USOO8409807B2

(12) United States Patent (10) Patent No.: US 8,409,807 B2 Neely et al. (45) Date of Patent: *Apr. 2, 2013

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Nanomaterials for the Life Sci tus and Future Goals.” Clin. Biochem. Rev. 27:173-184 (2006). ences vol. 4: Magnetic Nanomaterials. Weinheim: Wiley-VCH Pecket al., “RFMicrocoils Patterned Using Microlithographic Tech Verlag GmbH & Co. KgaA, pp. 1-53 (2009). niques for Use as Microsensors in NMR' Engineering in Medicine Lowery et al., “Application of Magnetics in Point of Care Testing.” in and Biology Society, Proceedings of the 15th annual international Point-of-Care Testing: Needs, Opportunity and Innovation 3 Edi conference of the IEEE, pp. 174-175 (Oct. 28-31, 1993). tion, AACC Press, pp. 85-95 (2010). Perez et al., “DNA-based Magnetic Nanoparticle Assembly Acts as a Lick et al., “Analysis of Plasma Protein Adsorption on Polymeric Magnetic Relaxation NanoSwitch Allowing Screening of DNA Nanoparticles with Different Surface Characteristics.” J. Biomed. cleaving Agents' J. Am. Chem. Soc. 124:2856-2857 (2002). Mater. Res. 39:478-485 (1998). Perez et al., “Peroxidase Substrate Nanosensors for MRImaging”. Lundqvist et al., “Nanoparticle Size and Surface Properties Deter Nano Lett 4:119-122 (2004). mine the Protein Corona with Possible Implications for Biological Perez et al., “Integrated Nanosensors to Determine Levels and Func Impacts.” Proc. Natl. Acad. Sci. 105:14265-14270 (2008). tional Activity of Human Telomerase” Neoplasia 10:1066-1072 Lustgarten and Wenk, “Simple, Rapid, Kinetic Method for Serum (2008). Measurement.” Clin. Chem. 18:1419-1422 (1972). Perez et al., “Magnetic Relaxation Switches Capable of Sensing Ma et al., “Rapid and Sensitive Detection of Microcystin by Molecular Interactions.” Nat. Biotechnol. 20:816-820 (2002). Immmunosensor Based on Nuclear Magnetic Resonance” Biosens Perez et al., “Viral-Induced Self Assembly of Magnetic Bioelectron. 25:240-243 (2009). Nanoparticles Allows the Detection of Viral Particles in Biological Media.” J. Am. Chem. Soc. 125: 10192-10193 (2003). Maaroufi et al., “Early Detection and Identification of Commonly Perez et al., “Use of Magnetic Nanoparticles as Nanosensors to Probe Encountered Candida Species from Simulated Blood Cultures by for Molecular Interactions.” ChembioChem. 5:261-264 (2004). Using a Real-Time PCR-Based Assay.” J. Mol. Diagn. 6: 108-114 Pryce et al., “Real-Time Automated Polymerase Chain Reaction (2004). (PCR) to Detect Candida albicans and Aspergillus fumigatus DNA in Maginet al., “Miniature Magnetic Resonance Machines' IEEE Spec Whole Blood from High-Risk Patients.” Diagn. Microbiol. Infect. trum 34:51-61 (1997). Dis. 47:487-496 (2003). Makiranta et al., “Modeling and Simulation of Magnetic Ramadanet al., "On-Chip Micro-electromagnets for Magnetic-based Nanoparticle Sensor' Conf. Proc. IEEE Eng. Med. Biol. Soc., Shang Bio-molecules Separation' J. Magn. Magn. Mater: 281: 150-172 hai, China, Sep. 1-4, 2005, pp. 1256-1259 (2005). (2004). Malba et al., “Laser-Lathe Lithography—A novel Method for Manu Renaud et al., “Implantable Planar rF Microcoils for NMR facturing Nuclear Magnetic Resonance Microcoils. Biomed. Microspectroscopy” Sens Actuators, A Phys. 99:244-248 (2002). Microdevices 5:21-27 (2003). Rida et al., "Long-Range Transport of Magnetic Microbeads Using Martin et al., “Strong Intrinsic Mixing in Vortex Magnetic Fields.” Simple Planar Coils Placed in a Uniform Magnetostatic Field' Appl. Phys. Rev. E80:016312 (2009) (6 pages). Phys. Lett. 83:2396-2398 (2003). Martin, “Theory of Strong Intrinsic Mixing of Particle Suspensions Rosenstraus et al., “An Internal Control for Routine Diagnostic PCR: in Vortex Magnetic Fields.” Phys. Rev. E 79:01 1503 (2009) (12 Design, Properties and Effect on Clinical Performance” J. Clin. pages). Microbiol. 36:191-197 (1998). Martin et al., “The Epidemiology of Sepsis in the United States from Routley et al., “The HALO System—A Light Weight Portable Imag 1979 Through 2000.” NEJM 348: 1546-1554 (2003). ing System’ Magn Reson Imaging. 22:1145-1151 (2004). Massin et al., “Planar Microcoil-Based Microfluidic NMR Probes.” Riittimann et al., “DNA Polymerases from the Extremely J. Magn. Reson. 164:242-255 (2003). Thermophilic Bacterium Thermus thermophilis HB-8.” Eur: J. Massin et al., “Planar Microcoil-based Magnetic Resonance Imaging Biochem. 149:41-46 (1985). of Cells' Transducers, Solid-state Sensors, Actuators and Seeber et al., “Triaxial Magnetic Field Gradient System for Microcoil Microsystems 12th Int’l conference vol. 2:967-970 (2003). Magnetic Resonance Imaging” Rev. Sci. Instrum. 71:4263-4272 Masson et al., “Combined Enzymic-Jaffe Method for Determination (2000). of Creatinine in Serum.” Clin. Chem. 27:18-21 (1981). Shapiro et al., “Dynamic Imaging with MRI Contrast Agents: Quan McCusker et al., “Improved Method for Direct PCR. Amplification titative Considerations' Magn Reson Imaging 24:449-462 (2006). from Whole Blood.” Nucleic Acids Res. 20:6747 (1992) (1 page). Siegel et al., "Affinity Maturation of Tacrolimus Antibody for McDowell et al., “Operating Nanoliter Scale NMR Microcoils in a 1 Improved Immunoassay Performance.” Clin. Chem. 54:1008-1017 Tesla Field”.J. Magn. Reson. 188:74-82 (2007). (2008). Mercier et al., “Direct PCR from Whole Blood, Without DNA Extrac Sillerud et al., "H NMR Detection of Superparamagnetic tion.” Nucleic Acids Res. 18:5908 (1990) (1 page). Nanoparticles at 1 TUsing a Microcoil and Novel Tuning Circuit.” J. Metwally et al., “Improving Molecular Detection of Candida DNA in Magn. Reson. 181:181-190 (2006). Whole Blood: Comparison of Seven Fungal DNA Extraction Proto Skurup et al., “New Creatinine Sensor for Point-of-Care Testing of cols Using Real-Time PCR.J. Med. Microbiol. 57:296-303 (2008). Creatinine Meets the National Kidney Disease Education Program Morrell et al., “Delaying the Empiric Treatment of Candida Blood Guidelines.” Clin. Chem. Lab Med. 46:3-8 (2008). stream Infection Until Positive Blood Culture Results are Obtained: Stöcklein et al., “Enzyme Kinetic Assays with Surface Plasmon A Potential Risk Factor for Hospital Mortality.” Antimicrob. Agents Resonance (BIAcore) Based on Competition Between Enzyme and Chemother: 49:3640-3645 (2005). Creatinine Antibody.” Biosen. Bioelectron. 15:377-382 (2000). Moser et al., "On-Chip Immune-Agglutination Assay with Analyte Sullivan and Irreverre, "A Highly Specific Test for Creatinine.” J. Capture by Dynamic Manipulation of Superparamagnetic Beads.” Biol. Chem. 233:530-534 (1958). Lab Chip 9:3261-3267 (2009). Sun et al., “Experimental Study on T. Relaxation Time of Protons in Niemeyer et al., “Self-Assembly of DNA-Streptavidin Water Suspensions of Iron-Oxide Nanoparticles: Waiting Time Nanostructures and Their Use as Reagents in Immuno-PCR” Nucleic Dependence” 321:2971-2975 Journal of Magnetism and Magnetic Acids Res. 27:4553-4561 (1999). Materials (2009). US 8,409,807 B2 Page 6

Syms et al., “MEMS Helmholtz Coils for Magnetic Resonance Imag Weetall and Lee "Antibodies Immobilized on Inorganic Supports.” ing” J. Micromech. Microeng. 15:S1-S9 (2005). App. Biochem. Biotechnol. 22:311-330 (1989). Taktak et al., “Multiparameter Magnetic Relaxation Switch Assays' Weissleder et al., “Cell-Specific Targeting of Nanoparticles by Anal. Chem. 79:8863-8869 (2007). Taktak et al., “Electrode Chemistry Yields a Nanoparticle-based Multivalent Attachment of Small Molecules.” Nat. Biotechnol. NMR Sensor for Calcium” Langmuir 24:7596-7598 (2008). 23: 1418-1423 (2005). Tanaka et al., “Properties of Superparamagnetic Iron Oxide Wensink et al., “High Signal to Noise Ratio in Low Field NMR on Nanoparticles Assembled on Nucleic Acids' Nucleic Acid Sympo chip Simulations and Experimental Results' Micro Electro Mechani sium Series, 52.693-694 (2008). cal Systems 17th IEEE International Conference, Netherlands, pp. Taur et al., “Effect of Antifungal Therapy Timing on Mortality in 407-410 (2004). Cancer Patients with Candidemia.” Antimicrob. Agents Chemother: Wildgruber et al., “Monocyte Subset Dynamics in Human Athero 54:184-190 (2010). sclerosis can be Profiled with Magentic Nano-Sensors' PloS One Thorne et al., “Analytic Validation of a Quantitative Real-Time PCR 4(5):e5663 (2009) (9 pages). Assay to Measure CMV Viral Load in Whole Blood.” Diagn. Mol. Wilson and Plass, “Creatine and Creatinine in Whole Blood and Pathol. 16:73-80 (2007). Plasma.” J. Biol. Chem. 29:413-423 (1917). 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PCR: Application to SNPs Affecting Homocysteine and Folate Zhang et al., “A Probe Design for the Acquisition of Homonuclear, Metabolism.” Clin. Chem, 47:2050-2053 (2001). Heteronuclear, and Inverse Detected NMR Spectra from Multiple van Bentum et al., “Towards Nuclear Magnetic Resonance Samples”.J. Magn. Reson. 153:254-258 (2001). u-spectrocopy and u-imaging” Analyst 129:793-803 (2004). Zhao et al., “Magnetic Sensors for Protease Assays' Angew. Chem. Vasseur and Ayyangar, “Inter-area and Inter-ASMPLS Traffic Engi Int. Ed. 42:1375-1378 (2003). neering” IETF Standard-working draft, internet Engineering Task International Search Report for International Application No. PCT/ Force, IETF, CH, Feb. 2004. US08/73346, completed Oct. 23, 2008, mailed Nov. 7, 2008. Vollenhofer-Schrumpfet al., “A Simple Nucleic Acid Hybridization? International Search Report for International Application No. PCT/ Latex Agglutination Assay for the Rapid Detection of Polymerase IB2008/052597; completed Oct. 31, 2008, mailed Nov. 11, 2008. Chain Reaction Amplicons' J. Microbiol Methods. 68:568-576 Blast data sheet analysis 1, downloaded from http://blast.ncbi.nlm. (2007). nih.gov/blast.cgi, Apr. 14, 2012 (4 pages). von Lilienfeld-Toal et al., “Utility of a Commercially Available Mul Blast data sheet analysis 2, downloaded from http://blast.ncbi.nlm. tiplex Real-Time PCR Assay to Detect Bacterial and Fungal Patho nih.gov/blast.cgi, Apr. 14, 2012 (3 pages). gens in Febrile Neutropenia.” J. Clin. Microbiol. 47:2405-2410 Buck et al., “Design Strategies and Performance of Custom DNA (2009). Sequencing Primers.” Biotechniques 27(3): 528-536 (1999). Wallemacq et al., “Improvement and Assessment of Enzyme-Linked U.S. Appl. No. 13/646,402, Neely et al. Immunosorbent Assay to Detect Low FK506 Concentrations in U.S. Appl. No. 13/649,839, Neely et al. Plasma or Whole Blood Within 6 Hours. Clin. Chem. 39:1045-1049 International Search Report and Written Opinion of the International (1993). Searching Authority for International Application No. PCT/US 11/ Wang et al., “A Novel Strategy to Engineer DNA Polymerases for 56933, dated May 10, 2012. Enhanced Processivity and Improved Performance in vitro” Nucleic International Search Report and Written Opinion of the International Acids Res. 32:1197-1207 (2004). Searching Authority for International Application No. PCT/US 11/ Weetall, "Preparation of Immobilized Proteins Covalently Coupled 56936, dated May 17, 2012. Through Silane Coupling Agents to Inorganic Supports. App. Biochem. Biotechnol. 41: 157-188 (1993). * cited by examiner U.S. Patent Apr. 2, 2013 Sheet 1 of 57 US 8,409,807 B2

