass through the capillary tively, the interrogation space can be determined by running a flow cell at a rate of about 15 uL/min. In some embodiments, calibration sample of known concentration through the ana the sample can pass through the capillary flow cell at a rate of lyZer system. 45 about 20 LL/min. In some embodiments, an electrokinetic When single molecules are detected in the sample concen force can be used to move the particle through the analyzer tration, the beam size and the depth of focus required for system. Such a method has been previously disclosed and is single molecule detection are set and thereby define the size incorporated by reference from previous U.S. patent applica of the interrogation space 314. The interrogation space 314 is tion Ser. No. 11/048,660. set Such that, with an appropriate sample concentration, only 50 In one aspect of the analyzer system 300, the detector 309 one particle is present in the interrogation space 314 during of the analyzer system detects the photons emitted by the each time interval over which time observations are made. fluorescent label. In one embodiment, the photon detector is a It will be appreciated that the detection interrogation vol photodiode. In a further embodiment, the detector is an ava ume as defined by the beam is not perfectly spherically lanche photodiode detector. In some embodiments, the pho shaped, and typically is a “bow-tie' shape. However, for the 55 todiodes can be silicon photodiodes with a wavelength detec purposes of definition, “volumes” of interrogation spaces are tion of 190 nm and 1100 nm. When germanium photodiodes defined herein as the Volume encompassed by a sphere of a are used, the wavelength of light detected is between 400 nm diameter equal to the focused spot diameter of the beam. The to 1700 nm. In other embodiments, when an indium gallium focused spot of the beam 311 may have various diameters arsenide photodiode is used, the wavelength of light detected without departing from the scope of the present invention. In 60 by the photodiode is between 800 nm and 2600 nm. When some embodiments, the diameter of the focused spot of the lead sulfide photodiodes are used as detectors, the wavelength beam is about 1 to about 5, 10, 15, or 20 microns, or about 5 of light detected is between 1000 nm and 3500 nm. to about 10, 15, or 20 microns, or about 10 to about 20 In some embodiments, the optics of the electromagnetic microns, or about 10 to about 15 microns. In some embodi radiation source 301 and the optics of the detector 309 are ments, the diameter of the focused spot of the beam is about 65 arranged in a conventional optical arrangement. In such an 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or arrangement, the electromagnetic radiation Source and the 20 microns. In some embodiments, the diameter of the detector are aligned on different focal planes. The arrange US 8,450,069 B2 67 68 ment of the laser and the detector optics of the analyzer types, as well as, tunable diode lasers (red to infrared regions), system as shown in FIGS. 1A and 1B is that of a conventional each with the possibility of frequency doubling. The lasers optical arrangement. provide continuous illumination with no accessory electronic In some embodiments, the optics of the electromagnetic or mechanical devices, such as shutters, to interrupt their radiation source and the optics of the detector are arranged in illumination. In an embodiment where a continuous wave a confocal optical arrangement. In such an arrangement, the laser is used, an electromagnetic radiation source of 3 mW electromagnetic radiation source 301 and the detector 309 are may be of sufficient energy to excite a fluorescent label. A aligned on the same focal plane. The confocal arrangement beam from a continuous wave laser of Such energy output renders the analyzer more robust because the electromagnetic may be between 2 to 5um in diameter. The time of exposure radiation source 301 and the detector optics 309 do not need 10 of the particle to laser beam in order to be exposed to 3 mW to be realigned if the analyzer System is moved. This arrange may be a time period of about 1 m.sec. In alternate embodi ment also makes the use of the analyzer more simplified ments, the time of exposure to the laser beam may be equal to because it eliminates the need to realign the components of or less than about 500 usec. In an alternate embodiment, the the analyzer system. The confocal arrangement for the ana time of exposure may be equal to or less than about 100 usec. lyzer 300 (FIG. 1A) and the analyzer 355 (FIG.1B) are shown 15 In an alternate embodiment, the time of exposure may be in FIGS. 3A and 3B respectively. FIG. 3A shows that the equal to or less than about 50 usec. In an alternate embodi beam 311 from an electromagnetic radiation source 301 is ment, the time of exposure may be equal to or less than about focused by the microscope objective 315 to form one inter 10 usec. rogation space 314 (FIG. 2A) within the capillary flow cell LEDs are another low-cost, high reliability illumination 313. A dichroic mirror 316, which reflects laser light but source. Recent advances in ultra-bright LEDs and dyes with passes fluorescent light, is used to separate the fluorescent high absorption cross-section and quantum yield Support the light from the laser light. Filter 317 that is positioned in front applicability of LEDs to single particle detection. Such lasers of the detector eliminates any non-fluorescent light at the could be used alone or in combination with other light sources detector. In some embodiments, an analyzer system config Such as mercury arc lamps, elemental arc lamps, halogen ured in a confocal arrangement can comprise two or more 25 lamps, arc discharges, plasma discharges, light-emitting interrogations spaces. Such a method has been previously diodes, or combination of these. disclosed and is incorporated by reference from previous U.S. In other embodiments, the EM source could be in the form patent application Ser. No. 11/048,660. of a pulse wave laser. In Such an embodiment, the pulse size The laser can be a tunable dye laser, Such as a helium-neon of the laser is an important factor. In Such an embodiment, the laser. The laser can be set to emit a wavelength of 632.8 nm. 30 size, focus spot, and the total energy emitted by the laser is Alternatively, the wavelength of the laser can be set to emit a important and must be of Sufficient energy as to be able to wavelength of 543.5 nm or 1523 mm. Alternatively, the elec excite the fluorescent label. When a pulse laser is used, a pulse tromagnetic laser can be an argon ion laser. In Such an of longer duration may be required. In some embodiments a embodiment, the argon ion laser can be operated as a con laser pulse of 2 nanoseconds may be used. In some embodi tinuous gas laser at about 25 different wavelengths in the 35 ments a laser pulse of 5 nanoseconds may be used. In some visible spectrum, the wavelength set between 408.9 and 686.1 embodiments a pulse of between 2 to 5 nanoseconds may be nm but at its optimum performance set between 488 and 514.5 used. . The optimal laser intensity depends on the photo bleaching 1. Electromagnetic Radiation Source characteristics of the single dyes and the length of time In some embodiments of the analyzer system a chemilu 40 required to traverse the interrogation space (including the minescent label may be used. In such an embodiment, it may speed of the particle, the distance between interrogation not be necessary to utilize an EM source for detection of the spaces if more than one is used and the size of the interroga particle. In another embodiment, the extrinsic label or intrin tion space(s)). To obtain a maximal signal, it is desirable to sic characteristic of the particle is a light-interacting label or illuminate the sample at the highest intensity which will not characteristic, such as a fluorescent label or a light-scattering 45 result in photo bleaching a high percentage of the dyes. The label. In such an embodiment, a source of EM radiation is preferred intensity is one such that no more that 5% of the used to illuminate the label and/or the particle. EM radiation dyes are bleached by the time the particle has traversed the sources for excitation of fluorescent labels are preferred. interrogation space. In some embodiments, the analyzer system consists of an The power of the laser is set depending on the type of dye electromagnetic radiation source 301. Any number of radia 50 molecules that need to be stimulated and the length of time the tion Sources may be used in any one analyzer System 300 dye molecules are stimulated, and/or the speed with which the without departing from the scope of the invention. Multiple dye molecules pass through the capillary flow cell. Laser Sources of electromagnetic radiation have been previously power is defined as the rate at which energy is delivered by the disclosed and are incorporated by reference from previous beam and is measured in units of Joules/second, or Watts. It U.S. patent application Ser. No. 11/048,660. In some embodi 55 will be appreciated that the greater the power output of the ments, all the continuous wave electromagnetic (EM) radia laser, the shorter the time that the laser illuminates the particle tion Sources emit electromagnetic radiation at the same wave may be, while providing a constant amount of energy to the lengths. In other embodiments, different sources emit interrogation space while the particle is passing through the different wavelengths of EM radiation. space. Thus, in Some embodiments, the combination of laser In one embodiment, the EM source(s) 301, 351, 352 are 60 power and time of illumination is such that the total energy continuous wave lasers producing wavelengths of between received by the interrogation space during the time of illumi 200 nm and 1000 nm. Such EM sources have the advantage of nation is more than about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, being Small, durable and relatively inexpensive. In addition, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 they generally have the capacity to generate larger fluorescent microJoule. In some embodiments, the combination of laser signals than other light Sources. Specific examples of suitable 65 power and time of illumination is such that the total energy continuous wave EM sources include, but are not limited to: received by the interrogation space during the time of illumi lasers of the argon, krypton, helium-neon, helium-cadmium nation is less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, US 8,450,069 B2 69 70 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or 110 micro illumination is such that the total energy received by the Joule. In some embodiments, the combination of laser power interrogation space during the time of illumination is about 80 and time of illumination is such that the total energy received microJoule. In some embodiments, the combination of laser by the interrogation space during the time of illumination is power and time of illumination is such that the total energy between about 0.1 and 100 microJoule. In some embodi received by the interrogation space during the time of illumi ments, the combination of laser power and time of illumina nation is about 90 microJoule. In some embodiments, the tion is such that the total energy received by the interrogation combination of laser power and time of illumination is such space during the time of illumination is between about 1 and that the total energy received by the interrogation space dur 100 microJoule. In some embodiments, the combination of ing the time of illumination is about 100 microJoule. laser power and time of illumination is such that the total 10 In some embodiments, the laser power output is set to at energy received by the interrogation space during the time of least about 1 mW, 2 mW, 3 mW, 4 mW, 5 mW, 6, mw, 7 mW. illumination is between about 1 and 50 microJoule. In some 8 mW, 9 mW. 10 mW, 13 mW, 15 mW, 20 mW, 25 mW, 30 embodiments, the combination of laser power and time of mW, 40 mW, 50 mW, 60 mW, 70 mW, 80 mW, 90 mW, 100 illumination is such that the total energy received by the mW, or more than 100 mW. In some embodiments, the laser interrogation space during the time of illumination is between 15 power output is set to at least about 1 mW. In some embodi about 2 and 50 microJoule. In some embodiments, the com ments, the laser power output is set to at least about 3 mW. In bination of laser power and time of illumination is such that Some embodiments, the laser power output is set to at least the total energy received by the interrogation space during the about 5 mW. In some embodiments, the laser power output is time of illumination is between about 3 and 60 microJoule. In set to at least about 10 mW. In some embodiments, the laser Some embodiments, the combination of laser power and time power output is set to at least about 15 mW. In some embodi of illumination is such that the total energy received by the ments, the laser power output is set to at least about 20 mW. In interrogation space during the time of illumination is between Some embodiments, the laser power output is set to at least about 3 and 50 microJoule. In some embodiments, the com about 30 mW. In some embodiments, the laser power output bination of laser power and time of illumination is such that is set to at least about 40 mW. In some embodiments, the laser the total energy received by the interrogation space during the 25 power output is set to at least about 50 mW. In some embodi time of illumination is between about 3 and 40 microJoule. In ments, the laser power output is set to at least about 60 mW. In Some embodiments, the combination of laser power and time Some embodiments, the laser power output is set to at least of illumination is such that the total energy received by the about 90 mW. interrogation space during the time of illumination is between The time that the laser illuminates the interrogation space about 3 and 30 microJoule. In some embodiments, the com 30 can be set to no less than about 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, bination of laser power and time of illumination is such that 50,60, 70, 80,90, 100,150, 200, 150,300,350,400,450,500, the total energy received by the interrogation space during the 600, 700, 800, 900, or 1000 microseconds. The time that the time of illumination is about 1 microJoule. In some embodi laser illuminates the interrogation space can be set to no more ments, the combination of laser power and time of illumina than about 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, tion is such that the total energy received by the interrogation 35 150, 200, 150, 300, 350, 400, 450, 500, 600, 700, 800, 900, space during the time of illumination is about 3 microJoule. In 1000, 1500, or 2000 microseconds. The time that the laser Some embodiments, the combination of laser power and time illuminates the interrogation space can be set between about of illumination is such that the total energy received by the 1 and 1000 microseconds. The time that the laser illuminates interrogation space during the time of illumination is about 5 the interrogation space can be set between about 5 and 500 microJoule. In some embodiments, the combination of laser 40 microseconds. The time that the laser illuminates the interro power and time of illumination is such that the total energy gation space can be set between about 5 and 100 microsec received by the interrogation space during the time of illumi onds. The time that the laser illuminates the interrogation nation is about 10 microJoule. In some embodiments, the space can be set between about 10 and 100 microseconds. The combination of laser power and time of illumination is such time that the laser illuminates the interrogation space can be that the total energy received by the interrogation space dur 45 set between about 10 and 50 microseconds. The time that the ing the time of illumination is about 15 microJoule. In some laser illuminates the interrogation space can be set between embodiments, the combination of laser power and time of about 10 and 20 microseconds. The time that the laser illu illumination is such that the total energy received by the minates the interrogation space can be set between about 5 interrogation space during the time of illumination is about 20 and 50 microseconds. The time that the laser illuminates the microJoule. In some embodiments, the combination of laser 50 interrogation space can be set between about 1 and 100 micro power and time of illumination is such that the total energy seconds. In some embodiments, the time that the laser illu received by the interrogation space during the time of illumi minates the interrogation space is about 1 microsecond. In nation is about 30 microJoule. In some embodiments, the some embodiments, the time that the laser illuminates the combination of laser power and time of illumination is such interrogation space is about 5 microseconds. In some embodi that the total energy received by the interrogation space dur 55 ments, the time that the laser illuminates the interrogation ing the time of illumination is about 40 microJoule. In some space is about 10 microseconds. In some embodiments, the embodiments, the combination of laser power and time of time that the laser illuminates the interrogation space is about illumination is such that the total energy received by the 25 microseconds. In some embodiments, the time that the interrogation space during the time of illumination is about 50 laser illuminates the interrogation space is about 50 micro microJoule. In some embodiments, the combination of laser 60 seconds. In some embodiments, the time that the laser illu power and time of illumination is such that the total energy minates the interrogation space is about 100 microseconds. In received by the interrogation space during the time of illumi some embodiments, the time that the laser illuminates the nation is about 60 microJoule. In some embodiments, the interrogation space is about 250 microseconds. In some combination of laser power and time of illumination is such embodiments, the time that the laser illuminates the interro that the total energy received by the interrogation space dur 65 gation space is about 500 microseconds. In some embodi ing the time of illumination is about 70 microJoule. In some ments, the time that the laser illuminates the interrogation embodiments, the combination of laser power and time of space is about 1000 microseconds. US 8,450,069 B2 71 72 For example, the time that the laser illuminates the inter interrogation space size depends on the brightness of the rogation space can be set to 1 millisecond, 250 microseconds, particles to be detected, the level of background signal, and 100 microseconds, 50 microseconds, 25 microseconds or 10 the concentration of the sample to be analyzed. microseconds with a laser that provides a power output of 3 The size of the interrogation space 314 can be limited by mW, 4 mw, 5 mW, or more than 5 mW. In some embodiments, adjusting the optics of the analyzer. In one embodiment, the a label is illuminated with a laser that provides a power output diameter of the beam 311 can be adjusted to vary the volume of 3 mW and illuminates the label for about 1000 microsec of the interrogation space 314. onds. In other embodiments, a label is illuminated for less In another embodiment, the field of view of the detector than 1000 milliseconds with a laser providing a power output 309 can be varied. Thus, the source 301 and the detector 309 of not more than about 20 mW. In other embodiments, the 10 can be adjusted so that single particles will be illuminated and label is illuminated with a laser power output of 20 mW for detected within the interrogation space 314. In another less than or equal to about 250 microseconds. In some embodiment, the width of aperture 306 (FIG. 1A) that deter embodiments, the label is illuminated with a laser power mine the field of view of the detector 309 is variable. This output of about 5 mW for less than or equal to about 1000 configuration allows for altering the interrogation space, in microseconds. 