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Figure 10

100 pul Pipette Tips Reagent Wells (Quantity either 3 or 5) (Quantity 7) 50 ul Pipette Tips - fits - Cutter (Optional) (Quantity Either 8 or 10) . 83 : 200 ul PCR (Reaction) Tubes (Quantity 7)

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Figure 11

U.S. Patent Apr. 2, 2013 Sheet 13 Of 57 US 8,409,807 B2

Figure 13

U.S. Patent Apr. 2, 2013 Sheet 14 Of 57 US 8,409,807 B2

Figure 14

Figure 14C U.S. Patent Apr. 2, 2013 Sheet 15 Of 57 US 8,409,807 B2

Figure 15

OR U.S. Patent Apr. 2, 2013 Sheet 16 Of 57 US 8,409,807 B2

Figure 16

U.S. Patent Apr. 2, 2013 Sheet 17 Of 57 US 8,409,807 B2

Figure 17

U.S. Patent US 8,409,807 B2

U.S. Patent Apr. 2, 2013 Sheet 19 Of 57 US 8,409,807 B2

Figure 19

Figure 19A

Figure 19B U.S. Patent Apr. 2, 2013 Sheet 20 Of 57 US 8,409,807 B2

Figure 20

- Sample Support

Main Plate Linkages (x4)

Linear Rail & Carriage System (x2)

Support for Driveshaft & Rails

Coupling & Driveshaft (hidden)

Mounting Plate

Die Motor U.S. Patent Apr. 2, 2013 Sheet 21 Of 57 US 8,409,807 B2

Figure 21

Sample Support

Main Piate Linkages (x2) Linear Rail & Carriage System (x1)

Support for Linear Rail

Coupling & Driveshaft (hidden) Support for Driveshaft Mounting Plate

Drive Motor U.S. Patent Apr. 2, 2013 Sheet 22 Of 57 US 8,409,807 B2

Figure 22

rive Shafe axis coaxial to motor shaft r1 axis offset from motor shaft - this creates orbital pattern riypical offset is A", but this can be optimized for sample volume & geometry

Couping sAllows for misalignment atypically helical coupling •Not necessarily required

Drivetter eTypically servo or stepper with encoder sThese motors have an "index" mark which allows the motor to find a specific point in it's rotation. These index marks are used to home the system, which enables its use in automated systems eExternal home switches could also be used Figure 22A

Main plate connected to offset axis of drive shaft (free to rotate). Plate follows orbital path and notor shaft. One end of linkage connected to Main plate (free to rotate). This end follows orbital path.

Other end of linkage connected to carriage of linear rail system free to rotate), his end follows linear path. Having 2 linkages connected to 1 carriage defines a line in the main plate, which prevents it from rotating around its own center, Having 4 linkages balances the system. Figure 22B U.S. Patent Apr. 2, 2013 Sheet 23 Of 57 US 8,409,807 B2

igure 23

Figure 23A Figure 23B

Figure 23C U.S. Patent Apr. 2, 2013 Sheet 24 Of 57 US 8,409,807 B2

Figure 24

a Free Analyte antibody

Analyte coated Particle U.S. Patent Apr. 2, 2013 Sheet 25 Of 57 US 8,409,807 B2

Figure 25 Creatinine Competitive Assay - in Serum SOO 4OO 3OO 2OO 100

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U.S. Patent Apr. 2, 2013 Sheet 26 Of 57 US 8,409,807 B2

Figure 26

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Figure 27

Creatinine Competitive Assay - sbgMAA Processing

Creat mg/dl. U.S. Patent Apr. 2, 2013 Sheet 28 Of 57 US 8,409,807 B2

Figure 28

Antigen:Protein 8 Conjugate

Antibody-coated Particle U.S. Patent Apr. 2, 2013 Sheet 29 Of 57 US 8,409,807 B2

Figure 29

Tacrolimus Competitive Assay, Extracted Whole Blood as a 5 PCurve Fit X Data

g 5 n as

U.S. Patent Apr. 2, 2013 Sheet 30 Of 57 US 8,409,807 B2

Figure 30

gMAA Temp = RT 1800 ...... 8

i: 1600 8 - ) 1 min dwell (12 min total) . 1400 345 sec dwell (9 min total) : 8 1200 . A 30 sec dwell (6 min total) : st 1000 8 - 8. -- 5, 800 -i-A- N : 600 --~x-axo~.: AOO ...... *...... 8 200o -....I. . . O 3. 10 1.OO OOO 1OOOO Analyte, pg/ml Figure 30A

E:45 sec dwell (9 min total) is 30 sec dwell (6 m in total)

Figure 30B U.S. Patent Apr. 2, 2013 Sheet 31 Of 57 US 8,409,807 B2

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500.00 c&c. in 400.00 to 300.00 cc 200.00 - 100.00 tox 0.00 -- U.S. Patent Apr. 2, 2013 Sheet 34 of 57 US 8,409,807 B2

Figure 34

1600.00 1400.00 1200.00 1OOOOO 3OOOO & 10K O.8:1 600.00 & 40K O.8:1 4OOOO 8.7OKO.8:1. 200.00 0.008---~ O.OO1 O.O1 0.1 1. Dextran Tac ug/ml U.S. Patent Apr. 2, 2013 Sheet 35 of 57 US 8,409,807 B2

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Figure 36

Titration of BSA-tac conjugates with anti-tacrolimus coated particles

1600 1400

1200 1OOO & C23 BSA:Tac 1:10 800

6OO 3 C2 BSA:Tac 1:30 400 X C2 BSA:TaC 1:50 2OO

OO1 0.1 1. 10 100 1OOO Conjugate conc. (ng/ml) U.S. Patent Apr. 2, 2013 Sheet 37 Of 57 US 8,409,807 B2

Figure 37

8AX B; & AXN 435

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anti-biotin Antibody gAm & AXN2B Biod : AXN3-18 Eid U.S. Patent Apr. 2, 2013 Sheet 39 Of 57 US 8,409,807 B2

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Figure 40 SSSADextra Particle Surface before protein block Figure 40A

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protein

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Figure 43

Figure 43B Figure 43C Figure 43D U.S. Patent Apr. 2, 2013 Sheet 44 of 57 US 8,409,807 B2

Figure 44

Thermal insulation

Forced air cooling

U.S. Patent Apr. 2, 2013 Sheet 45 of 57 US 8,409,807 B2

Figure 45B U.S. Patent Apr. 2, 2013 Sheet 46 of 57 US 8,409,807 B2

Figure 46

Figure 46A

8 day repeatibility (mean +f- 95% CI) 200

1000 800 600 400 200

00 101 iO2 103 04 105 106 C. albicans (CFU/mL whole blood) Figure 46B U.S. Patent Apr. 2, 2013 Sheet 47 of 57 US 8,409,807 B2

Figure 47

Workflow Summary: Aliquot 800 uL whole blood

Aliquot 50 ul of lysate Hypotonic lysis buffer into 50 unmaster mix + glass beads Remove supernatant

Measure T2

Add superparamagnetic Nanoparticles (can be added in closed tube format), incubate 20-30 min U.S. Patent Apr. 2, 2013 Sheet 48 of 57 US 8,409,807 B2

Figure 48

200 1000

note: do not detect at OCF for 37% of samples

00 10 02 103 104 105 C. albicans (CFU/mL whole blood) Figure 48A

100 Ol 02 103 104 105 C. krusei (CFU/mL whole blood) Figure 48B U.S. Patent Apr. 2, 2013 Sheet 49 of 57 US 8,409,807 B2

Figure 49

U.S. Patent Apr. 2, 2013 Sheet 50 of 57 US 8,409,807 B2

Figure 50

-- Sensitivity 0.2 --- dentity 0.0- S 9 S 1 - Specificity

Figure 50A

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Figure 50B U.S. Patent Apr. 2, 2013 Sheet 51 of 57 US 8,409,807 B2

Figure 51

200 OOO g 800 3. E 600 N 400 X standard thermocycle 200 X combined anneal/elongation

100 O1 102 103 104 105 C. albicans (CFU/mL whole blood) U.S. Patent Apr. 2, 2013 Sheet 52 of 57 US 8,409,807 B2

Figure 52

450

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250 -

200 -

50 -

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Figure 53

U.S. Patent Apr. 2, 2013 Sheet 54 Of 57 US 8,409,807 B2

Figure 54

U.S. Patent Apr. 2, 2013 Sheet 55 of 57 US 8,409,807 B2

Figures 55A-55D

Figure 55E U.S. Patent Apr. 2, 2013 Sheet 56 of 57 US 8,409,807 B2

Salia. afous Fieldsition laget Side Orientation Magnet Mounts

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Figure 56A

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Figure 56B U.S. Patent Apr. 2, 2013 Sheet 57 Of 57 US 8,409,807 B2