15 near real time, to compensate for more or less concentrated 2. Capillary Flow Cell samples, ensuring a low probability of two or more particles The capillary flow cell is fluidly connected to the sample simultaneously being within an interrogation space. Similar system. In one embodiment, the interrogation space 314 of an alterations for two or more interrogation spaces, 370 and 371, analyzer system is determined by the cross sectional area of may performed. the corresponding beam 311 and by a segment of the beam In another embodiment, the interrogation space can be within the field of view of the detector 309. In one embodi defined through the use of a calibration sample of known ment of the analyzer system, the interrogation space 314 has concentration that is passed through the capillary flow cell a volume, as defined herein, of between about between about prior to the actual sample being tested. When only one single 0.01 and 500 pIL, or between about 0.01 pland 100 pL, or particle is detected at a time in the calibration sample as the between about 0.01 pl. and 10 pl. or between about 0.01 p. 25 sample is passing through the capillary flow cell, the depth of and 1 pl. or between about 0.01 pl. and 0.5 pI, or between focus together with the diameter of the beam of the electro about 0.02 pl. and about 300 pI, or between about 0.02 pl. magnetic radiation source determines the size of the interro and about 50 pl. or between about 0.02 pl. and about 5 pl. or gation space in the capillary flow cell. between about 0.02 pl. and about 0.5 pI, or between about Physical constraints to the interrogation spaces can also be 0.02 pl. and about 2 pl. or between about 0.05 pl. and about 30 provided by a solid wall. In one embodiment, the wall is one 50 pl. or between about 0.05 pl. and about 5p, or between or more of the walls of a flow cell 313 (FIG. 2A), when the about 0.05 pland about 0.5 pL, or between about 0.05 pland sample fluid is contained within a capillary. In one embodi about 0.2 p, or between about 0.1 pI, and about 25 pl. In ment, the cell is made of glass, but other Substances transpar Some embodiments, the interrogation space has a volume ent to light in the range of about 200 to about 1,000 nm or between about 0.01 pland 10 pl. In some embodiments, the 35 higher, Such as quartz, fused silica, and organic materials such interrogation space 314 has a volume between about 0.01 p. as Teflon, nylon, plastics, such as polyvinylchloride, polysty and 1 p. In some embodiments, the interrogation space 314 rene, and polyethylene, or any combination thereof, may be has a volume between about 0.02 pl. and about 5 pl. In some used without departing from the scope of the present inven embodiments, the interrogation space 314 has a Volume tion. Although other cross-sectional shapes (e.g., rectangular, between about 0.02 pl. and about 0.5 pL. In some embodi 40 cylindrical) may be used without departing from the scope of ments, the interrogation space 314 has a Volume between the present invention, in one embodiment the capillary flow about 0.05 pl. and about 0.2 pl. In some embodiments, the cell 313 has a square cross section. In another embodiment, interrogation space 314 has a volume of about 0.1 pI. Other the interrogation space may be defined at least in part by a useful interrogation space Volumes are as described herein. It channel (not shown) etched into a chip (not shown). Similar should be understood by one skilled in the art that the inter 45 considerations apply to embodiments in which two interro rogation space 314 can be selected for maximum perfor gation spaces are used (370 and 371 in FIG. 2B). mance of the analyzer. Although very Small interrogation The interrogation space is bathed in a fluid. In one embodi spaces have been shown to minimize the background noise, ment, the fluid is aqueous. In other embodiments, the fluid is large interrogation spaces have the advantage that low con non-aqueous or a combination of aqueous and non-aqueous centration samples can be analyzed in a reasonable amount of 50 fluids. In addition the fluid may contain agents to adjust pH, time. In embodiments in which two interrogation spaces 370 ionic composition, or sieving agents, such as soluble macro and 371 are used, volumes such as those described hereinfor particles or polymers or gels. It is contemplated that valves or a single interrogation space 314 may be used. other devices may be present between the interrogation In one embodiment of the present invention, the interroga spaces to temporarily disrupt the fluid connection. Interroga tion spaces are large enough to allow for detection of particles 55 tion spaces temporarily disrupted are considered to be con at concentrations ranging from about 1000 femtomolar (fM) nected by fluid. to about 1 Zeptomolar (ZM). In one embodiment of the present In another embodiment of the invention, an interrogation invention, the interrogation spaces are large enough to allow space is the single interrogation space present within the flow for detection of particles at concentrations ranging from cell 313 which is constrained by the size of a laminar flow of about 1000 fM to about 1 attomolar (aM). In one embodiment 60 the sample material within a diluent Volume, also called of the present invention, the interrogation spaces are large sheath flow. In these and other embodiments, the interroga enough to allow for detection of particles at concentrations tion space can be defined by sheath flow alone or in combi ranging from about 10 fM to about 1 attomolar (aM). In many nation with the dimensions of the illumination source or the cases, the large interrogation spaces allow for the detection of field of view of the detector. Sheath flow can be configured in particles at concentrations of less than about 1 fM without 65 numerous ways, including: The sample material is the interior additional pre-concentration devices or techniques. One material in a concentric laminar flow, with the diluent volume skilled in the art will recognize that the most appropriate in the exterior; the diluent volume is on one side of the sample US 8,450,069 B2 73 74 volume; the diluent volume is on two sides of the sample other factors well known in the art. In some embodiments, material; the diluent volume is on multiple sides of the sample pumps of the system are driven by stepper motors, which are material, but not enclosing the sample material completely; easy to control very accurately with a microprocessor. the diluent Volume completely Surrounds the sample mate In preferred embodiments, the flush and analysis pumps rial; the diluent Volume completely surrounds the sample 5 are used in series, with special check valves to control the material concentrically; the sample material is the interior direction of flow. The plumbing is designed so that when the material in a discontinuous series of drops and the diluent analysis pump draws up the maximum sample, the sample Volume completely Surrounds each drop of sample material. does not reach the pump itself. This is accomplished by In some embodiments, single molecule detectors of the choosing the ID and length of the tubing between the analysis invention comprise no more than one interrogation space. In 10 pump and the analysis capillary Such that the tubing Volume Some embodiments, multiple interrogation spaces are used. is greater than the stroke Volume of the analysis pump. Multiple interrogation spaces have been previously disclosed 4. Detectors and are incorporated by reference from U.S. patent applica In one embodiment, light (e.g., light in the ultra-violet, tion Ser. No. 11/048,660. One skilled in the art will recognize visible or infrared range) emitted by a fluorescent label after that in some cases the analyzer will contain a plurality of 15 exposure to electromagnetic radiation is detected. The detec distinct interrogation spaces. In some embodiments, the ana tor 309 (FIG. 1A), or detectors (364,365, FIG.1B), is capable lyZer contains 2, 3, 4, 5, 6 or more distinct interrogation of capturing the amplitude and duration of photonbursts from Spaces. a fluorescent moiety, and further converting the amplitude 3. Motive Force and duration of the photon burst to electrical signals. Detec In one embodiment of the analyzer system, the particles are tion devices such as CCD cameras, video input module cam moved through the interrogation space by a motive force. In eras, and Streak cameras can be used to produce images with Some embodiments, the motive force for moving particles is contiguous signals. In another embodiment, devices such as a pressure. In some embodiments, the pressure is Supplied by a bolometer, a photodiode, a photodiode array, avalanche pho pump, and air pressure source, a vacuum source, a centrifuge, todiodes, and photomultipliers which produce sequential sig or a combination thereof. In some embodiments, the motive 25 nals may be used. Any combination of the aforementioned force for moving particles is an electrokinetic force. The use detectors may also be used. In one embodiment, avalanche of an electrokinetic force as a motive force has been previ photodiodes are used for detecting photons. ously disclosed in a prior application and is incorporated by Using specific optics between an interrogation space 314 reference from U.S. patent application Ser. No. 11/048,660. (FIG. 2A) and its corresponding detector 309 (FIG. 1A), In one embodiment, pressure can be used as a motive force 30 several distinct characteristics of the emitted electromagnetic to move particles through the interrogation space of the cap radiation can be detected including: emission wavelength, illary flow cell. In a further embodiment, pressure is supplied emission intensity, burst size, burst duration, and fluores to move the sample by means of a pump. Suitable pumps are cence polarization. In some embodiments, the detector 309 is known in the art. In one embodiment, pumps manufactured a photodiode that is used in reverse bias. A photodiode set in for HPLC applications, such as those made by Scivax, Inc. 35 reverse bias usually has an extremely high resistance. This can be used as a motive force. In other embodiments, pumps resistance is reduced when light of an appropriate frequency manufactured for microfluidics applications can be used shines on the P/Njunction. Hence, a reverse biased diode can when Smaller Volumes of sample are being pumped. Such be used as a detector by monitoring the current running pumps are described in U.S. Pat. Nos. 5,094,594, 5,730,187, through it. Circuits based on this effect are more sensitive to 6,033,628, and 6,533,553, which discloses devices which can 40 light than ones based on Zero bias. pump fluid volumes in the nanoliter or picoliter range. Pref In one embodiment of the analyzer system, the photodiode erably all materials within the pump that come into contact can be an avalanche photodiode, which can be operated with with sample are made of highly inert materials, e.g., poly much higher reverse bias than conventional photodiodes, thus etheretherketone (PEEK), fused silica, or sapphire. allowing each photo-generated carrier to be multiplied by A motive force is necessary to move the sample through the 45 avalanche breakdown, resulting in internal gain within the capillary flow cell to push the sample through the interroga photodiode, which increases the effective responsiveness tion space for analysis. A motive force is also required to push (sensitivity) of the device. The choice of photodiode is deter a flushing sample through the capillary flow cell after the mined by the energy or emission wavelength emitted by the sample has been passed through. A motive force is also fluorescently labeled particle. In some embodiments, the pho required to push the sample back out into a sample recovery 50 todiode is a silicon photodiode that detects energy in the range vessel, when sample recovery is employed. Standard pumps of 190-1100 nm; in another embodiment the photodiode is a come in a variety of sizes, and the proper size may be chosen germanium photodiode that detects energy in the range of to Suit the anticipated sample size and flow requirements. In 800-1700 nm; in another embodiment the photodiode is an Some embodiments, separate pumps are used for sample indium gallium arsenide photodiode that detects energy in the analysis and for flushing of the system. The analysis pump 55 range of 800-2600 nmi; and in yet other embodiments, the may have a capacity of approximately 0.000001 mL to photodiode is a lead sulfide photodiode that detects energy in approximately 10 mL, or approximately 0.001 mL to the range of between less than 1000 nm to 3500 nm. In some approximately 1 mL, or approximately 0.01 mL to approxi embodiments, the avalanche photodiode is a single-photon mately 0.2 mL, or approximately 0.005, 0.01, 0.05, 0.1, or 0.5 detector designed to detect energy in the 400 nm to 1100 nm mL. Flush pumps may be of larger capacity than analysis 60 wavelength range. Single photon detectors are commercially pumps. Flush pumps may have a volume of about 0.01 mL to available (e.g., Perkin Elmer, Wellesley, Mass.). about 20 mL, or about 0.1 mL to about 10 mL, or about 0.1 mL In some embodiments, the detector is an avalanche photo to about 2 mL, or about or about 0.05, 0.1, 0.5, 1, 5, or 10 mL. diode detector that detects energy between 300 nm and 1700 These pump sizes are illustrative only, and those of skill in the nm. In one embodiment, silicon avalanche photodiodes can art will appreciate that the pump size may be chosen accord 65 be used to detect wavelengths between 300 nm and 1100 nm. ing to the application, sample size, Viscosity of fluid to be Indium gallium arsenic photodiodes can be used to detect pumped, tubing dimensions, rate of flow, temperature, and wavelengths between 900 nm and 1700 nm. In some embodi US 8,450,069 B2 75 76 ments, an analyzer system can comprise at least one detector, can be drawn up using either a vacuum Suction created by a in other embodiments, the analyzer system can comprise at pump or by pressure applied to the sample that would push least two detectors, and each detector can be chosen and liquid into the tube, or by any other mechanism that serves to configured to detect light energy at a specific wavelength introduce the sample into the sampling tube. Generally, but range. For example, two separate detectors can be used to not necessarily, the sampling system introduces a sample of detect particles that have been tagged with different labels, known sample Volume into the single particle analyzer, in which upon excitation with an EM source, will emit photons Some embodiments where the presence or absence of a par with energy in different spectra. In one embodiment, an ana ticle or particles is detected, precise knowledge of the sample lyZer system can comprise a first detector that can detect size is not critical. In preferred embodiments the sampling fluorescent energy in the range of 450-700 nm such as that 10 system provides automated sampling for a single sample or a emitted by a green dye (e.g., Alexa Fluor 546); and a second plurality of samples. In embodiments where a sample of detector that can detect fluorescent energy in the range of known Volume is introduced into the system, the sampling 620-780 nm such as that emitted by a far-red dye (e.g., Alexa system provides a sample for analysis of more than about Fluor 647). Detectors for detecting fluorescent energy in the 0.0001, 0.001, 0.01, 0.1, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, range of 400-600 nm such as that emitted by blue dyes (e.g., 15 90, 100, 150, 200, 500, 1000, 1500, or 2000 ul. In some Hoechst 33342), and for detecting energy in the range of embodiments the sampling system provides a sample for 560-700 nm such as that emitted by reddyes (Alexa Fluor 546 analysis of less than about 2000, 1000, 500, 200, 100,90, 80, and Cy3) can also be used. 70, 60.50, 40,30, 20, 10, 5, 2, 1, 0.1, 0.01, or 0.001 ul. In some A system comprising two or more detectors can be used to embodiments the sampling system provides a sample for detect individual particles that are each tagged with two or analysis of between about 0.01 and 1500 ul, or about 0.1 and more labels that emit light in different spectra. For example, 1000 ul, or about 1 and 500 ul, or about 1 and 100 ul, or about two different detectors can detect an antibody that has been 1 and 50 ul, or about 1 and 20 ul. In some embodiments, the tagged with two different dye labels. Alternatively, an ana sampling system provides a sample for analysis between lyZer system comprising two detectors can be used to detect about 5ul and 200 ul, or about 5ul and about 100 ul, or about particles of different types, each type being tagged with dif 25 5ul and 50 ul. In some embodiments, the sampling system ferent dye molecules, or with a mixture of two or more dye provides a sample for analysis between about 10 ul and 200 molecules. For example, two different detectors can be used ul, or between about 10ul and 100 ul, or between about 10 ul to detect two different types of antibodies that recognize two and 50 ul. In some embodiments, the sampling system pro different proteins, each type being tagged with a different dye vides a sample for analysis between about 0.5ul and about 50 label or with a mixture of two or more dye label molecules. By 30 ul. varying the proportion of the two or more dye label mol Because of the sensitivity of the methods of the present ecules, two or more different particle types can be individu invention, very small sample volumes can be used. For ally detected using two detectors. It is understood that three or example, the methods here can be used to measure VEGF in more detectors can be used without departing from the scope Small sample Volumes, e.g., 10 Jul or less, compared to the of the invention. 35 standard sample volume of 100 ul. The present invention It should be understood by one skilled in the art that one or enables a greater number of samples to provide quantifiable more detectors can be configured at each interrogation space, results in Small Volume samples compared to other methods. whether one or more interrogation spaces are defined within For example, a lysate prepared from a typical 1 mm needle a flow cell, and that each detector may be configured to detect biopsy may have a Volume less than or equal to 10 ul. Using any of the characteristics of the emitted electromagnetic 40 the present invention, such sample can be assayed. In some radiation listed above. The use of multiple detectors, e.g., for embodiments, the present invention allows the use of sample multiple interrogation spaces, has been previously disclosed volume under 100 ul. In some embodiments, the present in a prior application and is incorporated by reference here invention allows the use of sample volume under 90 ul. In from U.S. patent application Ser. No. 11/048,660. Once a Some embodiments, the present invention allows the use of particle is labeled to render it detectable (or if the particle 45 sample volume under 80 ul. In some embodiments, the possesses an intrinsic characteristic rendering it detectable), present invention allows the use of sample volume under 70 any suitable detection mechanism known in the art may be ul. In some embodiments, the present invention allows the use used without departing from the scope of the present inven of sample volume under 60 ul. In some embodiments, the tion, for example a CCD camera, a video input module cam present invention allows the use of sample volume under 50 era, a Streak camera, abolometer, a photodiode, a photodiode 50 ul. In some embodiments, the present invention allows the use array, avalanche photodiodes, and photomultipliers produc of sample Volume under 40 ul. In some embodiments, the ing sequential signals, and combinations thereof. Different present invention allows the use of sample volume under 30 characteristics of the electromagnetic radiation may be ul. In some embodiments, the present invention allows the use detected including: emission wavelength, emission intensity, of sample Volume under 25 Jul. In some embodiments, the burst size, burst duration, fluorescence polarization, and any 55 present invention allows the use of sample volume under 20 combination thereof. ul. In some embodiments, the present invention allows the use C. Sampling System of sample Volume under 15 Jul. In some embodiments, the In a further embodiment, the analyzer system may include present invention allows the use of sample volume under 10 a sampling system to prepare the sample for introduction into ul. In some embodiments, the present invention allows the use the analyzer System. The sampling system included is capable 60 of sample Volume under 5 ul. In some embodiments, the of automatically sampling a plurality of samples and provid present invention allows the use of sample Volume under 1 ul. ing a fluid communication between a sample container and a In Some embodiments, the present invention allows the use of first interrogation space. sample volume under 0.05 ul. In some embodiments, the In some embodiments, the analyzer System of the invention present invention allows the use of sample volume under 0.01 includes a sampling system for introducing an aliquot of a 65 ul. In some embodiments, the present invention allows the use sample into the single particle analyzer for analysis. Any of sample volume under 0.005 ul. In some embodiments, the mechanism that can introduce a sample may be used. Samples present invention allows the use of sample Volume under US 8,450,069 B2 77 78 0.001 ul. In some embodiments, the present invention allows of diluents, addition of reagents, heating or cooling, addition the use of sample volume under 0.0005ul. In some embodi of label, binding of label, cross-linking with illumination, ments, the present invention allows the use of sample Volume separation of unbound label, inactivation and/or removal of under 0.0001 ul. interfering compounds and any other steps necessary for the In some embodiments, the sampling system provides a sample to be prepared for analysis by the single particle sample size that can be varied from sample to sample. In these analyzer. In some embodiments, blood is treated to separate embodiments, the sample size may be any one of the sample out plasma or serum. Additional labeling, removal of sizes described herein, and may be changed with every unbound label, and/or dilution steps may also be performed sample, or with sets of samples, as desired. on the serum or plasma sample. Sample Volume accuracy, and sample to sample Volume 10 In some embodiments, the analyzer system includes a precision of the sampling system, is required for the analysis sample preparation system that performs some or all of the at hand. In some embodiments, the precision of the sampling processes needed to provide a sample ready for analysis by Volume is determined by the pumps used, typically repre the single particle analyzer. This system may perform any or sented by a CV of less than about 50, 40,30, 20, 10, 5, 4,3,2, all of the steps listed above for sample preparation. In some 1,0.5,0.1, 0.05, or 0.01% of sample volume. In some embodi 15 embodiments samples are partially processed by the sample ments, the sample to sample precision of the sampling system preparation system of the analyzer system. Thus, in some is represented by a CV of less than about 50, 40, 30, 20, 10, 5, embodiments, a sample may be partially processed outside 4, 3, 2, 1, 0.5, 0.1, 0.05, or 0.01%. In some embodiments, the the analyzer System first. For example, the sample may be intra-assay precision of the sampling system is represented by centrifuged first. The sample may then be partially processed a CV of less than about 10, 5, 1, 0.5, or 0.1%. In some inside the analyzer by a sample preparation system. Process embodiments, the intra-assay precision of the sampling sys ing inside the analyzer includes labeling the sample, mixing tem shows a CV of less than about 10%. In some embodi the sample with a buffer and other processing steps that will ments, the interassay precision of the sampling system is be known to one in the art. In some embodiments, a blood represented by a CV of less than about 5%. In some embodi sample is processed outside the analyzer system to provide a ments, the interassay precision of the sampling system shows 25 serum or plasma sample, which is introduced into the ana a CV of less than about 1%. In some embodiments, the inter lyZer system and further processed by a sample preparation assay precision of the sampling system is represented by a CV system to label the particle or particles of interestand, option of less than about 0.5%. In some embodiments, the interassay ally, to remove unbound label. In other embodiments prepa precision of the sampling system shows a CV of less than ration of the sample can include immunodepletion of the about 0.1%. 30 sample to remove particles that are not of interestorto remove In some embodiments, the sampling system provides low particles that can interfere with sample analysis. In yet other sample carryover, advantageous in that an additional wash embodiments, the sample can be depleted of particles that can step is not required between samples. Thus, in Some embodi interfere with the analysis of the sample. For example, sample ments, sample carryover is less than about 1, 0.5, 0.1, 0.05, preparation can include the depletion of heterophilic antibod 0.04, 0.03, 0.02, 0.01, 0.005, or 0.001%. In some embodi 35 ies, which are known to interfere with immunoassays that use ments, sample carryover is less than about 0.02%. In some non-human antibodies to directly or indirectly detect a par embodiments, sample carryover is less than about 0.01%. ticle of interest. Similarly, other proteins that interfere with In Some embodiments the sampler provides a sample loop. measurements of the particles of interest can be removed In these embodiments, multiple samples are drawn into tub from the sample using antibodies that recognize the interfer ing sequentially and each is separated from the others by a 40 ing proteins. “plug of buffer. The samples typically are read one after the In Some embodiments, the sample can be subjected to Solid other with no flushing in between. Flushing is done once at phase extraction prior to being assayed and analyzed. For the end of the loop. In embodiments where a buffer “plug is example, a serum sample that is assayed for cAMP can first be used, the plug may be recovered ejecting the buffer plug into Subjected to Solid phase extraction using a c18 column to a separate well of a microtiter plate. 45 which it binds. Other proteins such as proteases, lipases and The sampling system may be adapted for use with standard phosphatases are washed from the column, and the cAMP is assay equipment, for example, a 96-well microtiter plate, or, eluted essentially free of proteins that can degrade or interfere preferably, a 384-well plate. In some embodiments the sys with measurements of cAMP Solid phase extraction can be tem includes a 96 well plate positioner and a mechanism to used to remove the basic matrix of a sample, which can dip the sample tube into and out of the wells, e.g., a mecha 50 diminish the sensitivity of the assay. In yet other embodi nism providing movementalong the X,Y, and Z axes. In some ments, the particles of interest present in a sample may be embodiments, the sampling system provides multiple sam concentrated by drying or lyophilizing a sample and solubi pling tubes from which samples may be stored and extracted lizing the particles in a smaller Volume than that of the origi from, when testing is commenced. In some embodiments, all nal sample. samples from the multiple tubes are analyzed on one detector. 55 In Some embodiments the analyzer System provides a In other embodiments, multiple single molecule detectors sample preparation system that provides complete prepara may be connected to the sample tubes. Samples may be tion of the sample to be analyzed on the system, such as prepared by Steps that include operations performed on complete preparation of a blood sample, a saliva sample, a sample in the wells of the plate prior to sampling by the urine sample, a cerebrospinal fluid sample, alymph sample, a sampling system, or sample may be prepared within the ana 60 BAL sample, a biopsy sample, a forensic sample, a bioterror lyZer system, or some combination of both. ism sample, and the like. In some embodiments the analyzer D. Sample Preparation System system provides a sample preparation system that provides Sample preparation includes the steps necessary to prepare Some or all of the sample preparation. In some embodiments, a raw sample for analysis. These steps can involve, by way of the initial sample is a blood sample that is further processed example, one or more steps of separation steps such as cen 65 by the analyzer System. In some embodiments, the sample is trifugation, filtration, distillation, chromatography; concen a serum or plasma sample that is further processed by the tration, cell lysis, alteration of pH, addition of buffer, addition analyzer system. The serum or plasma sample may be further US 8,450,069 B2 79 80 processed by, e.g., contacting with a label that binds to a TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP. particle or particles of interest; the sample may then be used CAPS, and CABS. The buffer can also be selected from the with or without removal of unbound label. group consisting of Gly-Gly, bicine, tricine, 2-morpholine In some embodiments, sample preparation is performed, ethanesulfonic acid (MES),4-morpholine propanesulfonic either outside the analysis system or in the sample preparation acid (MOPS) and 2-amino-2-methyl-1-propanol hydrochlo component of the analysis system, on one or more microtiter ride (AMP). A useful buffer is 2 mM Tris/borate at pH 8.1, but plates, such as a 96-well plate. Reservoirs of reagents, buffers, Tris/glycine and Tris/HCl are also acceptable. Other buffers and the like can be in intermittent fluid communication with are as described herein. the wells of the plate by means of tubing or other appropriate Buffers useful for electrophoresis are disclosed in a prior structures, as are well-known in the art. Samples may be 10 application and are incorporated by reference herein from prepared separately in 96 well plates or tubes. Sample isola U.S. patent application Ser. No. 11/048,660. tion, label binding and, if necessary, label separation steps E. Sample Recovery may be done on one plate. In some embodiments, prepared One highly useful feature of embodiments of the analyzers particles are then released from the plate and samples are and analysis systems of the invention is that the sample can be moved into tubes for sampling into the sample analysis sys 15 analyzed without consuming it. This can be especially impor tem. In some embodiments, all steps of the preparation of the tant when sample materials are limited. Recovering the sample are done on one plate and the analysis system acquires sample also allows one to do other analyses or reanalyze it. sample directly from the plate. Although this embodiment is The advantages of this feature for applications where sample described in terms of a 96-well plate, it will be appreciated size is limited and/or where the ability to reanalyze the sample that any vessel for containing one or more samples and Suit is desirable, e.g., forensic, drug screening, and clinical diag able for preparation of sample may be used. For example, nostic applications, will be apparent to those of skill in the art. standard microtiter plates of 384 or 1536 wells may be used. Thus, in Some embodiments, the analyzer system of the More generally, in Some embodiments, the sample prepara invention further provides a sample recovery system for tion system is capable of holding and preparing more than sample recovery after analysis. In these embodiments, the about 5, 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 200, 300, 500, 25 system includes mechanisms and methods by which the 1000, 5000, or 10,000 samples. In some embodiments, mul sample is drawn into the analyzer, analyzed and then tiple samples may be sampled for analysis in multiple ana returned, e.g., by the same path, to the sample holder, e.g., the lyZer systems. Thus, in Some embodiments, 2 samples, or sample tube. Because no sample is destroyed and because it more than about 2, 3, 4, 5, 7, 10, 1520, 50, or 100 samples are does not enter any of the valves or other tubing, it remains sampled from the sample preparation system and run in par 30 uncontaminated. In addition, because all the materials in the allel on multiple sample analyzer systems. sample path are highly inert, e.g., PEEK, fused silica, or Microfluidics systems may also be used for sample prepa sapphire, there is little contamination from the sample path. ration and as sample preparation systems that are part of The use of the stepper motor controlled pumps (particularly analyzer systems, especially for samples Suspected of con the analysis pump) allows precise control of the Volumes taining concentrations of particles high enough that detection 35 drawn up and pushed back out. This allows complete or requires Smaller samples. Principles and techniques of nearly complete recovery of the sample with little if any microfluidic manipulation are known in the art. See, e.g., U.S. dilution by the flush buffer. Thus, in some embodiments, Pat. Nos. 4,979,824; 5,770,029; 5,755,942; 5,746,901; 5,681, more than about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 751:5,658,413:5,653,939;5,653,859; 5,645,702; 5,605,662: 96%, 97%, 98%, 99%, 99.5%, or 99.9% of the sample is 5,571,410; 5,543,838; 5.480,614; 5,716,825; 5,603,351; 40 recovered after analysis. In some embodiments, the recovered 5,858, 195; 5,863,801; 5,955,028; 5,989,402: 6,041,515; sample is undiluted. In some embodiments, the recovered 6,071,478; 6,355,420; 6,495,104; 6,386.219; 6,606,609: sample is diluted less than about 1.5-fold, 1.4-fold, 1.3-fold, 6,802,342; 6,749,734; 6,623,613; 6,554,744; 6,361,671; 1.2-fold, 1.1-fold, 1.05-fold, 1.01-fold, 1.005-fold, or 1.001 6,143,152: 6,132,580: 5,274,240; 6,689,323; 6,783,992: fold. 6,537,437; 6,599,436; 6,811,668 and published PCT Patent 45 For sampling and/or sample recovery, any mechanism for Application No. WO9955461 (A1). Samples may be prepared transporting a liquid sample from a sample vessel to the in series or in parallel, for use in a single or multiple analyzer analyzer may be used. In some embodiments the inlet end of systems. the analysis capillary has attached a short length of tubing, In some embodiments, the sample comprises a buffer. The e.g., PEEK tubing that can be dipped into a sample container, buffer may be mixed with the sample outside the analyzer 50 e.g., a test tube or sample well, or can be held above a waste system, or it may be provided by the sample preparation container. When flushing, to clean the previous sample from mechanism. While any suitable buffer can be used, the pref the apparatus, this tube is positioned above the waste con erable buffer has low fluorescence background, is inert to the tainer to catch the flush waste. When drawing a sample in, the detectably labeled particle, can maintain the working pH and, tube is put into the sample well or test tube. Typically the in embodiments wherein the motive force is electrokinetic, 55 sample is drawn in quickly, and then pushed out slowly while has suitable ionic strength for electrophoresis. The buffer observing particles within the sample. Alternatively, in some concentration can be any suitable concentration, Such as in embodiments, the sample is drawn in slowly during at least the range from about 1 to about 