Figure 57

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US 8,409,807 B2 1. 2 NMR SYSTEMIS AND METHODS FOR THE tion device Suitable for use with magnetic nanosensors having RAPID DETECTION OF ANALYTES four unique features and qualities: 1) little to no sample prepa ration, 2) multiplex detection across multiple molecular CROSS-REFERENCE TO RELATED types, 3) rapid acquisition of diagnostic information, and 4) APPLICATIONS accurate information for point-of-care clinical decision mak 1ng. This application is a continuation of U.S. application Ser. No. 13/384,051, filed Jan. 13, 2012, a U.S. national phase SUMMARY OF THE INVENTION entry of International Application No. PCT/US2011/56936, filed Oct. 19, 2011, which is a continuation-in-part of U.S. 10 The invention features systems and methods for the detec application Ser. No. 12/910,594, filed Oct. 22, 2010, and tion of analytes. which claims benefit of U.S. Provisional Patent Application The invention features a method for detecting the presence No. 61/414,141, filed Nov. 16, 2010, U.S. Provisional Patent of an analyte in a liquid sample, the method including: (a) Application No. 61/418,465, filed Dec. 1, 2010, and U.S. contacting a solution with magnetic particles to produce a Provisional Patent Application No. 61/497,374, filed Jun. 15, 15 liquid sample including from 1x10° to 1x10" magnetic par 2011, each of which is incorporated herein by reference. ticles per milliliter of the liquid sample (e.g., from 1x10° to 1x10, 1x107 to 1x10, 1x107 to 1x10, 1x10 to 1x10', BACKGROUND OF THE INVENTION 1x10 to 1x10', or 1x10' to 1x10" magnetic particles per milliliter), wherein the magnetic particles have a mean diam This invention features assays and devices for the detection eter of from 150 nm to 699 nm (e.g., from 150 to 250, 200 to of analytes, and their use in the treatment and diagnosis of 350, 250 to 450, 300 to 500, 450 to 650, or from 500 to 699 disease. nm), a T, relaxivity per particle of from 1x10 to 1x10' Magnetic sensors have been designed to detect molecular mM's (e.g., from 1x10 to 1x10, 1x10 to 1x10, 1x10 interactions in a variety of media, including biofluids, food to 1x10', 1x10 to 1x10'', or from 1x10' to 1x10' products, and soil samples, among other media. Upon target 25 mM's'), and binding moieties on their surface, the binding binding, these sensors cause changes in properties of neigh moieties operative to alter aggregation of the magnetic par boring water molecules (or any solvent molecule with free ticles in the presence of the analyte or a multivalent binding hydrogens) of a sample, which can be detected by magnetic agent; (b) placing the liquid sample in a device, the device resonance (NMR/MRI) techniques. Thus, by using these sen including a Support defining a well holding the liquid sample sors in a liquid sample, it is possible to detect the presence, 30 including the magnetic particles, the multivalent binding and potentially quantify the amount, of an analyte at very low agent, and the analyte, and having an RF coil disposed about concentration. For example, small molecules, DNA, RNA, the well, the RF coil configured to detect a signal produced by proteins, carbohydrates, organisms, metabolites, and patho exposing the liquid sample to a bias magnetic field created gens (e.g., viruses) can be detected using magnetic sensors. using one or more magnets and an RF pulse sequence; (c) In general, magnetic sensors are magnetic particles that 35 exposing the sample to a bias magnetic field and an RF pulse bind or otherwise link to their intended molecular target to sequence; (d) following step (c), measuring the signal; and (e) form clusters (aggregates). It is believed that when magnetic on the basis of the result of step (d), detecting the analyte. In particles assemble into clusters and the effective cross sec certain embodiments, the magnetic particles are substantially tional area becomes larger (and the cluster number density is monodisperse; exhibit nonspecific reversibility in the smaller), the interactions with the water or other solvent mol 40 absence of the analyte and multivalent binding agent; and/or ecules are altered, leading to a change in the measured relax the magnetic particles further include a Surface decorated ation rates (e.g., T. T. T.), susceptibility, frequency of with a blocking agent selected from albumin, fish skingelatin, precession, among other physical changes. Additionally, gamma globulin, lysozyme, casein, peptidase, and an amine cluster formation can be designed to be reversible (e.g., by bearing moiety (e.g., amino polyethyleneglycol, glycine, eth temperature shift, chemical cleavage, pH shift, etc.) so that 45 ylenediamine, or amino dextran). In particular embodiments, “forward” or “reverse' (competitive and inhibition) assays the liquid sample further includes a buffer, from 0.1% to 3% can be developed for detection of specific analytes. Forward (w/w) albumin (e.g., from 0.1% to 0.5%, 0.3% to 0.7%, 0.5% (clustering) and reverse (declustering) types of assays can be to 1%, 0.8% to 2%, or from 1.5% to 3% (w/w) albumin), from used to detect a wide variety of biologically relevant materi 0.01% to 0.5% nonionic surfactant (e.g., from 0.01% to als. The MRS (magnetic resonance Switch) phenomenon was 50 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to previously described (see U.S. Patent Publication No. 0.4%, or from 0.3% to 0.5% nonionic surfactant), or a com 20090029392). bination thereof. In still other embodiments, the magnetic Many diagnostic assays require sensitivity in the picomo particles include a Surface decorated with 40 ug to 100 g lar or Subpicomolar range. In such assays an equally low (e.g., 40 ug to 60 ug, 50g to 70 g. 60 g to 80 ug, or 80 g concentration of paramagnetic particles is employed. As a 55 to 100 g.) of one or more proteins per milligram of the result, the binding events leading to cluster formation can magnetic particles. The liquid sample can include a multiva become a rate-limiting step in the completion of the assay as lent binding agent bearing a plurality of analytes conjugated the collision frequency of antigens, paramagnetic particles, to a polymeric scaffold. For example, the analyte can be and partially formed clusters is low in this concentration creatinine, the liquid sample can include a multivalent bind range (see Baudry et al., Proc Natl AcadSci USA, 103: 16076 60 ing agent bearing a plurality of creatinine conjugates, and the (2006)). The current detection of infectious agents, nucleic magnetic particles can include a Surface decorated with crea acids, Small molecules, biowarfare agents and organisms, and tinine antibodies. In another embodiment, the analyte can be molecular targets (biomarkers) or the combination of tacrolimus, the liquid sample can include a multivalent bind molecular and immunoassay targets usually requires up-front ing agent bearing a plurality oftacrolimus conjugates, and the sample preparation, time to analyze the sample, and single 65 magnetic particles can include a Surface decorated with tac tests for each of the individual analytes. There is a need for a rolimus antibodies. In particular embodiments of the method, rapid, commercially-realizable NMR-based analyte detec step (d) includes measuring the T relaxation response of the US 8,409,807 B2 3 4 liquid sample, and wherein increasing agglomeration in the including: (a) providing a whole blood sample from a Subject; liquid sample produces an increase in the observed T. relax (b) mixing the whole blood sample with an erythrocyte lysis ation rate of the sample. In certain embodiments, the analyte agent solution to produce disrupted red blood cells; (c) fol is a target nucleic acid (e.g., a target nucleic acid extracted lowing step (b), centrifuging the sample to form a Supernatant from a leukocyte, or a pathogen). 5 and a pellet, discarding some or all of the Supernatant, and The invention features a method for detecting the presence resuspending the pelletto forman extract, optionally washing of an analyte in a liquid sample, the method including (a) the pellet (e.g., with TE buffer) prior to resuspending the contacting a solution with magnetic particles to produce a pellet and optionally repeating step (c); (d) lysing cells of the liquid sample including from 1x10° to 1x10" magnetic par extract to form a lysate; (e) placing the lysate of step (d) in a ticles per milliliter of the liquid sample (e.g., from 1x10° to 10 detection tube and amplifying a target nucleic acid in the 1x10, 1x107 to 1x10, 1x107 to 1x10, 1x10 to 1x10', lysate to forman amplified lysate solution including the target 1x10 to 1x10'', or 1x10' to 1x10' magnetic particles per nucleic acid, wherein the target nucleic acid is characteristic milliliter), wherein the magnetic particles have a mean diam of the pathogen to be detected, (f) following step (e), adding eter of from 700 nm to 1200 nm (e.g., from 700 to 850, 800 to to the detection tube from 1x10° to 1x10" magnetic particles 950, 900 to 1050, or from 1000 to 1200 nm), a T. relaxivity 15 permilliliter of the amplified lysate solution (e.g., from 1x10' per particle of from 1x10 to 1x10' (e.g., from 1x10 to to 1x10, 1x107 to 1x10, 1x107 to 1x10, 1x10 to 1x109, 1x10', 1x10 to 1x10'', or from 1x10' to 1x10"? 1x10 to 1x10', or 1x10' to 1x10" magnetic particles per mM's'), and have binding moieties on their surface, the milliliter), wherein the magnetic particles have a mean diam binding moieties operative to alteran aggregation of the mag eter of from 700 nm to 1200 nm (e.g., from 700 to 850, 800 to netic particles in the presence of the analyte; (b) placing the 950, 900 to 1050, or from 1000 to 1200 nm), and binding liquid sample in a device, the device including a Support moieties on their surface, the binding moieties operative to defining a well holding the liquid sample including the mag alter aggregation of the magnetic particles in the presence of netic particles, the multivalent binding agent, and the analyte, the target nucleic acid or a multivalent binding agent; (g) and having an RF coil disposed about the well, the RF coil placing the detection tube in a device, the device including a configured to detect a signal produced by exposing the liquid 25 support defining a well for holding the detection tube includ sample to a bias magnetic field created using one or more ing the magnetic particles and the target nucleic acid, and magnets and an RF pulse sequence; (c) exposing the sample having an RF coil disposed about the well, the RF coil con to a bias magnetic field and an RF pulse sequence; (d) fol figured to detect a signal produced by exposing the liquid lowing step (c), measuring the signal; and (e) on the basis of sample to a bias magnetic field created using one or more the result of step (d), detecting the presence or concentration 30 magnets and an RF pulse sequence; (h) exposing the sample of an analyte. In certain embodiments, the magnetic particles to a bias magnetic field and an RF pulse sequence: (i) follow are substantially monodisperse; exhibit nonspecific revers ing step (h), measuring the signal from the detection tube; and ibility in the absence of the analyte and multivalent binding () on the basis of the result of step (i), detecting the pathogen. agent; and/or the magnetic particles further include a Surface In certain embodiments, steps (a) through (i) are completed decorated with a blocking agent selected from albumin, fish 35 within 4 hours (e.g., within 3.5 hours, 3.0 hours, 2.5 hours, 2 skin gelatin, gamma globulin, lysozyme, casein, peptidase, hours, 1.5 hours, or 1 hour). In another embodiment, step (i) and an amine-bearing moiety (e.g., amino polyethylenegly is carried out without any prior purification of the amplified col, glycine, ethylenediamine, or amino dextran). In particu lysate Solution (i.e., the lysate Solution is unfractionated after lar embodiments, the liquid sample further includes a buffer, it is formed). In particular embodiments, step c includes from 0.1% to 3% (w/w) albumin (e.g., from 0.1% to 0.5%, 40 washing the pellet prior to resuspending