200 mM. Any buffer system part of the draw-in cycle; the sample may be analyzed while may be used as long as it provides for Solubility, function, and being slowly drawn in. This can be followed by a quick return delectability of the molecules of interest. In some embodi 60 of the sample and a quick flush. In some embodiments, the ments, e.g., for application using pumping, the buffer is sample may be analyzed both on the inward (draw-in) and selected from the group consisting of phosphate, glycine, outward (pull out) cycle, which improves counting statistics, acetate, citrate, acidulate, carbonate/bicarbonate, imidazole, e.g., of Small and dilute samples, as well as confirming triethanolamine, glycine amide, borate, MES, Bis-Tris, ADA, results, and the like. If it is desired to save the sample, it can aces, PIPES, MOPSO, Bis-Tris Propane, BES, MOPS, TES, 65 be pushed back out into the same sample well it came from, or HEPES, DIPSO, MOBS, TAPSO, Trizma, HEPPSO, to another. If saving the sample is not desired, the tubing is POPSO, TEA, EPPS, Tricine, Gly-Gly, Bicine, HEPBS, positioned over the waste container. US 8,450,069 B2 81 82 VI. Methods Using Highly Sensitive Analysis of Molecules In some embodiments, the invention provides a method of The systems, system kits, and methods of the present establishing a marker for a biological state, by establishing a invention make possible measurement of molecules in range of concentrations for the marker in biological samples samples at concentrations far lower than previously mea obtained from a first population by measuring the concentra Sured. The high sensitivity of the instruments, kits, and meth tions of the marker the biological samples by detecting single ods of the invention allows the establishment of markers, e.g., molecules of the marker, e.g., by detecting a label that has biological markers, that have not previously been possible been attached to a single molecule of the marker. In some because of a lack of sensitivity of detection. The invention embodiments, the marker is a polypeptide or Small molecule. also includes the use of the compositions and methods The samples may be any sample type described herein, e.g., 10 blood, plasma, serum, or urine. described herein for the discovery of new markers. The method may utilize samples from a first population There are numerous markers currently available which, where the population is a population that does not exhibit the while potentially of use in determining a biological state, are biological state. In the case where the biological State is a not currently of practical use because their lower ranges are disease state, the first population may be a population that unknown. In some cases, abnormally high levels of the 15 does not exhibit the disease, e.g., a “normal population. In marker are detectable by current methodologies, but normal Some embodiments the method may further comprise estab ranges have not been established. In some cases, upper nor lishing a range of range of levels for the marker in biological mal ranges of the marker are detectable, but not lower normal samples obtained from a second population, where the mem ranges, or levels below normal. In some cases, for example, bers of the second population exhibit the biological state, by markers specific to tumors, or markers of infection, any level measuring the concentrations of the marker the biological of the marker indicates the potential presence of the biologi samples by detecting single molecules of the marker. In some cal state, and enhancing sensitivity of detection is an advan embodiments, e.g., cross-sectional studies, the first and sec tage for early diagnosis. In some cases, the rate of change, or ond populations are different. In some embodiments, at least lack of change, in the concentration of the marker over mul one member of the second population is a member of the first tiple timepoints provides the most useful information, but 25 population, or at least one member of said the population is a present methods of analysis do not permit determination of member of the second population. In some embodiments, levels of the marker at timepoint sampling in the early stages e.g., longitudinal studies, Substantially all the members of the of a condition, when it is typically at its most treatable. In second population are members of the first population who many cases, the marker may be detected at clinically useful have developed the biological state, e.g., a disease or patho levels only through the use of cumbersome methods that are 30 logical state. not practical or useful in a clinical setting, such as methods The detecting of single molecules of the marker is per that require complex sample treatment and time-consuming formed using a method as described herein, e.g., a method analysis. with a limit of detection for said marker of less than about In addition, there are potential markers of biological States 1000, 100, 50, 20, 10, 5, 1,0.5,0.1, 0.05, 0.01, 0.005, or 0.001 that existin sufficiently low concentrations that their presence 35 femtomolar of the marker in the samples, by detecting single remains extremely difficult or impossible to detect by current molecules of the marker. In some embodiments, the limit of methods. detection of said marker is thanabout 100, 50, 20, 10, 5, 1,0.5, The analytical methods and compositions of the present 0.1, 0.05, 0.01, 0.005, or 0.001 pg/ml of the marker in the invention provide levels of sensitivity and precision that allow samples, by detecting single molecules of the marker. the detection of markers for biological states at concentra 40 The biological State may be a phenotypic state; a condition tions at which the markers have been previously undetectable, affecting the organism; a state of development, age; health; thus allowing the “repurposing of Such markers from con pathology; disease; disease process; disease staging; infec firmatory markers, or markers useful only in limited research tion; toxicity; or response to chemical, environmental, or drug settings, to diagnostic, prognostic, treatment-directing, or factors (such as drug response phenotyping, drug toxicity other types of markers useful in clinical settings and/or in 45 phenotyping, or drug effectiveness phenotyping). large-scale clinical settings such as clinical trials. Such meth In Some embodiments, the biological state is a pathological ods allow, e.g., the determination of normal and abnormal state, including but not limited to inflammation, abnormal ranges for Such markers. cell growth, and abnormal metabolic state. In some embodi The markers thus repurposed can be used for, e.g., detec ments, the state is a disease state. Disease states include, but tion of normal state (normal ranges), detection of responder/ 50 are not limited to, cancer, cardiovascular disease, inflamma non-responder (e.g., to a treatment, Such as administration of tory disease, autoimmune disease, neurological disease, a drug); early disease or pathological occurrence detection infectious disease and pregnancy related disorders. In some (e.g., detection of cancer in its earliest stages, early detection embodiments the state is a disease stage state, e.g., a cancer of cardiac ischemia); disease staging (e.g., cancer); disease disease stage state. monitoring (e.g., diabetes monitoring, monitoring for recur 55 The methods may also be used for determination of a rence of cancer after treatment); study of disease mechanism; treatment response state. In some embodiments, the treatment and study of treatment toxicity, such as toxicity of drug treat is a drug treatment. The response may be a therapeutic effect ments (e.g., cardiotoxicity). or a side effect, e.g., an adverse effect. Markers fortherapeutic A. Methods effects will be based on the disease or condition treated by the The invention thus provides methods and compositions for 60 drug. Markers for adverse effects typically will be based on the sensitive detection of markers, and further methods of the drug class and specific structure and mechanism of action establishing values for normal and abnormal levels of the and metabolism. A common adverse effect is drug toxicity. markers. In further embodiments, the invention provides An example is cardiotoxicity, which can be monitored by the methods of diagnosis, prognosis, and/or treatment selection marker cardiac troponin. In some embodiments one or more based on values established for the markers. The invention 65 markers for the disease state and one or more markers for one also provides compositions for use in Such methods, e.g., or more adverse effects of a drug are monitored, typically in detection reagents for the ultrasensitive detection of markers. a population that is receiving the drug. Samples may be taken US 8,450,069 B2 83 84 at intervals and the respective values of the markers in the be measured separately in separate samples (e.g., aliquots of samples may be evaluated over time. a common sample) or simultaneously by multiplexing. The detecting of single molecules of the marker may com Examples of panels of markers and multiplexing are given in, prise labeling the marker with a label comprising a fluores e.g., U.S. patent application Ser. No. 11/048,660. cent moiety capable of emitting at least about 200 photons The establishment of Such markers and, e.g., reference when simulated by a laser emitting light at the excitation ranges for normal and/or abnormal states, allow for sensitive wavelength of the moiety, where the laser is focused on a spot and precise determination of the biological State of an organ not less than about 5 microns in diameter that contains the ism. Thus, in some embodiments, the invention provides a moiety, and wherein the total energy directed at the spot by method for detecting the presence or absence of a biological the laser is no more than about 3 microJoules. In some 10 state of an organism, comprisingi) measuring the concentra embodiments, the fluorescent moiety comprises a molecule tion of a marker in a biological sample from the organism, that comprises at least one Substituted indolium ring system wherein said marker is a marker established through estab in which the substituent on the 3-carbon of the indolium ring lishing a range of concentrations for said marker in biological contains a chemically reactive group or a conjugated Sub samples obtained from a first population by measuring the stance. In some embodiments, the fluorescent moiety may 15 concentrations of the marker the biological samples by comprise a dye selected from the group consisting of Alexa detecting single molecules of the marker; and ii) determining Fluor 488, Alexa Fluor 532, AlexaFluor 647, Alexa Fluor 680 the presence of absence of said biological state based on said or Alexa Fluor 700. In some embodiments, the moiety com concentration of said marker in said organism. prises Alexa Fluor 647. In some embodiments, the label fur In some embodiments, the invention provides a method for ther comprises a binding partner for the marker, e.g., an detecting the presence or absence of a biological state in an antibody specific for said marker, such as a polyclonal anti organism, comprising i) measuring the concentrations of a body or a monoclonal antibody. Binding partners for a variety marker in a plurality of biological samples from said organ of markers are described herein. ism, wherein said marker is a marker established through The method may further include establishing a threshold establishing a range of concentrations for said marker in level for the marker based on the first range, or the first and 25 biological samples obtained from a first population by mea second ranges, where the presence of marker in a biological Suring the concentrations of the marker the biological sample from an individual at a level above or below the samples by detecting single molecules of the marker, and ii) threshold level indicates an increased probability of the pres determining the presence of absence of said biological state ence of the biological state in said individual. An example of based on said concentrations of said marker in said plurality a threshold determined for a normal population is the Sug 30 of samples. In some embodiments, the samples are of differ gested threshold for cardiac troponin of greater than the 99th ent types, e.g., are samples from different tissue types. In this percentile value in a normal population. See Example 3. case, the determining is based on a comparison of the con Other threshold levels may be determined empirically, i.e., centrations of said marker in said different types of samples. based on data from the first and second populations regarding More commonly, the samples are of the same type, and the marker levels and the presence, absence, severity, rate of 35 samples are taken at intervals. The samples may be any progression, rate of regression, and the like, of the biological sample type described herein, e.g., blood, plasma, or serum; state being monitored. It will be appreciated that threshold or urine. Intervals between samples may be minutes, hours, levels may be established at either end of a range, e.g., a days, weeks, months, or years. In an acute clinical setting, the minimum below which the concentration of the marker in a intervals may be minutes or hours. In settings involving the sample indicates an increased probability of a biological 40 monitoring of an individual, the intervals may be days, weeks, state, and/or a maximum above which the concentration of months, or years. the marker in a sample indicates an increased probability of a In many cases, the biological state whose presence or biological state. In some embodiments, a risk stratification absence is to be detected is a disease phenotype. Thus, in one may be produced in which two or more ranges of marker embodiment, a phenotypic State of interest is a clinically concentrations correspond to two or more levels of risk. Other 45 diagnosed disease state. Such disease states include, for methods of analyzing data from two populations and for example, cancer, cardiovascular disease, inflammatory dis markers and producing clinically-relevant values for use by, ease, autoimmune disease, neurological disease, respiratory e.g., physicians and other health care professionals, are well disease, infectious disease and pregnancy related disorders. known in the art. Cancer phenotypes are included in some aspects of the For some biological markers, the presence of any marker at 50 invention. Examples of cancer herein include, but are not all is an indication of a disease or pathological state, and the limited to: breast cancer, skin cancer, bone cancer, prostate threshold is essentially Zero. An example is the use of prostate cancer, liver cancer, lung cancer, brain cancer, cancer of the specific antigen (PSA) to monitor cancer recurrence after larynx, gallbladder, pancreas, rectum, parathyroid, thyroid, removal of the prostate gland. As PSA is produced only by the adrenal, neural tissue, head and neck, colon, stomach, bron prostate gland, and as the prostate gland and all tumors are 55 chi, kidneys, basal cell carcinoma, squamous cell carcinoma presumed to be removed, PSA after removal is zero. Appear of both ulcerating and papillary type, metastatic skin carci ance of PSA at any level signals a possible recurrence of the noma, osteosarcoma, Ewing's sarcoma, Veticulum cell Sar cancer, e.g., at a metastatic site. Thus, the more sensitive the coma, myeloma, giant cell tumor, Small-cell lung tumor, non method of detection, the earlier an intervention may be made Small cell lung carcinoma gallstones, islet cell tumor, primary should such recurrence occur. 60 brain tumor, acute and chronic lymphocytic and granulocytic Other evaluations of marker concentration may also be tumors, hairy-cell tumor, adenoma, hyperplasia, medullary made. Such as in a series of samples, where change in value, carcinoma, pheochromocytoma, mucosal neuronms, intesti rate of change, spikes, decrease, and the like may all provide nal ganglloneuromas, hyperplastic corneal nerve tumor, useful information for determination of a biological state. In marfanoid habitus tumor, Wilm's tumor, seminoma, ovarian addition, panels of markers may be used if it is found that 65 tumor, leiomyomater tumor, cervical dysplasia and in situ more than one marker provides information regarding a bio carcinoma, neuroblastoma, retinoblastoma, Soft tissue sar logical state. If panels of markers are used, the markers may coma, malignant carcinoid, topical skin lesion, mycosis fun US 8,450,069 B2 85 86 goide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic Furthermore, after intervention (e.g., Surgery, chemotherapy, and other sarcoma, malignant hypercalcemia, renal cell or radiation to shrink or remove the tumor or tumors), it is tumor, polycythermia Vera, adenocarcinoma, glioblastoma often not possible to measure the tumor marker with sufficient multiforma, leukemias, lymphomas, malignant melanomas, sensitivity to determine if there has been a recurrence of the epidermoid carcinomas, and other carcinomas and sarcomas. cancer until residual disease has progressed to the point Cardiovascular disease may be included in other applica where further intervention is unlikely to be successful. Using tions of the invention. Examples of cardiovascular disease the analyzers, systems, and methods of the present invention, include, but are not limited to, congestive heart failure, high it is possible to both detect onset of tumor growth and return blood pressure, arrhythmias, atherosclerosis, cholesterol, of tumor growth at a point where intervention is more likely to Wolff-Parkinson-White Syndrome, long QT syndrome, 10 angina pectoris, tachycardia, bradycardia, atrial fibrillation, be successful, e.g., due to lower probability of metastasis. ventricular fibrillation, congestive heart failure, myocardial Markers for cancer that can be detected at levels not previ ischemia, myocardial infarction, cardiac tamponade, myo ously shown include markers disclosed herein. Examples of carditis, pericarditis, arrhythmogenic right ventricular dys assays for the detection of markers that can be repurposed to plasia, hypertrophic cardiomyopathy, Williams syndrome, 15 diagnostic markers include TGFB, Akt1, Fas ligand and IL-6, heart valve diseases, endocarditis, bacterial, pulmonary atre as described herein. sia, aortic valve Stenosis, Raynaud's disease, cholesterol B. Exemplary Markers embolism, Wallenberg syndrome, Hippel-Lindau disease, The instruments, labels, and methods of the invention have and telangiectasis. been used to establish ranges for markers in, e.g., serum and Inflammatory disease and autoimmune disease may be urine, at levels 10- to 100-fold lower than previous levels, or included in other embodiments of the invention. Examples of lower. The markers are indicative of a wide variety of bio inflammatory disease and autoimmune disease include, but logical states, e.g., cardiac disease and cardiotoxicity (tropo are not limited to, rheumatoid arthritis, non-specific arthritis, nin), infection (TREM-1), inflammation and other conditions inflammatory disease of the larynx, inflammatory bowel dis (LTE4, IL-6 and IL-8), asthma (LTE4), cancer (Akt1, TGF order, psoriasis, hypothyroidism (e.g., Hashimoto thyroid 25 beta, Fas ligand), and allograft rejection and degenerative ism), colitis, Type 1 diabetes, pelvic inflammatory disease, disease (Fasligand). inflammatory disease of the central nervous system, temporal Markers include protein and non-protein markers. The arteritis, polymyalgia rheumatica, ankylosing spondylitis, markers are described briefly here and procedures and results polyarteritis nodosa, Reiter's syndrome, Scleroderma, sys given in the Examples. temic lupus and erythematosus. 