the pellet to form the 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or from 1.5% to 3% extract. In particular embodiments step (d) includes combin (w/w) albumin), from 0.01% to 0.5% nonionic surfactant ing the extract with beads to form a mixture and agitating the (e.g., from 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, mixture to form a lysate. The magnetic particles can include 0.1% to 0.3%, 0.2% to 0.4%, or from 0.3% to 0.5% nonionic one or more populations having a first probe and a second surfactant), or a combination thereof. In still other embodi 45 probe conjugated to their Surface, the first probe operative to ments, the magnetic particles include a Surface decorated bind to a first segment of the target nucleic acid and the second with 40 ug to 100 ug (e.g., 40 ug to 60 Lig, 50 ug to 70 ug, 60 probe operative to bind to a second segment of the target ug to 80 ug, or 80 g to 100 ug.) of one or more proteins per nucleic acid, wherein the magnetic particles form aggregates milligram of the magnetic particles. The liquid sample can in the presence of the target nucleic acid. Alternatively, the include a multivalent binding agent bearing a plurality of 50 assay can be a disaggregation assay in which the magnetic analytes conjugated to a polymeric scaffold. For example, the particles include a first population having a first binding moi analyte can be creatinine, the liquid sample can include a ety on their surface and a second population having a second multivalent binding agent bearing a plurality of creatinine binding moiety on their surface, and the multivalent binding conjugates, and the magnetic particles can include a Surface moiety including a first probe and a second probe, the first decorated with creatinine antibodies. In another embodiment, 55 probe operative to bind to the first binding moiety and the the analyte can be tacrolimus, the liquid sample can include a second probe operative to bind to a second binding moiety, multivalent binding agent bearing a plurality of tacrolimus the binding moieties and multivalent binding moiety opera conjugates, and the magnetic particles can include a Surface tive to alter an aggregation of the magnetic particles in the decorated with tacrolimus antibodies. In particular embodi presence of the target nucleic acid. In certain embodiments, ments of the method, step (d) includes measuring the T 60 the magnetic particles are Substantially monodisperse; relaxation response of the liquid sample, and wherein increas exhibit nonspecific reversibility in the absence of the analyte ing agglomeration in the liquid sample produces an increase and multivalent binding agent; and/or the magnetic particles in the observed T. relaxation rate of the sample. In certain further include a surface decorated with a blocking agent embodiments, the analyte is a target nucleic acid (e.g., a target selected from albumin, fish skin gelatin, gamma globulin, nucleic acid extracted from a leukocyte, or a pathogen). 65 lysozyme, casein, peptidase, and an amine-bearing moiety The invention further features a method for detecting the (e.g., amino polyethyleneglycol, glycine, ethylenediamine, presence of a pathogen in a whole blood sample, the method or amino dextran). In particular embodiments, the lysate fur US 8,409,807 B2 5 6 ther includes a buffer, from 0.1% to 3% (w/w) albumin (e.g., 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to from 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, 0.4%, or from 0.3% to 0.5% nonionic surfactant), or a com or from 1.5% to 3% (w/w) albumin), from 0.01% to 0.5% bination thereof. In still other embodiments, the magnetic nonionic surfactant (e.g., from 0.01% to 0.05%, 0.05% to particles optionally include a surface decorated with 40 ug to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or from 100 Lug (e.g., 40 ug to 60 ug, 50 ug to 70 g. 60 g to 80 ug, or 0.3% to 0.5% nonionic surfactant), or a combination thereof. 80 ug to 100 ug.) of one or more proteins per milligram of the In still other embodiments, the magnetic particles include a magnetic particles. The lysate can include a multivalent bind Surface decorated with 40 ug to 100 Lug (e.g., 40 ug to 60 ug, ing agent bearing a plurality of analytes conjugated to a 50 ug to 70 ug, 60 ug to 80 ug, or 80 ug to 100 g.) of one or polymeric scaffold. more proteins per milligram of the magnetic particles. The 10 The invention further features a method for detecting the lysate can include a multivalent binding agent bearing a plu presence of a target nucleic acid in a whole blood sample, the rality of analytes conjugated to a polymeric scaffold. method including: (a) providing an extract produced by lys The invention features a method for detecting the presence ing the red blood cells in a whole blood sample from a subject, of a target nucleic acid in a whole blood sample, the method centrifuging the sample to form a Supernatant and a pellet, including: (a) providing one or more cells from a whole blood 15 discarding some or all of the Supernatant, and resuspending sample from a subject; (b) lysing the cells to form a lysate; (c) the pellet to form an extract, optionally washing the pellet amplifying a target nucleic acid in the lysate to form an (e.g., with TE buffer) prior to resuspending the pellet and amplified lysate Solution comprising the target nucleic acid; optionally repeating the centrifuging, discarding, and wash (d) following step (c), adding to a detection tube the amplified ing of step (a); (b) lysing cells in the extract to form a lysate; lysate solution and from 1x10° to 1x10" magnetic particles (c) placing the lysate of step (b) in a detection tube and per milliliter of the amplified lysate solution, wherein the amplifying nucleic acids therein to form an amplified lysate magnetic particles have a mean diameter of from 700 nm to solution including from 40% (w/w) to 95% (w/w) the target 1200 nm and binding moieties on their surface, the binding nucleic acid (e.g., from 40 to 60%, from 60 to 80%, or from 85 moieties operative to alter aggregation of the magnetic par to 95% (w/w) target nucleic acid) and from 5% (w/w) to 60% ticles in the presence of the target nucleic acid or a multivalent 25 (w/w) nontarget nucleic acid (e.g., from 5 to 20%, from 20 to binding agent; (e) placing the detection tube in a device, the 40%, or from 40 to 60% (w/w) nontarget nucleic acid); (d) device including a Support defining a well for holding the following step (c), adding to the detection tube from 1x10° to detection tube including the magnetic particles and the target 1x10" magnetic particles permilliliter of the amplified lysate nucleic acid, and having an RF coil disposed about the well, Solution, wherein the magnetic particles have a mean diam the RF coil configured to detect a signal produced by expos 30 eter of from 700 nm to 1200 nm and binding moieties on their ing the liquid sample to a bias magnetic field created using Surface, the binding moieties operative to alter aggregation of one or more magnets and an RF pulse sequence; (f) exposing the magnetic particles in the presence of the target nucleic the sample to a bias magnetic field and an RF pulse sequence; acid or a multivalent binding agent; (e) placing the detection (h) following step (f), measuring the signal from the detection tube inadevice, the device including a Support defining a well tube; and (i) on the basis of the result of step (h), detecting the 35 for holding the detection tube including the magnetic par target nucleic acid. In particular embodiments, the target ticles and the target nucleic acid, and having an RF coil nucleic acid is purified prior to step (d). In particular embodi disposed about the well, the RF coil configured to detect a ments, step (b) includes combining the extract with beads to signal produced by exposing the liquid sample to a bias mag form a mixture and agitating the mixture to form a lysate. The netic field created using one or more magnets and an RF pulse magnetic particles can include one or more populations hav 40 sequence; (f) exposing the sample to a bias magnetic field and ing a first probe and a second probe conjugated to their Sur an RF pulse sequence; (g) following step (f), measuring the face, the first probe operative to bind to a first segment of the signal from the detection tube; and (h) on the basis of the target nucleic acid and the second probe operative to bind to result of step (g), detecting the target nucleic acid, wherein a second segment of the target nucleic acid, wherein the step (g) is carried out without any prior purification of the magnetic particles form aggregates in the presence of the 45 amplified lysate Solution. In particular embodiments, step (b) target nucleic acid. Alternatively, the assay can be a disaggre includes combining the extract with beads to form a mixture gation assay in which the magnetic particles include a first and agitating the mixture to form a lysate. The magnetic population having a first binding moiety on their surface and particles can include one or more populations having a first a second population having a second binding moiety on their probe and a second probe conjugated to their surface, the first Surface, and the multivalent binding moiety including a first 50 probe operative to bind to a first segment of the target nucleic probe and a second probe, the first probe operative to bind to acid and the second probe operative to bind to a second the first binding moiety and the second probe operative to segment of the target nucleic acid, wherein the magnetic bind to a second binding moiety, the binding moieties and particles form aggregates in the presence of the target nucleic multivalent binding moiety operative to alter an aggregation acid. Alternatively, the assay can be a disaggregation assay in of the magnetic particles in the presence of the target nucleic 55 which the magnetic particles include a first population having acid. In certain embodiments, the magnetic particles are Sub a first binding moiety on their surface and a second population stantially monodisperse; exhibit nonspecific reversibility in having a second binding moiety on their surface, and the the absence of the analyte and multivalent binding agent; multivalent binding moiety including a first probe and a sec and/or the magnetic particles further include a surface deco ond probe, the first probe operative to bind to the first binding rated with a blocking agent selected from albumin, fish skin 60 moiety and the second probe operative to bind to a second gelatin, gamma globulin, lysozyme, casein, peptidase, and an binding moiety, the binding moieties and multivalent binding amine-bearing moiety (e.g., amino polyethyleneglycol, gly moiety operative to alter an aggregation of the magnetic par cine, ethylenediamine, or amino dextran). In particular ticles in the presence of the target nucleic acid. In certain embodiments, the lysate further includes a buffer, from 0.1% embodiments, the magnetic particles are Substantially mono to 3% (w/w) albumin (e.g., from 0.1% to 0.5%, 0.3% to 0.7%, 65 disperse; exhibit nonspecific reversibility in the absence of 0.5% to 1%, 0.8% to 2%, or from 1.5% to 3% (w/w) albumin), the analyte and multivalent binding agent; and/or the mag from 0.01% to 0.5% nonionic surfactant (e.g., from 0.01% to netic particles further include a surface decorated with a US 8,409,807 B2 7 8 blocking agent selected from albumin, fish skin gelatin, agent bearing a plurality of analytes conjugated to a poly gamma globulin, lysozyme, casein, peptidase, and an amine meric scaffold. The forward primer can include, for example, bearing moiety (e.g., amino polyethyleneglycol, glycine, eth the sequence 5'-GGCATG CCT GTTTGAGCGTC-3' (SEQ ylenediamine, or amino dextran). In particular embodiments, ID NO. 1). The reverse primer can include, for example, the the lysate further includes a buffer, from 0.1% to 3% (w/w) sequence 5'-GCTTATTGATAT GCT TAAGTT CAG CGG albumin (e.g., from 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%. GT-3' (SEQID NO. 2). In certain embodiments, (i) the Can 0.8% to 2%, or from 1.5% to 3% (w/w) albumin), from 0.01% dida species is Candida albicans, the first probe includes the to 0.5% nonionic surfactant (e.g., from 0.01% to 0.05%, oligonucleotide sequence 5'-ACC CAG CGG TTT GAG 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, GGA GAA AC-3' (SEQ ID NO. 3), and the second probe or from 0.3% to 0.5% nonionic surfactant), or a combination 10 thereof. In still other embodiments, the magnetic particles includes the oligonucleotide sequence 5'-AAA GTTTGA include a surface decorated with 40 ug to 100 ug (e.g., 40 ug AGATATACGTGGTGG ACGTTA-3' (SEQID NO.4); (ii) to 60 ug, 50 ug to 70 ug, 60 ug to 80 ug, or 80 ug to 100 ug.) the Candida species is Candida krusei and the first probe and of one or more proteins per milligram of the magnetic par the second probe include an oligonucleotide sequence Selected from: 5'-CGC ACG CGC AAG ATG GAA ACG-3' ticles. The lysate can include a multivalent binding agent 15 bearing a plurality of analytes conjugated to a polymeric (SEQ ID NO. 5), 5'-AAGTTC AGC GGG TAT TCC TAC scaffold. CT-3' (SEQ ID NO. 6), and 5'-AGC TTTTTG TTG TCT The invention features a method for detecting the presence CGC AAC ACT CGC-3 (SEQID NO. 32); (iii) the Candida of a Candida species in a liquid sample, the method includ species is Candida glabrata, the first probe includes the oli ing: (a) lysing the Candida cells in the liquid sample; (b) gonucleotide sequence: 5'-CTA CCAAACACAATG TGT amplifying a nucleic acid to be detected in the presence of a TTG. AGA AG-3 (SEQ ID NO. 7), and the second probe forward primer and a reverse primer, each of which is univer includes the oligonucleotide sequence: 5'-CCT GAT TTG sal to multiple Candida species to form a solution including a AGGTCA AACTTA AAG ACGTCT G-3' (SEQID NO. 8): Candida amplicon; (c) contacting the solution with magnetic and (iv) the Candida species is Candida parapsilosis or Can particles to produce a liquid sample including from 1x10' to 25 dida tropicalis and the first probe and the second probe 1x10" magnetic particles per milliliter of the liquid sample include an oligonucleotide sequence selected from: 5'-AGT (e.g., from 1x10° to 1x10, 1x107 to 1x10, 1x107 to 1x10, CCT ACC TGA TTT GAG GTCNitIndAA-3' (SEQ ID NO. 1x10 to 1x10, 1x10 to 1x10'', or 1x10' to 1x10' mag 9), 5'-CCGNitIndGGGTTTGAGGGAGAAAT-3' (SEQID netic particles per milliliter), wherein the magnetic particles NO. 10), AAA GTT ATG AAATAAATT GTG GTG GCC have a mean diameter of from 700 nm to 1200 nm (e.g., from 30 700 to 850, 800 to 950, 900 to 1050, or from 1000 to 1200 ACT AGC (SEQ ID NO. 33), ACC CGGGGGTTT GAG nm), a T, relaxivity per particle of from 1x10 to 1x10' GGAGAAA (SEQID NO. 34), AGT CCT ACC TGATTT mM's (e.g., from 1x10 to 1x10, 1x10 to 1x10", 1x10 GAG GTC GAA (SEQ ID NO. 35), and CCG AGGGTT to 1x10', 1x10 to 1x10', or from 1x10' to 1x10'’ TGA GGG AGA AAT (SEQID NO. 36). In certain embodi mM's'), and binding moieties on their surface, the binding 35 ments, steps (a) through (h) are completed within 4 hours moieties operative to alter aggregation of the magnetic par (e.g., within 3.5 hours, 3.0 hours, 2.5 hours, 2 hours, 1.5 ticles in the presence of the Candida amplicon or a multiva hours, or 1 hour or less). In particular embodiments, the lent binding agent; (d) placing the liquid sample in a device, magnetic particles include two populations, a first population the device including a Support defining a well for holding the bearing the first probe on its Surface, and the second popula liquid sample including the magnetic particles and the Can 40 tion bearing the second probe on its surface. In another dida amplicon, and having an RF coil disposed about the well, embodiment, the magnetic particles are a single population the RF coil configured to detect a signal produced by expos bearing both the first probe and the second probe on the ing the liquid sample to a bias magnetic field created using Surface of the magnetic particles. The magnetic particles can one or more magnets and an RF pulse sequence; (e) exposing include one or more populations having a first probe and a the sample to a bias magnetic field and an RF pulse sequence; 45 second probe conjugated to their surface, the first probe (f) following step (e), measuring the signal; and (g) on the operative to bind to a first segment of the Candida amplicon basis of the result of step (f), determining whether the Can and the second probe operative to bind to a second segment of dida species was present in the sample. In certain embodi the Candida amplicon, wherein the magnetic particles form ments, the magnetic particles are Substantially monodisperse; aggregates in the presence of the target nucleic acid. Alterna exhibit nonspecific reversibility in the absence of the analyte 50 tively, the assay can be a disaggregation assay in which the and multivalent binding agent; and/or the magnetic particles magnetic particles include a first population having a first further include a surface decorated with a blocking agent binding moiety on their surface and a second population selected from albumin, fish skin gelatin, gamma globulin, having a second binding moiety on their surface, and the lysozyme, casein, peptidase, and an amine-bearing moiety multivalent binding moiety including a first probe and a sec (e.g., amino polyethyleneglycol, glycine, ethylenediamine, 55 ond probe, the first probe operative to bind to the first binding or amino dextran). In particular embodiments, the liquid moiety and the second probe operative to bind to a second sample further includes a buffer, from 0.1% to 3% (w/w) binding moiety, the binding moieties and multivalent binding albumin (e.g., from 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%. moiety operative to alter an aggregation of the magnetic par 0.8% to 2%, or from 1.5% to 3% (w/w) albumin), from 0.01% ticles in the presence of the Candida amplicon. In particular to 0.5% nonionic surfactant (e.g., from 0.01% to 0.05%, 60 embodiments, the method can produce (i) a coefficient of 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, variation in the T2 value of less than 20% on Candida positive or from 0.3% to 0.5% nonionic surfactant), or a combination samples; (ii) at least 95% correct detection at less than or thereof. In still other embodiments, the magnetic particles equal to 5 cells/mL in samples spiked into 50 individual include a surface decorated with 40 ug to 100 ug (e.g., 40 ug healthy patient blood samples; (iii) at least 95% correct detec to 60 ug, 50 ug to 70 ug, 60 ug to 80 ug, or 80 ug to 100 ug.) 65 tion less than or equal to 5 cells/mL in samples spiked into 50 of one or more proteins per milligram of the magnetic par individual unhealthy patient blood samples; and/or (iv) ticles. The liquid sample can include a multivalent binding greater than or equal to 80% correct detection in clinically US 8,409,807 B2 9 10 positive patient samples (i.e., Candida positive by another the extract to form a lysate; (e) placing the lysate of step (d) in technique, such as by cell culture) starting with 2 mL of a container and amplifying a target nucleic acid in the lysate blood. to form an amplified lysate solution including the target The invention features a method for detecting the presence nucleic acid, wherein the target nucleic acid is characteristic of a Candida species in a whole blood sample sample, the of the pathogen to be detected; (f) following step (e), mixing method including: (a) providing an extract produced by lys the amplified lysate solution with from 1x10° to 1x10" mag ing the red blood cells in a whole blood sample from a subject; netic particles per milliliter of the amplified lysate solution to (b) centrifuging the sample to form a Supernatant and a pellet, form a liquid sample (e.g., from 1x10° to 1x10, 1x107 to discarding some or all of the Supernatant; (c) washing the 1x10, 1x107 to 1x10, 1x10 to 1x10, 1x10 to 1x10'', or pellet (e.g., with TE buffer) by mixing the pellet with a buffer, 10 1x10" to 1x10" magnetic particles per milliliter), wherein agitating the sample (e.g., by Vortexing), centrifuging the the magnetic particles have a mean diameter of from 150 nm sample to form a Supernatant and a pellet, discarding some or to 1200 nm (e.g., from 150 to 250, 200 to 350,250 to 450,300 all of the Supernatant; (d) optionally repeating steps (b) and to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950,900 (c); (e) bead beating the pellet to form a lysate in the presence to 1050, or from 1000 to 1200 nm), a T. relaxivity perparticle of a buffer (e.g., TE buffer); (f) centrifuging the sample to 15 offrom 1x10 to 1x10'’ mM's' (e.g., from 1x10 to 1x10, form a Supernatant containing the lysate; (g) amplifying 1x10 to 1x10, 1x10 to 1x10, 1x10 to 1x10'', or from nucleic acids in the lysate of step (f) to form a Candida 1x10' to 1x10' mM's'), and binding moieties on their amplicon; and (h) detecting the presence of the Candida Surface, the binding moieties operative to alter aggregation of amplicon, wherein, the method can produce (i) at least 95% the magnetic particles in the presence of the target nucleic correct detection at less than or equal to 5 cells/mL in Samples acid or a multivalent binding agent, (g) placing the liquid spiked into 50 individual healthy patient blood samples; (ii) at sample in a device, the device including a Support defining a least 95% correct detection less than or equal to 5 cells/mL in well for holding the detection tube including the magnetic samples spiked into 50 individual unhealthy patient blood particles and the target nucleic acid, and having an RF coil samples; and/or (iii) greater than or equal to 80% correct disposed about the well, the RF coil configured to detect a detection in clinically positive patient samples (i.e., Candida 25 signal produced by exposing the liquid sample to a bias mag positive by cell culture) starting with 2 mL of blood at step (a). netic field created using one or more magnets and an RF pulse The invention features a method for detecting the presence sequence: (h) exposing the sample to a bias magnetic field and of a pathogen in a whole blood sample, the method including an RF pulse sequence: (i) following step (h), measuring the the steps of: (a) providing from 0.05 to 4.0 mL of the whole signal from the liquid sample; and () on the basis of the result blood sample (e.g., from 0.05 to 0.25, 0.25 to 0.5,0.25 to 0.75, 30 of step (i), detecting the pathogen, wherein the pathogen is 0.4 to 0.8, 0.5 to 0.75, 0.6 to 0.9, 0.65 to 1.25, 1.25 to 2.5, 2.5 selected from bacteria and fungi, and wherein the method is to 3.5, or 3.0 to 4.0 mL of whole blood); (b) placing an aliquot capable of detecting a pathogen concentration of 10 cells/mL of the sample of step (a) in a container and amplifying a target (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or nucleic acid in the sample to form an amplified Solution 50 cells/mL) in the whole blood sample. In certain embodi including the target nucleic acid, wherein the target nucleic 35 ments, steps (a) through (i) are completed within 3 hours (e.g., acid is characteristic of the pathogen to be detected; (c) plac within 3.2, 2.9, 2.7, 2.5, 2.3, 2.2, 2.1, 2.0, 19, 1.8, 1.7, 1.6, ing the amplified liquid sample in a detecting device; (d) on 1.5, or 1 or less hours). In still other embodiments, step (i) is the basis of the result of step (c), detecting the pathogen, carried out without any prior purification of the amplified wherein the pathogen is selected from bacteria and fungi, and lysate Solution, and/or the liquid sample of step (i) includes wherein the method is capable of detecting a pathogen con 40 whole blood proteins and non-target oligonucleotides. In cer centration of 10 cells/mL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, tain embodiments, the pathogen is selected from bacteria and 20, 25, 30, 35, 40, 45, or 50 cells/mL) in the whole blood fungi. The pathogen can be any bacterial or fungal pathogen sample. The detecting device can detect the pathogen via an described herein. In particular embodiments