30 1. Cardiac Damage The methods and compositions of the invention can also Cardiac troponin is an example of a marker that is previ provide laboratory information about markers of infectious ously detectable only in abnormally high amounts. Cardiac disease including markers of Adenovirus, Bordella pertussis, troponin is a marker of cardiac damage, useful in diagnosis, Chlamydia pneumoiea, Chlamydia trachomais, Cholera prognosis, and determination of method of treatment in a Toxin, Cholera Toxin B, Campylobacter jejuni, Cytomega 35 number of diseases and conditions, e.g., acute myocardial lovirus, Diptheria Toxin, Epstein-Barr NA, Epstein-Barr EA, infarct (AMI). In addition, cardiac troponin is a useful marker Epstein-Barr VCA, Helicobacter Pylori, Hepatitis B virus of cardiotoxicity due to treatment, e.g., drug treatment. (HBV) The troponin complex in muscle consists of troponin I, C Core, Hepatitis B virus (HBV) Envelope, Hepatitis B virus and T. Troponin C exists as two isoforms, one from cardiac (HBV) Survace (Ay), Hepatitis C virus (HCV) Core, Hepati 40 and slow-twitch muscle and one from fast-twitch muscle; tis C virus (HCV) NS3, Hepatitis C virus (HCV) NS4, Hepa because it is found in virtually all striated muscle, its use as a titis C virus (HCV) NS5, Hepititis A, Hepititis D, Hepatitis E specific marker is limited. In contrast, troponin I and T are virus (HEV) orf23 KD, Hepatitis E virus (HEV) orf26 KD, expressed as different isoforms in slow-twitch, fast-twitch Hepatitis E virus (HEV) orf3 3 KD, Human immunodefi and cardiac muscle. The unique cardiac isoforms of troponin ciency virus (HIV)-1 p24. Human immunodeficiency virus 45 I and Tallow them to be distinguished immunologically from (HIV)-1 gp41, Human immunodeficiency virus (HIV)-1 the other troponins of skeletal muscle. Therefore, the release gp120, Human papilloma virus (HPV), Herpes simplex virus into the blood of cardiac troponin I and T is indicative of HSV-1/2. Herpes simplex virus HSV-1 g). Herpes simplex damage to cardiac muscle, and provides the basis for their use virus HSV-2 gG, Human T-cell leukemia virus (HTLV)-1/2, as diagnostic or prognostic markers, or to aid in determination Influenza A, Influenza A HN, Influenza B, Leishmanina 50 of treatment. donovani, Lyme disease, Mumps, M. pneumoniae, M. teber Currently used markers for cardiac damage suffer disad culosis, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, Vantages that limit their clinical usefulness. Cardiac enzyme Polio Virus, Respiratory syncytial virus (RSV), Rubella, assays have formed the basis for determining whether or not Rubeola, Streptolysin O, Tetanus Toxin, T. pallidum 15 kd, T. there is damage to the cardiac muscle. Unfortunately, the pallidum p47. T. Cruzi, Toxoplasma, and Varicella Zoater. 55 standard creatine kinase-MB (CK-MB) assay is not reliable Detection and monitoring of cancers often depends on the in excluding infarction until 10 to 12 hours after the onset of use of crude measurements of tumor growth, such as visual chest pain. Earlier diagnosis would have very specific advan ization of the tumor itself, that are either inaccurate or that tages with regard to fibrinolytic therapy and triage. must reach high levels before they become detectable, e.g., in Because the level of troponin found in the circulation of a practical clinical setting by present methods. At the point of 60 healthy individuals is very low, and cardiac specific troponins detection, the tumor has often grown to Sufficient size that do notarise from extra-cardiac sources, the troponins are very intervention is unlikely to occur before metastasis. For sensitive and specific markers of cardiac injury. In addition to example, detection of lung cancer by X-ray requires a tumor cardiac infarct, a number of other conditions can cause dam of >1 cm in diameter, and by CT scan of >2-3 mm. Alterna age to the heart muscle, and early detection of Such damage tively, a biomarker of tumor growth may be used, but, again, 65 would prove useful to clinicians. However, present methods often the tumor is well-advanced by the time the biomarker is of detection and quantitation of cardiac troponin do not pos detectable at levels accessible to current clinical technology. sess sufficient sensitivity to detect the release of cardiac US 8,450,069 B2 87 88 troponin into the blood until levels have reached abnormally an endpoint; (4) understand how cTnI levels change from high concentrations, e.g., 0.1 ng/ml or greater. normal levels in a variety of cardiac-related diseases; and/or The methods and compositions of the invention thus (5) understand the utility of cTnI as a biomarker to serve as a include methods and compositions for the highly sensitive Surrogate endpoint for clinical events. detection and quantitation of cardiac troponin, and composi In some embodiments, the method is capable of detecting tions and methods for diagnosis, prognosis, and/or determi cTnI at a limit of detection of less than about 100, 80, 60, 50, nation of treatment based on Such highly sensitive detection 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 or and quantitation. A standard curve for cardiac troponin I was 0.0001 pg/ml, e.g., less than about 100 pg/ml. In some established with a limit of detection less than about 1 pg/ml embodiments, the method is capable of detecting the cTnI at (Example 1). Levels of cardiac troponin I were established in 10 a limit of detection of less than about 100 pg/ml. In some normal individuals and a threshold value at the 99" percentile embodiments, the method is capable of detecting the cTnI a of normal established (Example 3). Serial samples from indi limit of detection of less than about 80 pg/ml. In some viduals who suffered acute myocardial infarct were analyzed, embodiments, the method is capable of detecting the cTnI a and time courses for cardiac troponin I concentrations, limit of detection of less than about 60 pg/ml. In some including deviations from baseline, were determined (Ex 15 embodiments, the method is capable of detecting the cTnI a ample 4). Thus, cardiac troponin I serves as an example of a limit of detection of less than about 50 pg/ml. In some marker that can be detected by the systems and methods of the embodiments, the method is capable of detecting the cTnI a invention at levels to provide diagnostic and prognostic infor limit of detection of less than about 30 pg/ml. In some mation of use in clinical and research settings. See also U.S. embodiments, the method is capable of detecting the cTnI a patent application Ser. No. 1 1/784.213, filed Mar. 5, 2008 and limit of detection of less than about 25 pg/ml. In some entitled “Highly Sensitive System and Methods for Analysis embodiments, the method is capable of detecting the cTnI a of Troponin, which is incorporated by reference herein in its limit of detection of less than about 10 pg/ml. In some entirety. embodiments, the method is capable of detecting the cTnI a Cardiac troponin-I (cTnI) is specific to cardiomyocytes limit of detection of less than about 5 pg/ml. In some embodi and is released into blood following heart damage. Extensive 25 ments, the method is capable of detecting the cTnI a limit of studies have shown that cTnI is slowly released from dam detection of less than about 1 pg/ml. In some embodiments, aged cardiomyocytes and often requires 4-8 hours post the method is capable of detecting the cTnI a limit of detec trauma to be detectable. Measurement of cTnI concentrations tion of less than about 0.5 pg/ml. In some embodiments, the in plasma/serum are the standard of care for diagnosing non method is capable of detecting the cTnI at a limit of detection STEMI acute myocardial infarction (AMI). In addition this 30 of less than about 0.1 pg/ml. In some embodiments, the biomarker has been widely accepted in pre-clinical and clini method is capable of detecting the cTnI at a limit of detection cal drug development settings as an indicator of myocardial of less than about 0.05 pg/ml. In some embodiments, the damage and hence heart damage. TnI is accepted as a biom method is capable of detecting the cTnI at a limit of detection arker to assess potential cardiotoxicity of experimental thera of less than about 0.01 pg/ml. In some embodiments, the pies. It is extensively studied in pre-clinical setting and 35 method is capable of detecting the cTnI at a limit of detection included in clinical drug development programs when pre of less than about 0.005 pg/ml. In some embodiments, the clinical data Suggests a potential of cardiac-related adverse method is capable of detecting the cTnI at a limit of detection eVentS. of less than about 0.001 pg/ml. In some embodiments, the Even though cTnI is used as the standard of care for diag method is capable of detecting the cTnI at a limit of detection nosing AMI, as well as in pre-clinical and clinical develop 40 of less than about 0.0005 pg/ml. In some embodiments, the ment, until recently its concentration in the plasma of appar method is capable of detecting the cTnI at a limit of detection ently healthy humans and preclinical animal models had not of less than about 0.0001 pg/ml. been reported. Thus it was impossible to benchmark a “nor 2. Infection mal level within a given animal or human and measure Small Recent reports have established TREM-1 as a biomarker of increases (velocity) of cTnI which might be associated with 45 bacterial or fungal infections. See, e.g., Bouchon et al. (2000) Subtle cardiac damage. Furthermore, many assays do not J. Immunol. 164:4991-95; Colonna (2003) Nat. Rev. Immu equally quantify cTnI across different species and require nol 3:445-53; Gibot et al. (2004) N. Engl. J. Med. 350:451 large plasma sample Volumes, limiting their use in pre-clini 58; Gibot et al. (2004) Ann. Intern. Med. 141:9-15. Assays of cal settings, especially in rodent model systems. Using the the invention suggest that TREM-1 may routinely be mea methods of the present invention, normal levels of endog 50 sured at a concentration of 100 fM or less. See Example 9. enous cTnI and Small changes in plasma cTnI can be quanti In some embodiments, the method is capable of detecting fied in humans, rats, dogs and monkeys providing previously TREM-1 at a limit of detection of less than about 100, 80, 60, intractable answers around cardiomyocyte pathophysiology. 50, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 See Examples 1-4. or 0.0001 pg/ml, e.g., less than about 100 pg/ml. In some In Some embodiments, the cTnI assay of the present inven 55 embodiments, the method is capable of detecting the tion is used to: (1) define the concentration of plasma and TREM-1 at a limit of detection of less than about 100 pg/ml. serum cTnI in healthy humans, rats, dogs and monkeys; (2) In some embodiments, the method is capable of detecting the identify AMIs earlier; (3) measure heart damage earlier under TREM-1 a limit of detection of less than about 80 pg/ml. In physical stress or known cardiotoxins; and/or (4) study cTnI Some embodiments, the method is capable of detecting the concentrations in a single rat using only 10 LIL plasma. In 60 TREM-1a limit of detection of less than about 60 pg/ml. In other embodiments, the cTnI assay of the present invention is Some embodiments, the method is capable of detecting the used to: (1) measure the potential cardiac safety and dosing of TREM-1a limit of detection of less than about 50 pg/ml. In therapeutics in both pre-clinical and clinical settings; (2) per Some embodiments, the method is capable of detecting the form studies using individual small animals or precious TREM-1a limit of detection of less than about 30 pg/ml. In samples, when sample Volume is an issue; (3) design more 65 Some embodiments, the method is capable of detecting the robust clinical and preclinical studies when velocity of cTnI TREM-1a limit of detection of less than about 25 pg/ml. In concentration change from a baseline normal level is used as Some embodiments, the method is capable of detecting the US 8,450,069 B2 89 90 TREM-1a limit of detection of less than about 10 pg/ml. In are able to detect IL-6 only in the upper part of normal ranges. Some embodiments, the method is capable of detecting the In contrast, the analyzers and analyzer systems of the inven TREM-1a limit of detection of less than about 5 pg/ml. In tion allow the detection of IL-6 down to a concentration Some embodiments, the method is capable of detecting the below about 0.01 pg/ml, or less than one-tenth to one-hun TREM-1a limit of detection of less than about 1 pg/ml. In 5 dredth of normal range values. Thus, the analyzers and ana Some embodiments, the method is capable of detecting the lyzer systems of the invention allow a far broader and more TREM-1a limit of detection of less than about 0.5 pg/ml. In nuanced database to be produced for a biomarker, e.g., for Some embodiments, the method is capable of detecting the IL-6, and also allow screening for that biomarker both within TREM-1 at a limit of detection of less than about 0.1 pg/ml. and outside of the normal range, allowing earlier detection. In some embodiments, the method is capable of detecting the 10 Thus, the analyzers and analyzer Systems of the invention TREM-1 at a limit of detection of less than about 0.05 pg/ml. allow a far broader and more nuanced database to be pro In some embodiments, the method is capable of detecting the duced for a biomarker, e.g., for IL-6, and also allow screening TREM-1 at a limit of detection of less than about 0.01 pg/ml. for that biomarker both within and outside of the normal In some embodiments, the method is capable of detecting the range, allowing earlier detection of conditions in which the TREM-1 at a limit of detection of less than about 0.005 pg/ml. 15 biomarker, e.g., IL-6, is implicated. In some embodiments, the method is capable of detecting the a. Interleukin 1 TREM-1 at a limit of detection of less than about 0.001 pg/ml. IL-1C. and -B are pro-inflammatory cytokines involved in In some embodiments, the method is capable of detecting the immune defense against infection, and are part of the IL-1 TREM-1 at a limit of detection of less than about 0.0005 superfamily of cytokines. Both IL-1C. and IL-1B are produced pg/ml. In some embodiments, the method is capable of 20 by macrophages, monocytes and dendritic cells. IL-1 is detecting the TREM-1 at a limit of detection of less than about involved in various immune responses with a primary role in 0.0001 pg/ml. inflammation, making IL-1 a target for Rheumatoid Arthritis 3. Cytokines (RA). IL-1C. and IL-1B are produced as precursor peptides, The normal level of many cytokines, chemokines and which are proteolytically processed and released in response growth factors is not known primarily because of the inability 25 to cell injury, and thus induce apoptosis. IL-1B production in of existing technology to detect levels that are below those peripheral tissue has also been associated with hyperalgesia found in Samples from diseased patients. For example, the (increased sensitivity to pain) associated with fever. basal level of other cytokines such as IL-10, TNF-alpha, IL-4, Amgen currently markets Kineret (anakinra), a synthetic IL-1beta, IL-2, IL-12 and IFN-gamma cannot be detected by form of the human interleukin-1 receptor antagonist (IL routine assays that are performed in a clinical setting, whereas 30 1Ra). IL-1Ra blocks the biologic activity of IL-1 alpha and the analyzer systems of the invention can readily determine beta by competitively inhibiting IL-1 from binding to the the level of these and other cytokines. Knowing the level of interleukin-1 type I receptor (IL1-RI), which is expressed in a cytokines and growth factors aids clinicians with the diagno wide variety of tissues and organs. IL-1Ra inhibits the bio sis, prognosis and treatment of a variety of diseases including logical activities of IL-1 both in vitro and in vivo, and has cancer, and respiratory, infectious, and cardiovascular dis- 35 been shown to be effective in animal models of septic shock, eases. Early cytokine detection to monitor normal and disease rheumatoid arthritis, graft versus host disease, stroke, and states in clinical specimens can be achieved using the ana cardiac ischemia. Also in the Amgen pipeline is AMG 108, a lyZer systems of the invention to analyze samples Such as fully human monoclonal antibody that targets inhibition of plasma, serum, and urine as well as other fluid samples to the action of interleukin-1 (IL-1). A Phase 2 clinical study is provide for better translational medicine. For example, deter- 40 under way to assess long-term safety of treating rheumatoid mining levels of cytokines for which a normal range of con arthritis with AMG 108. centration is not known, would aid clinicians with diagnosis i. Interleukin 1, Alpha (IL-1C...) and treatment of the following conditions and diseases. Bone The broad involvement of inflammation in human disease Morphogenetic Proteins would be useful to monitor the treat ensures that this protein will remain an attractive diagnostic ment for fractures, spinal fusions, orthopedic Surgery, and 45 target. Elevated levels of IL-1C. will continue to be a diagnos oral surgery; Interleukin-10 (IL-10) would be useful for tic target for inflammatory diseases like rheumatoid arthritis. detecting and monitoring for the presence of cancers includ Thus, there is a need to develop assays with sensitivity to ing non-Hodgkin’s lymphoma, multiple myeloma, mela quantify low normal levels of IL-1C. in order to differentiate noma, and ovarian cancer, as well as for detecting and moni between low and high levels of IL-1C. which indicate disease. toring the effect of anti-inflammatory therapy, organ 50 Also, there is a need to evaluate the potential of IL-1C. as a transplantation, immunodeficiencies, and parasitic infec therapeutic drug target to decrease elevated levels of IL-1C. as tions; Interleukin-11 (IL-11) is useful for the detection and a treatment for IL-1C. associated disease. This will present a monitoring for the presence of cancers such as breast cancer, need to detect velocity of decreasing IL-1C. levels to evaluate Interleukin-12 (IL-12) for cancer and HIV infections; TNFC. effectiveness and dosing of therapies. This may prevent an inflammatory cytokine, alone or in combination with IL-6, 55 adverse events like Neutropenia that develop after co-admin can be used as a good predictor of sepsis, acute pancreatitis, istration of drugs targeted to inflammatory cytokine path tuberculosis, and autoimmune disease Such as rheumatoid ways, like Kineret (IL-1Ra antagonist) and enteracept (TNF arthritis and lupus. alpha antagonist). To meet these goals, it is essential to have Alternatively, databases may already exist for normal and an assay that can detect IL-1C. to below normal levels in abnormal values but present methods may not be practical for 60 human plasma. screening individuals on a routine basis to determine with The present invention provides an IL-1C. assay sensitive sufficient sensitivity whether the value of the individual for enough to quantify IL-1C. concentration in plasma from the marker is within the normal range. For example, most healthy, normal human Subjects with previously unattainable present methods for the determination of IL-6 concentration levels of accuracy and precision. See Example 23. It enables in a sample are capable of detecting IL-6 only down to a 65 differentiation between IL-1C. concentrations in healthy and concentration of about 5 pg/ml; the normal range of IL-6 diseased States, allowing efficient pre-clinical and clinical values is about 1 to about 10 pg/ml; hence, present methods study design. The IL-1C. assay increases the utility of IL-1C. US 8,450,069 B2 91 92 by allowing quantification at very low levels and differentia enteracept (TNF-alpha antagonist). To meet these goals, it is tion between Small changes in concentration that can provide essential to have an assay that can detect IL-1B to below insights into drug efficacy or disease progression. The IL-1C. normal levels in human plasma. assay enables the accurate quantification of IL-1C. in human The present invention provides an IL-1B assay that plasma with a broad dynamic range. In various embodiments, increases the utility of IL-1B by allowing quantification at the assay allows investigators to: (1) measure the efficacy and very low levels and differentiation between small changes in dosing of therapeutics designed to interfere with the IL-1 concentration that can provide insights into drug efficacy or mediated inflammatory response, such as Kineret; (2) design disease progression. See Example 24. The IL-1B assay is more robust clinical and preclinical studies when IL-1C. con sensitive enough to quantify IL-1B concentration in plasma centration can be used as a therapeutic endpoint, as in the 10 from healthy, normal human Subjects with previously unat clinical trial of AMG 108; and/or (3) understand how IL-1C. tainable levels of accuracy and precision. The IL-1B assay levels change in patients as they transition from a healthy to a enables the accurate quantification of IL-1B in human plasma diseased state. with a broad dynamic range. In various embodiments, this In some embodiments, the method is capable of detecting assay will allow investigators to: (1) measure the efficacy and IL-1C. at a limit of detection of less than about 100, 80, 60, 50, 15 dosing of therapeutics designed interfere with the IL-1 medi 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 or ated inflammatory response. Such as Kineret; (2) design more 0.0001 pg/ml, e.g., less than about 100 pg/ml. In some robust clinical and preclinical studies when IL-1B concentra embodiments, the method is capable of detecting the IL-1C. at tion can be used as a therapeutic endpoint, as in the clinical a limit of detection of less than about 100 pg/ml. In some trial of AMG 108; and (3) understand how IL-1B levels embodiments, the method is capable of detecting the IL-1C. a change in patients as they transition from a healthy to dis limit of detection of less than about 80 pg/ml. In some eased State. embodiments, the method is capable of detecting the IL-1C. a In some embodiments, the method is capable of detecting limit of detection of less than about 60 pg/ml. In some the at a limit of detection of less than about 100, 80, 60, 50, 30, embodiments, the method is capable of detecting the IL-1C. a 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 or limit of detection of less than about 50 pg/ml. In some 25 0.0001 pg/ml, e.g., less than about 100 pg/ml. In some embodiments, the method is capable of detecting the IL-1C. a embodiments, the method is capable of detecting the IL-1B at limit of detection of less than about 30 pg/ml. In some a limit of detection of less than about 200 pg/ml. In some embodiments, the method is capable of detecting the IL-1C. a embodiments, the method is capable of detecting the IL-1B at limit of detection of less than about 25 pg/ml. In some a limit of detection of less than about 150 pg/ml. In some embodiments, the method is capable of detecting the IL-1C. a 30 embodiments, the method is capable of detecting the IL-1B at limit of detection of less than about 10 pg/ml. In some a limit of detection of less than about 100 pg/ml. In some embodiments, the method is capable of detecting the IL-1C. a embodiments, the method is capable of detecting the IL-1B a limit of detection of less than about 5 pg/ml. In some embodi limit of detection of less than about 80 pg/ml. In some ments, the method is capable of detecting the IL-1C. a limit of embodiments, the method is capable of detecting the IL-1B a detection of less than about 1 pg/ml. In some embodiments, 35 limit of detection of less than about 60 pg/ml. In some the method is capable of detecting the IL-1C. a limit of detec embodiments, the method is capable of detecting the IL-1B a tion of less than about 0.5 pg/ml. In some embodiments, the limit of detection of less than about 50 pg/ml. In some method is capable of detecting the IL-1C. at a limit of detec embodiments, the method is capable of detecting the IL-1B a tion of less than about 0.1 pg/ml. In some embodiments, the limit of detection of less than about 30 pg/ml. In some method is capable of detecting the IL-1C. at a limit of detec 40 embodiments, the method is capable of detecting the IL-1B a tion of less than about 0.05 pg/ml. In some embodiments, the limit of detection of less than about 25 pg/ml. In some method is capable of detecting the IL-1C. at a limit of detec embodiments, the method is capable of detecting the IL-1B a tion of less than about 0.01 pg/ml. In some embodiments, the limit of detection of less than about 10 pg/ml. In some method is capable of detecting the IL-1C. at a limit of detec embodiments, the method is capable of detecting the IL-1B a tion of less than about 0.005 pg/ml. In some embodiments, the 45 limit of detection of less than about 5 pg/ml. In some embodi method is capable of detecting the IL-1C. at a limit of detec ments, the method is capable of detecting the IL-1B a limit of tion of less than about 0.001 pg/ml. In some embodiments, the detection of less than about 1 pg/ml. In some embodiments, method is capable of detecting the IL-1C. at a limit of detec the method is capable of detecting the IL-1B a limit of detec tion of less than about 0.0005 pg/ml. In some embodiments, tion of less than about 0.5 pg/ml. In some embodiments, the the method is capable of detecting the IL-1C. at a limit of 50 method is capable of detecting the IL-1B at a limit of detection detection of less than about 0.0001 pg/ml. of less than about 0.1 pg/ml. In some embodiments, the ii. Interleukin 1, Beta (IL-1B) method is capable of detecting the IL-1B at a limit of detection Like IL-1C, the broad involvement of inflammation in of less than about 0.05 pg/ml. In some embodiments, the human disease ensures that IL-1B will remain an attractive method is capable of detecting the IL-1B at a limit of detection diagnostic target. Elevated levels of IL-1B will continue to be 55 of less than about 0.01 pg/ml. In some embodiments, the a diagnostic target for inflammatory diseases like rheumatoid method is capable of detecting the IL-1B at a limit of detection arthritis. Thus, there is a need to develop assays with sensi of less than about 0.005 pg/ml. In some embodiments, the tivity to quantify low normal levels of IL-1B in order to method is capable of detecting the IL-1B at a limit of detection differentiate between low and high levels of IL-1B which of less than about 0.001 pg/ml. In some embodiments, the indicate disease. Also, there is a need to evaluate the potential 60 method is capable of detecting the IL-1B at a limit of detection of IL-1B as a therapeutic drug target to decrease elevated of less than about 0.0005 pg/ml. In some embodiments, the levels of IL-1B as a treatment for IL-1B associated disease. method is capable of detecting the IL-1B at a limit of detection This will present a need to detect velocity of decreasing IL-1B of less than about 0.0001 pg/ml. levels to evaluate effectiveness and dosing of therapies. This b. Interleukin 4 (IL-4) may prevent adverse events like Neutropenia that develop 65 Interleukin-4 (IL-4) is a cytokine that is a key regulator in after co-administration of drugs targeted to inflammatory humoral and adaptive immunity IL-4 induces differentiation cytokine pathways, like Kineret (IL-1Ra antagonist) and of naive helper T cells (Tho cells) to Th2 cells. It has many US 8,450,069 B2 93 94 biological roles, including the stimulation of activated B-cell of less than about 0.5 pg/ml. In some embodiments, the and T-cell proliferation, and the differentiation of CD4+ method is capable of detecting the IL-4 at a limit of detection T-cells into Th2 cells IL-4 plays an important role in the of less than about 0.1 pg/ml. In some embodiments, the development of allergic inflammatory responses. IL-4 con method is capable of detecting the IL-4 at a limit of detection trols the production of IgE, expands IL-4 producing T cell 5 of less than about 0.05 pg/ml. In some embodiments, the subsets and stabilizes effector cell functions. method is capable of detecting the IL-4 at a limit of detection IL-4 has therapeutic potential due to its role in the devel of less than about 0.01 pg/ml. In some embodiments, the opment of allergic inflammatory responses. IL-4 also has method is capable of detecting the IL-4 at a limit of detection shown to have promise in drug targeting for cancer. For of less than about 0.005 pg/ml. In some embodiments, the example, PRX321 (Protox) is a targeted therapeutic toxin in 10 which IL-4 is linked to a Pseudomonas exo-toxin, a potent method is capable of detecting the IL-4 at a limit of detection Substance that can destroy cancer cells. Besides brain, kidney of less than about 0.001 pg/ml. In some embodiments, the and lung cancer, PRX321 has shown promising pre-clinical method is capable of detecting the IL-4 at a limit of detection results in a number of cancers over-expressing IL-4 receptors of less than about 0.0005 pg/ml. In some embodiments, the including pancreatic, ovarian, breast, head and neck, mela 15 method is capable of detecting the IL4 at a limit of detection noma, prostate and blood cancers such as chronic lympho of less than about 0.0001 pg/ml. cytic leukemia (CLL) and Hodgkin’s lymphoma. c. Interleukin 6 (IL-6) The concentration of plasma IL-4 in healthy human Sub Interleukin-6 (IL-6) is a pro-inflammatory cytokine jects has yet to be defined. Thus it is difficult to understand the secreted by T cells and macrophages to stimulate immune role that differences in IL-4 concentrations play between response to trauma, especially burns or other tissue damage disease and healthy States. In addition, measuring the efficacy leading to inflammation. IL-6 is also secreted by macroph of experimental therapeutics that target lowering IL-4 by ages in response to specific microbial molecules, referred to measuring the Velocity of IL-4 decreases is hindered by lack as pathogen associated molecular patterns (PAMPs), which of assay sensitivity. Furthermore the reading range of the trigger the innate immune response and initiate inflammatory most sensitive ELISAs is limited to less than two logs, which 25 cytokine production. IL-6 is one of the most important media forces sample retesting and wastage. Thus there is need for a tors of fever and of the acute phase response. IL-6 is also highly sensitive assay that can detect the velocity of subtle called a “myokine, a cytokine produced from muscle, and is changes in concentration, and that can measure baseline con elevated in response to muscle contraction. Additionally, centration of IL-4 in normal Subjects. osteoblasts secrete IL-6 to stimulate osteoclast formation. The IL-4 Assay provided by the present invention is sen 30 IL-6-related disorders include but are not limited to sepsis, sitive enough to quantify IL-4 concentrations in plasma from peripheral arterial disease, and chronic obstructive pulmo healthy, normal human subjects with a level of accuracy and nary disease. Interleukin-6 mediated inflammation is also the precision currently unobtainable using other high sensitivity common causative factor and therapeutic target for athero assays. See Example 25. This assay enables the quantification Sclerotic vascular disease and age-related disorders including of very low levels of plasma IL-4. In some embodiments, the 35 osteoporosis and type 2 diabetes. In addition, IL-6 can be assay allows the measurement of Small changes in IL-4 level measured in combination with other cytokines, for example that can provide insights into therapeutic efficacy. In various TNFC. to diagnose additional diseases such as septic shock. embodiments, this assay allows investigators to: (1) measure IL-6 has therapeutic potential as a drug target which would the efficacy and dosing of therapeutics designed interfere result in an anti-inflammatory and inhibition of the acute with general inflammatory and allergic responses; (2) design 40 phase response. In terms of host response to a foreign patho more robust clinical and preclinical studies when IL-4 con gen, IL-6 has been shown, in mice, to be required for resis centration is used as a therapeutic endpoint; and (3) under tance against the bacterium, Streptococcus pneumoniae stand how IL-4 levels change in patients as they transition Inhibitors of IL-6 (including estrogen) are used to treat post from a healthy to diseased state. menopausal osteoporosis. There is also therapeutic potential In some embodiments, the method of the present invention 45 for cancer, as IL-6 is essential for hybridoma growth and is is capable of detecting IL-4 at a limit of detection of less than found in many Supplemental cloning media Such as briclone. about 100, 80, 60, 50, 30, 20, 10, 5, 1,0.5,0.01, 0.005, 0.001, Circulating levels of IL-6 in the plasma of healthy subjects 0.0005 or 0.0001 pg/ml, e.g., less than about 100 pg/ml. In is difficult to determine with many currently available assays, Some embodiments, the method is capable of detecting the thus it is difficult to differentiate disease from healthy states. IL-4 at a limit of detection of less than about 100 pg/ml. In 50 Furthermore, when used as a therapeutic target, it is desirous Some embodiments, the method is capable of detecting the to measure therapeutic efficacy by measuring IL-6 levels as IL-4 a limit of detection of less than about 80 pg/ml. In some they decrease below normal state levels. This can not be embodiments, the method is capable of detecting the IL-4 a achieved with assays currently available. An IL-6 assay is limit of detection of less than about 60 pg/ml. In some currently available outside the U.S. for diagnostic use, and for embodiments, the method is capable of detecting the IL-4 a 55 research use only (RUO) in the U.S. and Japan. limit of detection of less than about 50 pg/ml. In some The present invention provides an IL-6 assay that enables embodiments, the method is capable of detecting the IL-4 a the quantification of very low levels of plasma IL-6 and limit of detection of less than about 30 pg/ml. In some allows for accurate measurement of Small changes in its level embodiments, the method is capable of detecting the IL-4 a due to disease processes or therapeutic intervention. See limit of detection of less than about 25 pg/ml. In some 60 Example 26. This high level of sensitivity can provide embodiments, the method is capable of detecting the IL-4 a insights into therapeutic efficacy. In various embodiments, limit of detection of less than about 10 pg/ml. In some this assay will allow investigators to: (1) measure the efficacy embodiments, the method is capable of detecting the IL-4 a and dosing of therapeutics designed interfere with the inflam limit of detection of less than about 5 pg/ml. In some embodi matory response; (2) design more robust clinical and preclini ments, the method is capable of detecting the IL-4 a limit of 65 cal studies when IL-6 concentration is used as a therapeutic detection of less than about 1 pg/ml. In some embodiments, endpoint; and (3) understand how IL-6 levels change in the method is capable of detecting the IL-4 a limit of detection patients as they transition from a healthy to diseased state. US 8,450,069 B2 95 96 In some embodiments, the present invention provides a detecting the IL-8 a limit of detection of less than about 0.5 method capable of detecting IL-6 at a limit of detection of less pg/ml. In some embodiments, the method is capable of than about 100, 80, 60, 50, 30, 20, 10, 5, 1, 0.5,0.01, 0.005, detecting the IL-8 at a limit of detection of less than about 0.1 0.001, 0.0005 or 0.0001 pg/ml, e.g., less than about 100 pg/ml. In some embodiments, the method is capable of pg/ml. In some embodiments, the method is capable of detecting the IL-8 at a limit of detection of less than about detecting the IL-6 at a limit of detection of less than about 100 0.05 pg/ml. In some embodiments, the method is capable of pg/ml. In some embodiments, the method is capable of detecting the IL-8 at a limit of detection of less than about detecting the IL-6 a limit of detection of less than about 80 0.01 pg/ml. In