the method is optical, fluorescent, mass, density, magnetic, chromato capable of measuring a pathogen concentration of 10 cells/ graphic, and/or electrochemical measurement of the ampli 45 mL in the whole blood sample with a coefficient of variation fied liquid sample. In certain embodiments, steps (a) through of less than 15% (e.g., 10 cells/mL with a coefficient of (d) are completed within 3 hours (e.g., within 3.2, 2.9, 2.7, variation of less than 15%, 10%, 7.5%, or 5%; or 25 cells/mL 2.5, 2.3.2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, or 1.5 hours or 1 hour). with a coefficient of variation of less than 15%, 10%, 7.5%, or In still other embodiments, step (c) is carried out without any 5%; or 50 cells/mL with a coefficient of variation of less than prior purification of the amplified solution, and/or the liquid 50 15%, 10%, 7.5%, or 5%; or 100 cells/mL with a coefficient of sample of step (c) includes whole blood proteins and non variation of less than 15%, 10%, 7.5%, or 5%). In certain target oligonucleotides. In certain embodiments, the patho embodiments, the magnetic particles are Substantially mono gen is selected from bacteria and fungi. The pathogen can be disperse; exhibit nonspecific reversibility in the absence of any bacterial or fungal pathogen described herein. the analyte and multivalent binding agent; and/or the mag The invention also features a method for detecting the 55 netic particles further include a surface decorated with a presence of a pathogen in a whole blood sample, the method blocking agent selected from albumin, fish skin gelatin, including the steps of: (a) providing a whole blood sample gamma globulin, lysozyme, casein, peptidase, and an amine from a subject; (b) mixing from 0.05 to 4.0 mL of the whole bearing moiety (e.g., amino polyethyleneglycol, glycine, eth blood sample (e.g., from 0.05 to 0.25, 0.25 to 0.5,0.25 to 0.75, ylenediamine, or amino dextran). In particular embodiments, 0.4 to 0.8, 0.5 to 0.75, 0.6 to 0.9, 0.65 to 1.25, 1.25 to 2.5, 2.5 60 the liquid sample further includes a buffer, from 0.1% to 3% to 3.5, or 3.0 to 4.0 mL of whole blood) with an erythrocyte (w/w) albumin (e.g., from 0.1% to 0.5%, 0.3% to 0.7%, 0.5% lysis agent Solution to produce disrupted red blood cells; (c) to 1%, 0.8% to 2%, or from 1.5% to 3% (w/w) albumin), from following step (b), centrifuging the sample to form a Super 0.01% to 0.5% nonionic surfactant (e.g., from 0.01% to natant and a pellet, discarding some or all of the Supernatant, 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to and resuspending the pellet to form an extract, optionally 65 0.4%, or from 0.3% to 0.5% nonionic surfactant), or a com washing the pellet (e.g., with TE buffer) prior to resuspending bination thereof. In still other embodiments, the magnetic the pellet and optionally repeating step (c); (d) lysing cells of particles include a Surface decorated with 40 ug to 100 g US 8,409,807 B2 11 12 (e.g., 40 ug to 60 ug, 50g to 70 g. 60 ug to 80 ug, or 80 g magnetic particles are substantially monodisperse; exhibit to 100 ug.) of one or more proteins per milligram of the nonspecific reversibility in the absence of the analyte and magnetic particles. The liquid sample can include a multiva multivalent binding agent, and/or the magnetic particles fur lent binding agent bearing a plurality of analytes conjugated ther include a Surface decorated with a blocking agent to a polymeric scaffold. The method for monitoring can selected from albumin, fish skin gelatin, gamma globulin, include any of the magnetic assisted agglomeration methods lysozyme, casein, peptidase, and an amine-bearing moiety described herein. The magnetic particles can include one or (e.g., amino polyethyleneglycol, glycine, ethylenediamine, more populations having a first probe and a second probe or amino dextran). In particular embodiments, the liquid conjugated to their surface, the first probe operative to bind to sample further includes a buffer, from 0.1% to 3% (w/w) a first segment of the target nucleic acid and the second probe 10 operative to bind to a second segment of the target nucleic albumin (e.g., from 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%. acid, wherein the magnetic particles form aggregates in the 0.8% to 2%, or from 1.5% to 3% (w/w) albumin), from 0.01% presence of the target nucleic acid. Alternatively, the assay to 0.5% nonionic surfactant (e.g., from 0.01% to 0.05%, can be a disaggregation assay in which the magnetic particles 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, include a first population having a first binding moiety on 15 or from 0.3% to 0.5% nonionic surfactant), or a combination their surface and a second population having a second binding thereof. In still other embodiments, the magnetic particles moiety on their Surface, and the multivalent binding moiety include a surface decorated with 40 ug to 100 ug (e.g., 40 ug including a first probe and a second probe, the first probe to 60 ug, 50g to 70 ug. 60 g to 80 ug, or 80 ug to 100 ug.) operative to bind to the first binding moiety and the second of one or more proteins per milligram of the magnetic par probe operative to bind to a second binding moiety, the bind ticles. The liquid sample can include a multivalent binding ing moieties and multivalent binding moiety operative to alter agent bearing a plurality of analytes conjugated to a poly an aggregation of the magnetic particles in the presence of the meric scaffold. The method for monitoring can include any of target nucleic acid. the magnetic assisted agglomeration methods described The invention further features a method for detecting the herein. The magnetic particles can include one or more popu presence of a virus in a whole blood sample, the method 25 lations having a first probe and a second probe conjugated to including the steps of: (a) providing a plasma sample from a their surface, the first probe operative to bind to a first segment subject; (b) mixing from 0.05 to 4.0 mL of the plasma sample of the target nucleic acid and the second probe operative to (e.g., from 0.05 to 0.25, 0.25 to 0.5,0.25 to 0.75, 0.4 to 0.8,0.5 bind to a second segment of the target nucleic acid, wherein to 0.75, 0.6 to 0.9, 0.65 to 1.25, 1.25 to 2.5, 2.5 to 3.5, or 3.0 the magnetic particles form aggregates in the presence of the to 4.0 mL of whole blood) with a lysis agent to produce a 30 target nucleic acid. Alternatively, the assay can be a disaggre mixture comprising disrupted viruses; (c) placing the mixture gation assay in which the magnetic particles include a first of step (b) in a container and amplifying a target nucleic acid population having a first binding moiety on their surface and in the filtrate to form an amplified filtrate solution including a second population having a second binding moiety on their the target nucleic acid, wherein the target nucleic acid is Surface, and the multivalent binding moiety including a first characteristic of the virus to be detected; (d) following step 35 probe and a second probe, the first probe operative to bind to (c), mixing the amplified filtrate solution with from 1x10° to the first binding moiety and the second probe operative to 1x10" magnetic particles per milliliter of the amplified fil bind to a second binding moiety, the binding moieties and trate solution to form a liquid sample (e.g., from 1x10' to multivalent binding moiety operative to alter an aggregation 1x10, 1x107 to 1x10, 1x107 to 1x10, 1x10 to 1-10', of the magnetic particles in the presence of the target nucleic 1x10 to 1x10'', or 1x10' to 1x10' magnetic particles per 40 acid. milliliter), wherein the magnetic particles have a mean diam In any of the systems and methods of the invention in which eter of from 150 nm to 1200 nm (e.g., from 150 to 250, 200 to a PCR amplification is performed, the PCR method can be 350, 250 to 450, 300 to 500, 450 to 650, 500 to 700 nm, 700 real time PCR for quantifying the amount of a target nucleic to 850, 800 to 950,900 to 1050, or from 1000 to 1200 nm), a acid present in a sample. T. relaxivity per particle of from 1x10 to 1x10" mM's 45 The invention features a method of quantifying a target (e.g., from 1x10 to 1x10, 1x10 to 1x10', 1x10 to 1x10', nucleic acid molecule in a sample by amplifying the target 1x10 to 1x10'', or from 1x10' to 1x10 mM's), and nucleic acid molecule (e.g., using PCR or isothermal ampli binding moieties on their surface, the binding moieties opera fication) in an amplification reaction mixture in a detection tive to alter aggregation of the magnetic particles in the pres tube resulting in the production of amplicons corresponding ence of the target nucleic acid or a multivalent binding agent; 50 to the target nucleic acid molecule, wherein the amplification (e) placing the liquid sample in a device, the device including reaction mixture includes (1) a target nucleic acid molecule, a support defining a well for holding the detection tube (2) biotin labeled amplification primers specific for the target including the magnetic particles and the target nucleic acid, nucleic acid molecule, and (3) avidin labeled Superparamag and having an RF coil disposed about the well, the RF coil netic particles. In this method, the amplification is performed configured to detect a signal produced by exposing the liquid 55 in a device including a Support defining a well for holding the sample to a bias magnetic field created using one or more detection tube including the Superparamagnetic particles and magnets and an RF pulse sequence, (f) exposing the liquid the target nucleic acid molecule, and having an RF coil dis sample to a bias magnetic field and an RF pulse sequence; (g) posed about the well, the RF coil configured to detect a signal following step (f), measuring the signal from the liquid produced by exposing the sample to a bias magnetic field sample; and (h) on the basis of the result of step (g), detecting 60 created using one or more magnets and an RF pulse sequence. the virus, wherein the method is capable of detecting fewer In this method, the amplification includes the following steps: than 100 virus copies (e.g., fewer than 80, 70, 60, 50, 40, 30, (a) performing one or more cycles of amplification; 20, or 10 copies) in the whole blood sample. In certain (b) exposing the amplification reaction mixture to condi embodiments, steps (a) through (g) are completed within 3 tions permitting the aggregation or disaggregation of the hours (e.g., within 3.2, 2.9, 2.7, 2.5.2.3, 2.2.2.1.2.0, 1.9, 1.8, 65 avidin labeled Superparamagnetic particles, 1.7, 1.6, 1.5 hours, or 1 hour or less). The virus can be any (c) exposing the sample to a bias magnetic field and an RF viral pathogen described herein. In certain embodiments, the pulse sequence; US 8,409,807 B2 13 14 (d) following step (c), measuring the signal from the detec magnets and an RF pulse sequence. This amplification of this tion tube; method includes the following steps: (e) repeating steps (a)-(d) until a desired amount of ampli (a) performing one or more cycles of amplification; fication is obtained; and (b) exposing the amplification reaction mixture to condi (f) on the basis of the result of step (d), quantifying the 5 tions permitting the hybridization of the portion of the amplicons present at the corresponding cycle of ampli hairpin structure of (3) with the amplicons; fication. (c) exposing the sample to a bias magnetic field and an RF In this method, the initial quantity of target nucleic acid pulse sequence; molecule in the sample is determined based on the quantity of (d) following step (c), measuring the signal from the detec amplicons determined at each cycle of the PCR. 10 tion tube; The invention further features a method of quantifying a (e) repeating steps (a)-(d) until a desired amount of ampli target nucleic acid molecule in a sample by amplifying the fication is obtained; and target nucleic acid molecule (e.g., using PCR or isothermal (f) on the basis of the result of step (d), quantifying the amplification) in an amplification reaction mixture in a detec amplicons present at the corresponding cycle of ampli tion tube resulting in the production of amplicons corre 15 fication. sponding to the target nucleic acid molecule. In this method, In this method, the initial quantity of target nucleic acid the amplification reaction mixture includes (1) a target molecule in the sample is determined based on the quantity of nucleic acid molecule, (2) amplification primers including a amplicons determined at each cycle of the amplification. 