some embodiments, the method is capable of pg/ml. In some embodiments, the method is capable of detecting the IL-8 at a limit of detection of less than about detecting the IL-6 a limit of detection of less than about 60 10 0.005 pg/ml. In some embodiments, the method is capable of pg/ml. In some embodiments, the method is capable of detecting the IL-8 at a limit of detection of less than about detecting the IL-6 a limit of detection of less than about 50 0.001 pg/ml. In some embodiments, the method is capable of pg/ml. In some embodiments, the method is capable of detecting the IL-8 at a limit of detection of less than about detecting the IL-6 a limit of detection of less than about 30 0.0005 pg/ml. In some embodiments, the method is capable pg/ml. In some embodiments, the method is capable of 15 of detecting the IL-8 at a limit of detection of less than about detecting the IL-6 a limit of detection of less than about 25 0.0001 pg/ml. pg/ml. In some embodiments, the method is capable of 4. Inflammatory Markers detecting the IL-6 a limit of detection of less than about 10 Other cytokines that can be useful in detecting early onset pg/ml. In some embodiments, the method is capable of of inflammatory disease include markers and panels of mark detecting the IL-6 a limit of detection of less than about 5 ers of inflammation as described herein. Examples of cytok pg/ml. In some embodiments, the method is capable of ines that can be used to detect inflammatory disorders are detecting the IL-6 a limit of detection of less than about 1 Leukotriene 4 (LTE4), which can be an early marker of pg/ml. In some embodiments, the method is capable of asthma, and TGFB, which can be used to detect and monitor detecting the IL-6 a limit of detection of less than about 0.5 the status of inflammatory disorders including fibrosis, scle pg/ml. In some embodiments, the method is capable of 25 rosis. Some markers can be used to detect more than one detecting the IL-6 at a limit of detection of less than about 0.1 disorder, e.g., TGFB, can also be used to detect the presence pg/ml. In some embodiments, the method is capable of of cancer. detecting the IL-6 at a limit of detection of less than about a. Leukotriene E4 0.05 pg/ml. In some embodiments, the method is capable of Cysteinyl leukotrienes (LTC4, LTD4, LTE4) play an detecting the IL-6 at a limit of detection of less than about 30 important role in the pathogenesis of asthma. Leukotrienes 0.01 pg/ml. In some embodiments, the method is capable of are produced by mast cells, eosinophils, and other airway detecting the IL-6 at a limit of detection of less than about inflammatory cells and increase vascular permeability, con 0.005 pg/ml. In some embodiments, the method is capable of strict bronchial Smooth muscle, and mediate bronchial hyper detecting the IL-6 at a limit of detection of less than about responsiveness. Levels of urinary LTE4, the stable metabolite 0.001 pg/ml. In some embodiments, the method is capable of 35 of LTC4 and LTD4, are increased in children and adults with detecting the IL-6 at a limit of detection of less than about asthma compared with healthy controls and in asthmatics 0.0005 pg/ml. In some embodiments, the method is capable after bronchial challenge with antigen, after oral challenge of detecting the IL-6 at a limit of detection of less than about with aspirin in aspirin sensitive asthmatic Subjects, and dur 0.0001 pg/ml. ing exercise induced bronchospasm. The importance of leu d. Interleukin 8 (IL-8) 40 kotrienes in the pathology of asthma has been further dem Like IL-6, the present invention provides an Interleukin 8 onstrated in large clinical trials with agents that block the (IL-8) assay that enables the quantification of very low levels actions of leukotrienes. For example, montelukast, a potent of plasma IL-8 and allows for accurate measurement of small leukotriene receptor antagonist taken orally once daily, sig changes in its level due to disease processes or therapeutic nificantly improves asthma control in both children (aged intervention. See FIG. 17. In some embodiments, the present 45 2-14 years) and adults and attenuates exercise induced bron invention provides a method capable of detecting IL-8 at a choconstriction. limit of detection of less than about 100, 80, 60, 50, 30, 20, 10, Activation of the leukotriene pathways is accompanied by 5, 1,0.5,0.01, 0.005, 0.001, 0.0005 or 0.0001 pg/ml, e.g., less rises in urinary levels of LTE4, and acute exacerbations of than about 100 pg/ml. In some embodiments, the method is asthma are accompanied by increased levels of LTE4 in urine capable of detecting the IL-8 at a limit of detection of less than 50 followed by a significant decrease during resolution. The about 100 pg/ml. In some embodiments, the method is degree of airflow limitation correlates with levels of urinary capable of detecting the IL-8 a limit of detection of less than LTE4 during the exacerbation and follow up periods, thus about 80 pg/ml. In some embodiments, the method is capable indicating that the leukotriene pathway is activated during of detecting the IL-8 a limit of detection of less than about 60 acute asthma. In addition, inhalation of bronchoconstricting pg/ml. In some embodiments, the method is capable of 55 doses of LTC4 or LTE4 alter urinary LTE4 excretion in a detecting the IL-8 a limit of detection of less than about 50 dose-dependent manner thus indicating that urinary LTE4 pg/ml. In some embodiments, the method is capable of can be used as a marker of Sulphidopeptide leukotriene Syn detecting the IL-8 a limit of detection of less than about 30 thesis in the lungs of patients with asthma. pg/ml. In some embodiments, the method is capable of The methods of the invention can be used to detect changes detecting the IL-8 a limit of detection of less than about 25 60 in LTE4 in biological samples Such as urinary samples. See pg/ml. In some embodiments, the method is capable of Example 5. Measurements of subnanogram levels of LTE4 detecting the IL-8 a limit of detection of less than about 10 can be useful as a reference for detecting and monitoring pg/ml. In some embodiments, the method is capable of Sulphidopeptide leukotriene synthesis in the lungs of patients detecting the IL-8 a limit of detection of less than about 5 with chronic or acute asthma. pg/ml. In some embodiments, the method is capable of 65 In some embodiments, the methods of the present inven detecting the IL-8 a limit of detection of less than about 1 tion are capable of detecting LTE4 at a limit of detection of pg/ml. In some embodiments, the method is capable of less than about 100, 80, 60, 50, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, US 8,450,069 B2 97 98 0.01, 0.005, 0.001, 0.0005 or 0.0001 pg/ml, e.g., less than TGFB is also a marker for cancers including prostate can about 100 pg/ml. In some embodiments, the method is cer, cervical cancer, lung carcinoma, and Hodgkin’s disease. capable of detecting LTE4 at a limit of detection of less than Plasma levels of TGFB in patients with lung cancer are often about 100 pg/ml. In some embodiments, the method is elevated. It has been shown that in patients with an elevated capable of detecting the LTE4 a limit of detection of less than plasma TGF beta 1 level at diagnosis, monitoring this level about 80 pg/ml. In some embodiments, the method is capable may be useful in detecting both disease persistence and recur of detecting the LTE4 a limit of detection of less than about 60 rence after radiotherapy. pg/ml. In some embodiments, the method is capable of Transforming growth factor-beta (TGFB) is also a multi detecting the LTE4 a limit of detection of less than about 50 potent growth factor affecting development, homeostasis, 10 and tissue repair. Increased expression of TGFB has been pg/ml. In some embodiments, the method is capable of reported in different malignancies, suggesting a role for this detecting the LTE4 a limit of detection of less than about 30 growth factor in tumorigenesis. In particular, it has been pg/ml. In some embodiments, the method is capable of demonstrated that the presence of TGFB in the endothelial detecting the LTE4 a limit of detection of less than about 25 and perivascular layers of small vessels in the tumor stroma of pg/ml. In some embodiments, the method is capable of 15 osteosarcomas Suggests an angiogenic activity of this growth detecting the LTE4 a limit of detection of less than about 10 factor, and that increased expression of TGF-beta isoforms pg/ml. In some embodiments, the method is capable of have been suggested to play a role in the progression of detecting the LTE4 a limit of detection of less than about 5 osteosarcoma (Kloen et al., Cancer, 80:2230-39 (1997)). pg/ml. In some embodiments, the method is capable of TGFB is one of the few known proteins that can inhibit cell detecting the LTE4 a limit of detection of less than about 1 growth. However, although the notion is controversial, some pg/ml. In some embodiments, the method is capable of researchers believe that some human malignancies such as detecting the LTE4 a limit of detection of less than about 0.5 breast cancer subvert TGFB for their own purposes. In a pg/ml. In some embodiments, the method is capable of paradox that is not understood, these cancers make TGFB and detecting the LTE4 at a limit of detection of less than about steadily increase its expression until it becomes a marker of 0.1 pg/ml. In some embodiments, the method is capable of 25 advancing metastasis and decreased Survival. For example, detecting the LTE4 at a limit of detection of less than about levels of plasma TGFB are markedly elevated in men with 0.05 pg/ml. In some embodiments, the method is capable of prostate cancer metastatic to regional lymph nodes and bone. detecting the LTE4 at a limit of detection of less than about In men without clinical or pathologic evidence of metastases, 0.01 pg/ml. In some embodiments, the method is capable of the preoperative plasma TGF-B level is a strong predictor of detecting the LTE4 at a limit of detection of less than about 30 biochemical progression after Surgery, presumably because 0.005 pg/ml. In some embodiments, the method is capable of of an association with occult metastatic disease present at the detecting the LTE4 at a limit of detection of less than about time of radical prostatectomy. 0.001 pg/ml. In some embodiments, the method is capable of In some embodiments, the method is capable of detecting detecting the LTE4 at a limit of detection of less than about TGF-B at a limit of detection of less than about 100, 80, 60. 0.0005 pg/ml. In some embodiments, the method is capable 35 50, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 of detecting the LTE4 at a limit of detection of less than about or 0.0001 pg/ml, e.g., less than about 100 pg/ml. In some 0.0001 pg/ml. embodiments, the method is capable of detecting the TGF-B b. TGFB at a limit of detection of less than about 100 pg/ml. In some The methods of the invention can also be performed to embodiments, the method is capable of detecting the TGF-B detect the early onset of diseases for which TGFB is a marker. 40 a limit of detection of less than about 80 pg/ml. In some Examples of TGFB-related diseases include fibrotic diseases. embodiments, the method is capable of detecting the TGF-B Fibrosis refers to the excessive and persistent formation of a limit of detection of less than about 60 pg/ml. In some scar tissue, which is responsible for morbidity and mortality embodiments, the method is capable of detecting the TGF-B associated with organ failure in a variety of chronic diseases a limit of detection of less than about 50 pg/ml. In some affecting the lungs, kidneys, eyes, heart, liver, and skin. TGFB 45 embodiments, the method is capable of detecting the TGF-B is well known for its role as a mediator of chronic fibrotic a limit of detection of less than about 30 pg/ml. In some effects. For example, TGFB is implicated in promoting fibro embodiments, the method is capable of detecting the TGF-B blastic proliferation and matrix accumulation in fibrotic lung a limit of detection of less than about 25 pg/ml. In some disease. Inhibition of TGFB has been proposed as a potential embodiments, the method is capable of detecting the TGF-B therapeutic avenue for the management of lung fibrosis. 50 a limit of detection of less than about 10 pg/ml. In some TGFB not only stimulates the synthesis of many extracellular embodiments, the method is capable of detecting the TGF-B matrix molecules, including fibronectin and type I collagen a limit of detection of less than about 5 pg/ml. In some and their receptors, but also decreases matrix degradation via embodiments, the method is capable of detecting the TGF-B differential effects on the expression of proteases and their a limit of detection of less than about 1 pg/ml. In some inhibitors, strongly promoting generation of extracellular 55 embodiments, the method is capable of detecting the TGF-B matrix. Thus the analyzer Systems of the invention can detect a limit of detection of less than about 0.5 pg/ml. In some abnormal levels of TGFB, e.g., associated with fibrotic dis embodiments, the method is capable of detecting the TGF-B eases, including but not limited to idiopathic pulmonary at a limit of detection of less than about 0.1 pg/ml. In some fibrosis, diabetic nephropathy, progressive nephropathies embodiments, the method is capable of detecting the TGF-B including glomerulosclerosis and IgA nephropathy (causes of 60 at a limit of detection of less than about 0.05 pg/ml. In some kidney failure and the need for dialysis and retransplant); embodiments, the method is capable of detecting the TGF-B diabetic retinopathy and advanced macular degeneration (fi at a limit of detection of less than about 0.01 pg/ml. In some brotic diseases of the eye and leading causes of blindness); embodiments, the method is capable of detecting the TGF-B cirrhosis and biliary atresia (leading causes of liver fibrosis at a limit of detection of less than about 0.005 pg/ml. In some and failure); congestive heart failure; myocardiopathy asso 65 embodiments, the method is capable of detecting the TGF-B ciated with progressive fibrosis in Chagas disease; lung fibro at a limit of detection of less than about 0.001 pg/ml. In some sis; and Scleroderma. embodiments, the method is capable of detecting the TGF-B US 8,450,069 B2 99 100 at a limit of detection of less than about 0.0005pg/ml. In some pg/ml. In some embodiments, the method is capable of embodiments, the method is capable of detecting the TGF-B detecting the Akt1 a limit of detection of less than about 60 at a limit of detection of less than about 0.0001 pg/ml. pg/ml. In some embodiments, the method is capable of Other markers of abnormal cell growth that are detected by detecting the Akt1 a limit of detection of less than about 50 the methods of the invention include Akt1, Fas ligand, VEGF, pg/ml. In some embodiments, the method is capable of Af3-40, AB-42, cTnI, IL-1C., IL-1B, IL-4, and IL-6 as detecting the Akt1 a limit of detection of less than about 30 described herein. pg/ml. In some embodiments, the method is capable of 5. Akt1 detecting the Akt1 a limit of detection of less than about 25 Akt1 is v-akt murine thymoma viral oncogene homolog 1 pg/ml. In some embodiments, the method is capable of and is a serine-threonine protein kinase encoded by the AKT1 10 detecting the Akt1 a limit of detection of less than about 10 gene. Akt kinases have been implicated in disparate cell pg/ml. In some embodiments, the method is capable of responses, including inhibition of apoptosis and promotion of detecting the Akt1 a limit of detection of less than about 5 cell proliferation, angiogenesis, and tumor cell invasiveness. pg/ml. In some embodiments, the method is capable of Best known for its ability to inhibit apoptotic and non detecting the Akt1 a limit of detection of less than about 1 apoptotic cell death, Akt can be monitored to predict tumor 15 pg/ml. In some embodiments, the method is capable of response to anticancer treatment. Predicting tumor response detecting the Akt1 a limit of detection of less than about 0.5 by assessing the influence of apoptosis and nonapoptotic cell pg/ml. In some embodiments, the method is capable of death, would allow for developing a more efficient strategy detecting the Akt1 at a limit of detection of less than about 0.1 for enhancing the therapeutic effect of anticancer treatment. pg/ml. In some embodiments, the method is capable of Anticancer treatment-induced apoptosis is regulated by the detecting the Akt1 at a limit of detection of less than about balance of proapoptotic and antiapoptotic proteins through 0.05 pg/ml. In some embodiments, the method is capable of mitochondria, and resistance to apoptosis is mediated by detecting the Akt1 at a limit of detection of less than about Akt-dependent and Bcl-2-dependent pathways. Bcl-2 par 0.01 pg/ml. In some embodiments, the method is capable of tially inhibits nonapoptotic cell death as well as apoptosis, detecting the Akt1 at a limit of detection of less than about whereas Akt inhibits both apoptotic and nonapoptotic cell 25 0.005 pg/ml. In some embodiments, the method is capable of death through several target proteins. Since drug sensitivity is detecting the Akt1 at a limit of detection of less than about likely correlated with the accumulation of apoptotic and non 0.001 pg/ml. In some embodiments, the method is capable of apoptotic cell deaths, which may influence overall tumor detecting the Akt1 at a limit of detection of less than about response in anticancer treatment. The ability to predict overall 0.0005 pg/ml. In some embodiments, the method is capable tumor response from the modulation of several important cell 30 of detecting the Akt1 at a limit of detection of less than about death-related proteins may result in a more efficient strategy 0.0001 pg/ml. for improving the therapeutic effect. 6. Fas Ligand Akt1 is also involved in Epithelial-mesenchymal transition Fas Ligand (FasL), also known as CD95L, is a member of (EMT), which is an important process during development the TNF family and induces apoptosis via binding to Fas and oncogenesis by which epithelial cells acquire fibroblast 35 (CD95). The protein exists in two forms; either membrane like properties and show reduced intercellular adhesion and FasL or soluble FasL, which migrate at molecular weight of increased motility. AKT is activated in many human carcino 45 kDa and 26 kDa, respectively. FasL is expressed on a mas, and the AKT-driven EMT may confer the motility variety of cells including activated lymphocytes, natural required for tissue invasion and metastasis. Thus future thera killer cells and monocytes. Interaction of FasL and Fas plays pies based on AKT inhibition may complement conventional 40 an important role in physiological apoptotic processes. Mal treatments by controlling tumor cell invasion and metastasis. function of the Fas-FasL system causes hyperplasia in periph Akt is constitutively activated in most melanoma cell lines eral lymphoid organs and accelerates autoimmune disease and tumor samples of different progression stages, and acti progression and tumorigenesis. There are limited data about vation of AKT has been linked to the expression of invasion/ the levels of soluble apoptotic factors in general, and more metastasis-related melanoma cell adhesion molecule (Mel 45 specifically about their modulation with therapeutic regi CAM), which in turn is strongly associated with the CS. acquisition of malignancy by human melanoma. Akt1 is also The systems and methods of the invention can detect con activated in pancreatic cancer, and AKT activation has been centrations of Fas ligand that are as low as 2.4 pg/ml. Thus, in shown to correlate with higher histologic tumor grade. Thus, Some embodiments, the analyzer systems and methods of the AKT activation is associated with tumor grade, an important 50 invention provide for the detection of Fas ligand to identify prognostic factor. Akt1 is also upregulated in prostate cancer pathological conditions such as abnormal levels of apoptosis. and that expression is correlated with tumor progression. Measurements of Fas in patient samples can be used to diag Thus, Akt1 could be targeted for therapeutic intervention of nose conditions such as polycystic ovarian syndrome, tumors cancer while at its earliest stages. In some embodiments, the Such as testicular germ cell tumors, bladder cancer, lung analyzer systems of the invention provide a method for pro 55 cancer, and rare tumors such as follicular dendritic cell viding an early diagnosis of a cancer by determining the tumors. In addition, Fas measurements of Fas ligand can be presence or concentration of Akt1 in a sample from a patient used to diagnose allograft rejection and degenerative disease when the level of Akt1 is less than about 100, 50, or 25 pg/ml. Such as osteoarthritis. Thus, in Some embodiments, the ana See Example 6. lyZer systems and methods of the invention can be used to In some embodiments, the methods of the present inven 60 determine the concentration of Fas ligand in a sample from a tion are capable of detecting Akt1 at a limit of detection of less patient Suspected of Suffering from Fas ligand related disor than about 100, 80, 60, 50, 30, 20, 10, 5, 1,0.5,0.1, 0.05, 0.01, der to diagnose the disorder, or the concentration of Fas 0.005, 0.001, 0.0005 or 0.0001 pg/ml, e.g., less than about ligand can be used to monitor the progress or status of a Fas 100 pg/ml. In some embodiments, the method is capable of ligand related disorder in a patient undergoing therapy for the detecting Akt1 at a limit of detection of less than about 100 65 disorder. In some embodiments, the assay is capable of deter pg/ml. In some embodiments, the method is capable of mining the level of Fas ligand in the sample at a concentration detecting the Akt1 a limit of detection of less than about 80 less than about 100, 50, 25, 10, or 5 pg/ml. See Example 8. US 8,450,069 B2 101 102 In some embodiments, the methods of the present inven changes that occur as disease progresses. However, current tion are capable of detecting FasLata limit of detection of less commercially available immunoassays can only measure than about 100, 80, 60, 50, 30, 20, 10, 5, 1,0.5,0.1, 0.05, 0.01, elevated concentrations of VEGF. They are not sensitive 0.005, 0.001, 0.0005 or 0.0001 pg/ml, e.g., less than about enough to measure VEGF in plasma obtained from healthy 100 pg/ml. In some embodiments, the method is capable of 5 human subjects or detect the small changes in VEGF levels detecting FasL at a limit of detection of less than about 100 that may be indicative of an early disease state. However, the pg/ml. In some embodiments, the method is capable of plasma VEGF assay according to the present invention pro detecting the FasL a limit of detection of less than about 80 vides the power needed to use VEGF as a biomarker for pg/ml. In some embodiments, the method is capable of disease and the sensitivity to quantify VEGF in healthy detecting the FasL a limit of detection of less than about 60 10 human Subjects as well as those undergoing anti-VEGF pg/ml. In some embodiments, the method is capable of therapy. In some embodiments, the human VEGF assay has detecting the FasL a limit of detection of less than about 50 an LOD of about 0.1 pg/ml and a lower limit of quantitation pg/ml. In some embodiments, the method is capable of (LLOQ) of 0.3 pg/ml, making it 90x more sensitive than the detecting the FasL a limit of detection of less than about 30 commonly used ELISA assay. See Examples 11-21. pg/ml. In some embodiments, the method is capable of 15 The present invention increases the clinical utility of VEGF detecting the FasL a limit of detection of less than about 25 by allowing scientists to detect very low levels of VEGF and pg/ml. In some embodiments, the method is capable of measure Small changes in its level that can provide insights detecting the FasL a limit of detection of less than about 10 into drug efficacy or disease progression. Among other pg/ml. In some embodiments, the method is capable of improvements, the assay allows investigators to: (1) measure detecting the FasL a limit of detection of less than about 5 the efficacy and dosing of therapeutics designed to lower the pg/ml. In some embodiments, the method is capable of levels of VEGF, particularly when VEGF levels should go detecting the FasL a limit of detection of less than about 1 much lower than that seen in normal states; (2) design more pg/ml. In some embodiments, the method is capable of robust clinical and preclinical studies when VEGF concen detecting the FasL a limit of detection of less than about 0.5 tration is used as a therapeutic endpoint; and (3) understand pg/ml. In some embodiments, the method is capable of 25 how VEGF levels change in patients as they transition from a detecting the FasLata limit of detection of less than about 0.1 healthy to diseased state with cancer and other diseases pg/ml. In some embodiments, the method is capable of involving angiogenesis. detecting the FasL at a limit of detection of less than about In some embodiments, the present invention provides 0.05 pg/ml. In some embodiments, the method is capable of methods to quantify normal levels of VEGF, and identify detecting the FasL at a limit of detection of less than about 30 abnormally elevated levels of VEGF indicative of the pres 0.01 pg/ml. In some embodiments, the method is capable of ence of an early stage cancer/tumor. Typical healthy levels of detecting the FasL at a limit of detection of less than about VEGF inhumans are less than 50 pg/mL, and are significantly 0.005 pg/ml. In some embodiments, the method is capable of elevated (>100 pg/mL, often 200-500 pg/mL) in subjects with detecting the FasL at a limit of detection of less than about cancer. In other embodiments, the methods described herein 0.001 pg/ml. In some embodiments, the method is capable of 35 can be used to indicate the presence of other cancers, such as detecting the FasL at a limit of detection of less than about prostate and lymphoma. The method can be used to indicate 0.0005 pg/ml. In some embodiments, the method is capable the presence of solid tumors that are undergoing vasculariza of detecting the FasL at a limit of detection of less than about tion, which will have increased levels of VEGF. 0.0001 pg/ml. In some embodiments, the present invention provides 7. VEGF 40 methods to quantify normal levels of VEGF, and identify Vascular endothelial growth factor-A (VEGF-A), com abnormally elevated levels of VEGF indicative of the pres monly known as VEGF, is a member of a family of secreted ence of vascular inflammation. This measurement can be glycoproteins that promote endothelial cell growth, Survival, augmented by co-measurement of other inflammatory cytok migration, and vascular permeability, all of which contribute ines in healthy individuals, where elevated levels are indica to angiogenesis. The binding of VEGF to its receptor triggers 45 tive of inflammation. In some embodiments, because of the the activation of a cell signaling pathway that is critical for the role of VEGF in angiogenesis and artherosclerosis, the inven growth of blood vessels from pre-existing vasculature. VEGF tion can also be used to quantify abnormally elevated levels of is implicated in a variety of diseases including cancer, age VEGF as indicative of cardiac disease in conjunction with related macular degeneration, diabetic retinopathy and rheu elevated levels of cTnI which is the gold standard for detect matoid arthritis. As such, it is an attractive candidate for the 50 ing myocardial infarction. This measurement can be aug development of therapies to these diseases, particularly can mented by co-measurement of other cardiac markers (i.e., C. pro-BNP) or inflammatory markers (i.e., hsCRP, cytokines) The first anti-VEGF drug, the monoclonal antibody Avas in healthy individuals, where elevated levels are indicative of tin, was approved by the FDA in 2004 and is approved to treat cardiac disease. In some embodiments, the method described metastatic colon and non Small-cell lung cancer. The drug is 55 can be used to quantify normal levels of VEGF, and identify also under study for the treatment of many other cancers. abnormally elevated levels of VEGF indicative of the pres Other compounds that target VEGF-mediated cell signaling ence of artherosclerosis in subjects with diabetes. This mea include the monoclonal antibody fragment Lucentis, Surement can be augmented by co-measurement of other approved to treat age related macular degeneration, and two markers for diabetes (i.e., insulin) and for metabolic disease Small molecules, Sutent and Nexavar, which target receptor 60 (i.e., glucagon like peptide-1 (GLP-1)). tyrosine kinases, including the VEGF receptor. Other drug The present invention provides methods to measure VEGF candidates targeting this path are in development. in very Small sample Volumes that are less than the standard With the efficacy seen with drugs that target VEGF and its sample volume of 100 ul. The methods are enabled by the pathway, VEGF is an attractive development target. In addi sensitivity of the assay and enable a greater number of tion, as researchers study various cancers and other diseases 65 samples to provide quantifiable results in Small volume where VEGF signaling is implicated, measuring Small samples compared to other methods. In one embodiment, the changes in VEGF levels will help them understand biological methods measure VEGF in human or mouse plasma samples US 8,450,069 B2 103 104 of less than or equal to 10 ul. In another embodiment, the the brainforming plaques. Thus the concentration of AB-42 is methods measure VEGF in tissue lysates from human or decreased in the CSF of many AD patients. Recent studies mouse plasma samples of less than or equal to 10 ul. These Suggest that a decrease in AB-42 concentrations (with a par methods have been tested in lysates from human breast cancer alleled change in the ratio of AB-40/AB-42) in CSF and tissue biopsies, as well as in mouse tissue lysates from several plasma are predictive of the onset of AD. strains of mice. In another embodiment, the methods measure There is no cure for Alzheimer's disease and currently VEGF in lysates prepared from tissue biopsies in healthy and available therapeutics minimize some of the symptoms asso diseased individuals. Based on a typical 1 mm needle biopsy, ciated with AD but do not slow disease progression. Numer and resulting lysates Volume of less than or equal to 10 ul this ous experimental approaches focus on minimizing AB-42 method enables quantification of VEGF from a needle biopsy. 10 levels by preventing production of or lowering AB-42 con Small volume sample sizes are also provided with other centrations, stimulating the immune system to attack AB pro markers of the present invention. teins as well as preventing AB proteins from aggregating and In one aspect, the present invention provides a method for forming plaques. An important component in designing determining the presence or absence of a single molecule of therapeutic trials is to identify patients that are at risk for VEGF or a fragment or complex thereof in a sample, by i) 15 developing ADSuch that studies can be performed in a cost labeling the molecule, fragment, or complex, if present, with effective timely manner. Hence biomarkers would be invalu a label; and ii) detecting the presence or absence of the label, able for both understanding AB levels as Surrogate endpoints where the detection of the presence of the label indicates the as well as in efficient study design. presence of the single molecule, fragment, or complex of Preventive therapy is a major focus as the best way to VEGF in the sample. In some embodiments, the methods of manage AD. Guidelines describe the need for non-invasive the present invention are capable of detecting VEGF at a limit biomarkers that can be used to predict and diagnose the for of detection of less than about 115, 100, 80, 60, 50, 30, 20, 10, mation of AD. Such information will be invaluable for clini 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 or 0.0001 cal study design, as well as the evaluation of therapeutic pg/ml, e.g., less than about 115 pg/ml. In some embodiments, effectiveness. Measuring AB-40 and AB-42 concentrations in the method is capable of detecting VEGF at a limit of detec 25 plasma provide promise for Such information. In healthy nor tion of less than about 115 pg/ml. In some embodiments, the mal humans, plasma concentrations range from 200-400 method is capable of detecting VEGF at a limit of detection of pg/ml (AB-40) and 15-30 pg/ml (AB-42). However with AD, less than about 100 pg/ml. In some embodiments, the method Af3-42 levels decrease, and are often undetectable by cur is capable of detecting the VEGF a limit of detection of less rently available EIA technology. Furthermore, interventional than about 80 pg/ml. In some embodiments, the method is 30 strategies based on depleting AB-42 formation require meth capable of detecting the VEGF a limit of detection of less than ods that measure decreases in AB-42. Thus there is a need to about 60 pg/ml. In some embodiments, the method is capable accurately and precisely quantify low concentrations of amy of detecting the VEGF a limit of detection of less than about loid proteins in plasma. 50 pg/ml. In some embodiments, the method is capable of The AB-40 and AB-42 assays according to the present detecting the VEGF a limit of detection of less than about 30 35 invention allow the quantification of amyloid beta proteins pg/ml. In some embodiments, the method is capable of from human plasma with exceptional sensitivity, enabling the detecting the VEGF a limit of detection of less than about 25 use of A-40/AB-42 as a velocity biomarker in Alzheimer's pg/ml. In some embodiments, the method is capable of disease studies and to evaluate therapeutic interventions. See detecting the VEGF a limit of detection of less than about 10 Example 22. Among other advantages, this assay allows pg/ml. In some embodiments, the method is capable of 40 investigators to: (1) identify Subjects with potential high risk detecting the VEGF a limit of detection of less than about 5 for developing AD and hence design interventional studies pg/ml. In some embodiments, the method is capable of that include high risk for disease development; (2) design detecting the VEGF a limit of detection of less than about 1 more robust clinical and preclinical studies when AB protein pg/ml. In some embodiments, the method is capable of concentrations are used as a therapeutic endpoint; and (3) detecting the VEGF a limit of detection of less than about 0.5 45 understand how AB protein levels change in humans as they pg/ml. In some embodiments, the method is capable of transition from a healthy to a diseased state. detecting the VEGF at a limit of detection of less than about In some embodiments, the methods of the present inven 0.1 pg/ml. In some embodiments, the method is capable of tion are capable of detecting the AB-40 at a limit of detection detecting the VEGF at a limit of detection of less than about of less than about 100, 80, 60, 50, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05 pg/ml. In some embodiments, the method is capable of 50 0.05, 0.01, 0.005, 0.001, 0.0005 or 0.0001 pg/ml, e.g., less detecting the VEGF at a limit of detection of less than about than about 100 pg/ml. In some embodiments, the method is 0.01 pg/ml. In some embodiments, the method is capable of capable of detecting AB-40 at a limit of detection of less than detecting the VEGF at a limit of detection of less than about about 100 pg/ml. In some embodiments, the method is 0.005 pg/ml. In some embodiments, the method is capable of capable of detecting the AB-40 a limit of detection of less than detecting the VEGF at a limit of detection of less than about 55 about 80 pg/ml. In some embodiments, the method is capable 0.001 pg/ml. In some embodiments, the method is capable of of detecting the AB-40 a limit of detection of less than about detecting the VEGF at a limit of detection of less than about 60 pg/ml. In some embodiments, the method is capable of 0.0005 pg/ml. In some embodiments, the method is capable detecting the AB-40 a limit of detection of less than about 50 of detecting the VEGF at a limit of detection of less than about pg/ml. In some embodiments, the method is capable of 0.0001 pg/ml. 60 detecting the AB-40 a limit of detection of less than about 30 pg/ml. In some embodiments, the method is capable of Amyloid beta proteins (40 and 42 amino acids) are the detecting the AB-40 a limit of detection of less than about 25 main constituent of amyloid plaques in the brains of Alzhe pg/ml. In some embodiments, the method is capable of imer's disease (AD) patients. In healthy and diseased States detecting the AB-40 a limit of detection of less than about 10 Af3-40 is the more common form (10-20x higher than AB-42) 65 pg/ml. In some embodiments, the method is capable of of the two in both cerebrospinal fluid (CSF) and plasma. In detecting the AB-40 a limit of detection of less than about 5 patients with AD, AB-42 primarily aggregates and deposits in pg/ml. In some embodiments, the method is capable of