5' overhang, wherein the amplification primers are specific The invention also features a method of quantifying a target for the target nucleic acid molecule, and (3) oligonucleotide nucleic acid molecule in a sample by amplifying the target labeled Superparamagnetic particles, wherein the oligonucle nucleic acid molecule using PCR in an amplification reaction otide label is substantially complementary to the 5' overhang mixture in a detection tube resulting in the production of of the amplification primers. In this method, the amplification amplicons corresponding to the target nucleic acid molecule. is performed in a device including a Support defining a well In this method, the amplification reaction mixture includes for holding the detection tube including the Superparamag 25 (1) a target nucleic acid molecule, (2) a polymerase with 5' netic particles and the target nucleic acid molecule, and hav exonuclease activity, (3) amplification primers specific for ing an RF coil disposed about the well, the RF coil configured the target nucleic acid molecule, and (4) oligonucleotide teth to detect a signal produced by exposing the sample to a bias ered Superparamagnetic particles, wherein the oligonucle magnetic field created using one or more magnets and an RF otide tether connects at least two Superparamagnetic particles pulse sequence. In this method, the amplification includes the 30 and the oligonucleotide tether is Substantially complemen following steps: tary to a portion of the nucleic acid sequence of the amplicons. (a) performing one or more cycles of amplification; In this method, the amplification is performed in a device (b) exposing the amplification reaction mixture to condi including a Support defining a well for holding the detection tions permitting the hybridization of the oligonucleotide tube including the Superparamagnetic particles and the target labeled superparamagnetic particles with the 5' over 35 nucleic acid molecule, and having an RF coil disposed about hang; the well, the RF coil configured to detect a signal produced by (c) exposing the sample to a bias magnetic field and an RF exposing the sample to a bias magnetic field created using one pulse sequence; or more magnets and an RF pulse sequence. The amplifica (d) following step (c), measuring the signal from the detec tion of this method includes the following steps: tion tube; 40 (a) performing one or more cycles of PCR under conditions (e) repeating steps (a)-(d) until a desired amount of ampli permitting the hybridization of the oligonucleotide fication is obtained; and tether to an amplicon during the extension phase of the (f) on the basis of the result of step (d), quantifying the PCR, wherein during the extension phase of the PCR, amplicons present at the corresponding cycle of ampli the 5' exonuclease activity of the polymerase untethers fication. 45 the at least two Superparamagnetic particles permitting a In this method, the initial quantity of target nucleic acid decrease in Superparamagnetic particle aggregation; molecule in the sample is determined based on the quantity of (b) exposing the sample to a bias magnetic field and an RF amplicons determined at each cycle of the amplification. pulse sequence; The invention further features a method of quantifying a (c) following step (b), measuring the signal from the detec target nucleic acid molecule in a sample by amplifying the 50 tion tube; target nucleic acid molecule (e.g., using PCR or isothermal (d) repeating steps (a)-(c) until the PCR is complete; and amplification) in an amplification reaction mixture in a detec (e) on the basis of the result of step (c), quantifying the tion tube resulting in the production of amplicons corre amplicons present at the corresponding cycle of PCR. sponding to the target nucleic acid molecule. In this method In this method, the initial quantity of target nucleic acid the amplification reaction mixture includes (1) a target 55 molecule in the sample is determined based on the quantity of nucleic acid molecule, (2) amplification primers specific for amplicons determined at each cycle of the PCR. the target nucleic acid molecule, and (3) oligonucleotide The invention also features a method of quantifying a target labeled Superparamagnetic particles, wherein the oligonucle nucleic acid molecule in a sample by amplifying the target otide label contains a hairpin structure and a portion of the nucleic acid molecule (e.g., using PCR or isothermal ampli hairpin structure is Substantially complementary to a portion 60 fication) in an amplification reaction mixture in a detection of the nucleic acid sequence of the amplicons. In this method, tube resulting in the production of amplicons corresponding the amplification is performed in a device including a Support to the target nucleic acid molecule. In this method, the ampli defining a well for holding the detection tube including the fication reaction mixture includes (1) a target nucleic acid Superparamagnetic particles and the target nucleic acid mol molecule, (2) amplification primers specific for the target ecule, and having an RF coil disposed about the well, the RF 65 nucleic acid molecule, and (3) Superparamagnetic particles coil configured to detect a signal produced by exposing the labeled with a plurality of oligonucleotides, wherein a first sample to a bias magnetic field created using one or more group of the plurality of oligonucleotides are substantially US 8,409,807 B2 15 16 complementary to a portion of the nucleic acid sequence of In any of the foregoing methods of quantifying a target the amplicons and Substantially complementary to a second nucleic acid molecule, the detection tube can remained sealed group of the plurality of oligonucleotides, wherein the first throughout the amplification reaction. The Superparamag group of the plurality of oligonucleotides has a lesser hybrid netic particles of these methods can be greater or less than 100 ization affinity for the second group of the plurality of oligo nm in diameter (e.g., 30 nm in diameter). nucleotides than for the amplicons. In this method, the ampli Also, in any of the foregoing methods of quantifying a fication is performed in a device including a Support defining target nucleic acid molecule, the methods can further include a well for holding the detection tube including the Superpara applying a magnetic field to the detection tube following the magnetic particles and the target nucleic acid molecule, and measuring the signal from the detection tube, resulting in the 10 sequestration of the Superparamagnetic particles to the side of having an RF coil disposed about the well, the RF coil con the detection tube, and releasing the magnetic field Subse figured to detect a signal produced by exposing the sample to quent to the completion of one or more additional cycles of a bias magnetic field created using one or more magnets and amplification. an RF pulse sequence. The amplification of this method Also, in any of the foregoing methods of quantifying a includes the following steps: 15 target nucleic acid molecule, the sample can, e.g., not include (a) performing one or more cycles of amplification; isolated nucleic acid molecules prior to step (a) (e.g., the (b) exposing the amplification reaction mixture to condi sample can be whole blood or not contain a target nucleic acid tions permitting the preferential hybridization of the first molecule prior to step (a)). group of the plurality of oligonucleotides with the The invention features a method of monitoring one or more amplicons thereby permitting disaggregation of the analytes in a liquid sample derived from a patient for the Superparamagnetic particles; diagnosis, management, or treatment of a medical condition (c) exposing the sample to a bias magnetic field and an RF in a patient, the method including (a) combining with the pulse sequence; liquid sample from 1x10° to 1x10" magnetic particles per (d) following step (c), measuring the signal from the detec milliliter of the liquid sample (e.g., from 1x10° to 1x10, tion tube; 25 1x107 to 1x10, 1x107 to 1x10, 1x10 to 1x10', 1x10 to (e) repeating steps (a)-(d) until a desired amount of ampli 1x10'', or 1x10' to 1x10' magnetic particles permilliliter), fication is obtained; and wherein the magnetic particles have a mean diameter of from (f) on the basis of the result of step (d); quantifying the 150 nm to 1200 nm (e.g., from 150 to 250, 200 to 350,250 to amplicons present at the corresponding cycle of ampli 450, 300 to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 fication. 30 to 950, 900 to 1050, or from 1000 to 1200 nm), and a T, In this method, the initial quantity of target nucleic acid relaxivity perparticle of from 1x10 to 1x10' mM's' (e.g., molecule in the sample is determined based on the quantity of from 1x10 to 1x10, 1x10 to 1x10', 1x10 to 1x10', amplicons determined at each cycle of the amplification. 1x10 to 1x10'', or from 1x10' to 1x10 mM's), and The invention further features a method of quantifying a wherein the magnetic particles have binding moieties on their target nucleic acid molecule in a sample by amplifying the 35 Surfaces, the binding moieties operative to alter the specific target nucleic acid molecule (e.g., using PCR or isothermal aggregation of the magnetic particles in the presence of the amplification) in an amplification reaction mixture in a detec one or more analytes or a multivalent binding agent; (b) tion tube resulting in the production of amplicons corre placing the liquid sample in a device, the device including a sponding to the target nucleic acid molecule. In this method, Support defining a well for holding the liquid sample includ the amplification reaction mixture includes (1) a target 40 ing the magnetic particles and the one or more analytes, and nucleic acid molecule, (2) amplification primers specific for having an RF coil disposed about the well, the RF coil con the target nucleic acid molecule, and (3) Superparamagnetic figured to detect a signal produced by exposing the liquid particles. In this method, the amplification is performed in a sample to a bias magnetic field created using one or more device including a Support defining a well for holding the magnets and an RF pulse sequence; (c) exposing the sample detection tube including the Superparamagnetic particles and 45 to the bias magnetic field and the RF pulse sequence; (d) the target nucleic acid molecule, and having an RF coil dis following step (c), measuring the signal; (e) on the basis of the posed about the well, the RF coil configured to detect a signal result of step (d), monitoring the one or more analytes; and (f) produced by exposing the sample to a bias magnetic field using the result of step (e) to diagnose, manage, or treat the created using one or more magnets and an RF pulse sequence. medical condition. In one embodiment, the one or more ana The amplification of this method including the following 50 lytes include creatinine. In another embodiment, the patientis steps: immunocompromised, and the one or more analytes include (a) performing one or more cycles of amplification; an analyte selected from pathogen-associated analytes, anti (b) exposing the amplification reaction mixture to condi biotic agents, antifungal agents, and antiviral agents (e.g., the tions permitting the aggregation or disaggregation of the one or more analytes can include Candida spp., tacrolimus, Superparamagnetic particles, 55 fluconazole, and/or creatinine). In still another embodiment, (c) exposing the sample to a bias magnetic field and an RF the patient has cancer, and the one or more analytes are pulse sequence; selected from anticancer agents, and genetic markers present (d) following step (c), measuring the signal from the detec in a cancer cell. The patient can have, or be at risk of an tion tube; infection, and the one or more analytes include an analyte (e) repeating steps (a)-(d) until a desired amount of ampli 60 selected from pathogen-associated analytes, antibiotic fication is obtained; and agents, antifungal agents, and antiviral agents. The patient (f) on the basis of the result of step (d), quantifying the can have an immunoinflammatory condition, and the one or amplicons present at the corresponding cycle of ampli more analytes include an analyte selected from antiinflam fication. matory agents and TNF-alpha. The patient can have heart In this method, the initial quantity of target nucleic acid 65 disease, and the one or more analytes can include a cardiac molecule in the sample is determined based on the quantity of marker. The patient can have HIV/AIDS, and the one or more amplicons determined at each cycle of the amplification. analytes can include CD3, viral load, and AZT. In certain US 8,409,807 B2 17 18 embodiments, the method is used to monitor the liver function the liquid sample from 1x10° to 1x10" magnetic particles per of the patient, and wherein the one or more analytes are milliliter of the liquid sample (e.g., from 1x10° to 1x10, selected from albumin, aspartate transaminase, alanine tran 1x107 to 1x10, 1x107 to 1x10, 1x10 to 1x10', 1x10 to saminase, alkaline phosphatase, gamma glutamyl transpepti 1x10', or 1x10' to 1x10" magnetic particles permilliliter), dase, bilirubin, alpha fetoprotein, lactase dehydrogenase, wherein the magnetic particles have a mean diameter of from mitochondrial antibodies, and cytochrome P450. For 150 nm to 1200 nm (e.g., from 150 to 250, 200 to 350,250 to example, the one or more analytes include cytochrome P450 450, 300 to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 polymorphisms, and the ability of the patient to metabolize a to 950, 900 to 1050, or from 1000 to 1200 nm), and a T. drug is evaluated. The method can include identifying the relaxivity perparticle of from 1x10 to 1x10' mM's' (e.g., patient as a poor metabolizer, a normal metabolizer, an inter 10 from 1x10 to 1x10, 1x10 to 1x10', 1x10 to 1x10', mediate metabolizer, or an ultra rapid metabolizer. The 1x10 to 1x10'', or from 1x10' to 1x10 mM's'), and method can be used to determine an appropriate dose of a wherein the magnetic particles have binding moieties on their therapeutic agent in a patient by (i) administering the thera Surfaces, the binding moieties operative to alter the specific peutic agent to the patient; (ii) following step (i), obtaining a aggregation of the magnetic particles in the presence of one or sample including the therapeutic agent or metabolite thereof 15 more analytes characteristic of sepsis selected from GRO from the patient; (iii) contacting the sample with the magnetic alpha, High mobility group-box 1 protein (HMGB-1), IL-1 particles and exposing the sample to the bias magnetic field receptor, IL-1 receptor antagonist, IL-1b, IL-2, IL-4, IL-6, and the RF pulse sequence and detecting a signal produced by IL-8, IL-10, IL-12, IL-13, IL-18, macrophage inflammatory the sample; and (iv) on the basis of the result of step (iii), protein (MIP-1), macrophage migration inhibitory factor determining the concentration of the therapeutic agent or (MIF), osteopontin, RANTES (regulated on activation, nor metabolite thereof. The therapeutic agent can be an antican mal T-cell expressed and secreted; or CCL5), TNF-C, C-re cer agent, antibiotic agent, antifungal agent, or any therapeu active protein (CRP), CD64, monocyte chemotactic protein 1 tic agent described herein. In any of the above methods of (MCP-1), adenosine deaminase binding protein (ABP-26), monitoring, the monitoring can be intermittent (e.g., peri inducible nitric oxide synthetase (iNOS), lipopolysaccharide odic), or continuous. In certain embodiments, the magnetic 25 binding protein, and procalcitonin; (d) placing each assay particles are substantially monodisperse; exhibit nonspecific sample in a device, the device including a Support defining a reversibility in the absence of the analyte and multivalent well for holding the liquid sample including the magnetic binding agent; and/or the magnetic particles further include a particles and the one or more analytes, and having an RF coil Surface decorated with a blocking agent selected from albu disposed about the well, the RF coil configured to detect a min, fish skin gelatin, gamma globulin, lysozyme, casein, 30 signal produced by exposing the liquid sample to a bias mag peptidase, and an amine-bearing moiety (e.g., amino polyeth netic field created using one or more magnets and an RF pulse yleneglycol, glycine, ethylenediamine, or amino dextran). In sequence; (e) exposing each assay Sample to the bias mag particular embodiments, the liquid sample further includes a netic field and the RF pulse sequence: (f) following step (e), buffer, from 0.1% to 3% (w/w) albumin (e.g., from 0.1% to measuring the signal produced by the first assay sample and 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or from 1.5% 35 the signal produced by the second assay sample; (g) on the to 3% (w/w) albumin), from 0.01% to 0.5% nonionic surfac basis of the result of step (f), monitoring the one or more tant (e.g., from 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to analytes of the first assay sample and monitoring the one or 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or from 0.3% to 0.5% more analytes of the second assay Sample; and (h) using the nonionic Surfactant), or a combination thereof. In still other results of step (g) to diagnose the Subject. In one embodiment, embodiments, the magnetic particles include a surface deco 40 the one or more pathogen-associated analytes of the first rated with 40 ug to 100 Lug (e.g., 40 ug to 60 ug, 50 ug to 70 ug, assay sample are derived from a pathogen associated with 60 g to 80 ug, or 80 g to 100g.) of one or more proteins per sepsis selected from Acinetobacter baumannii, Aspergillus milligram of the magnetic particles. The liquid sample can filmigatis, Bacteroides fragilis, B. fragilis, blaSHV, include a multivalent binding agent bearing a plurality of Burkholderia cepacia, Campylobacter jejuni/coli, Candida analytes conjugated to a polymeric scaffold. The method for 45 guilliermondii, C. albicans, C. glabrata, C. krusei, C. lusita monitoring can include any of the magnetic assisted agglom niae, C. parapsilosis, C. tropicalis, Clostridium pefringens, eration methods described herein. Coagulase negative Staph, Enterobacter aeraogenes, E. cloa The invention features a method of diagnosing sepsis in a cae, Enterobacteriaceae, Enterococcus faecalis, E. faecium, Subject, the method including (a) obtaining a liquid sample Escherichia coli, Haemophilus influenzae, Kingella Kingae, derived from the blood of a patient; (b) preparing a first assay 50 Klebsiella Oxytoca, K. pneumoniae, Listeria monocytogenes, sample by combining with a portion of the liquid sample from Mec A gene (MRSA), Morganella morgana, Neisseria men 1x10° to 1x10" magnetic particles per milliliter of the liquid ingitidis, Neisseria spp. non-meningitidis, PrevOtella buccae, sample (e.g., from 1x10° to 1x10, 1x107 to 1x10, 1x107 to P intermedia, P. melaminogenica, Propionibacterium acnes, 1x10, 1x10 to 1x10, 1x10 to 1x10'', or 1x100 to 1x10' Proteus mirabilis, P. vulgaris, Pseudomonas aeruginosa, Sal magnetic particles per milliliter), wherein the magnetic par 55 monella enterica, Serratia marcescens, Staphylococcus ticles have a mean diameter of from 150 nm to 1200 nm (e.g., aureus, S. haemolyticus, S. maltophilia, S. saprophyticus, from 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to Stenotrophomonas maltophilia, S. maltophilia, Streptococ 650, 500 to 700 nm, 700 to 850, 800 to 950, 900 to 1050, or cus agalactie, S. bovis, S. dysgalactie, S. mitis, S. mutans, S. from 1000 to 1200 nm), and a T. relaxivity per particle of pneumoniae, S. pyogenes, and S. sanguinis. The one or more from 1x10 to 1x10' mM's (e.g., from 1x10 to 1x10, 60 pathogen-associated analytes can be derived from treatment 1x10 to 1x10, 1x10 to 1x10, 1x10 to 1x10'', or from resistant strains of bacteria, Such as penicillin-resistant, 1x10" to 1x10" mM's'), and wherein the magnetic par methicillin-resistant, quinolone-resistant, macrollide-resis ticles have binding moieties on their Surfaces, the binding tant, and/or Vancomycin-resistant bacterial strains (e.g., moieties operative to alter the specific aggregation of the methicillin resistant Staphylococcus aureus or Vancomycin magnetic particles in the presence of one or more pathogen 65 resistant enterococci). In certain embodiments, the one or associated analytes or a multivalent binding agent; (c) pre more analytes of the second assay Sample are selected from paring a second assay sample by combining with a portion of GRO-alpha, High mobility group-box 1 protein (HMGB-1), US 8,409,807 B2 19 20 IL-1 receptor, IL-1 receptor antagonist, IL-1b, IL-2, IL-4, include (i) monitoring a pathogen-associated analyte, and (ii) IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, macrophage inflam monitoring a second analyte characteristic of sepsis selected matory protein (MIP-1), macrophage migration inhibitory from GRO-alpha, High mobility group-box 1 protein factor (MIF), osteopontin, RANTES (regulated on activation, (HMGB-1), IL-1 receptor, IL-1 receptor antagonist, IL-1b, normal T-cell expressed and secreted; or CCL5), TNF-C. IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, macroph C-reactive protein (CRP), CD64, and monocyte chemotactic age inflammatory protein (MIP-1), macrophage migration protein 1 (MCP-1). In a particular embodiment, the method inhibitory factor (MIF), osteopontin, RANTES (regulated on further includes preparing a third assay sample to monitor the activation, normal T-cell expressed and secreted; or CCL5), concentration of an antiviral agent, antibiotic agent, or anti TNF-O, C-reactive protein (CRP), CD64, monocyte chemo fungal agent circulating in the blood stream of the Subject. In 10 tactic protein 1 (MCP-1), adenosine deaminase binding pro certain embodiments, the Subject can be an immunocompro tein (ABP-26), inducible nitric oxide synthetase (iNOS), mised Subject, or a subject at risk of becoming immunocom lipopolysaccharide binding protein, and procalcitonin. In cer promised. In any of the above methods of monitoring, the tain embodiments, the pathogen-associated analyte is derived monitoring can be intermittent (e.g., periodic), or continuous. from a pathogen associated with sepsis selected from Acine In certain embodiments, the magnetic particles are Substan 15 tobacter baumannii, Aspergillus fumigatis, Bacteroides fra tially monodisperse; exhibit nonspecific reversibility in the gilis, B. fragilis, blaSHV. Burkholderia cepacia, Campylo absence of the analyte and multivalent binding agent; and/or bacter jejuni/coli, Candida guilliermondii, C. albicans, C. the magnetic particles further include a surface decorated glabrata, C. krusei, C. Lusitaniae, C. parapsilosis, C. tropi with a blocking agent selected from albumin, fish skingelatin, calis, Clostridium pefringens, Coagulase negative Staph, gamma globulin, lysozyme, casein, peptidase, and an amine Enterobacter aeraogenes, E. cloacae, Enterobacteriaceae, bearing moiety (e.g., amino polyethyleneglycol, glycine, eth Enterococcus faecalis, E. faecium, Escherichia coli, Haemo ylenediamine, or amino dextran). In particular embodiments, philus influenzae, Kingella Kingae, Klebsiella Oxytoca, K. the liquid sample further includes a buffer, from 0.1% to 3% pneumoniae, Listeria monocytogenes, Mec A gene (MRSA), (w/w) albumin (e.g., from 0.1% to 0.5%, 0.3% to 0.7%, 0.5% Morganella morgana, Neisseria meningitidis, Neisseria spp. to 1%, 0.8% to 2%, or from 1.5% to 3% (w/w) albumin), from 25 non-meningitidis, PrevOtella buccae, P intermedia, P 0.01% to 0.5% nonionic surfactant (e.g., from 0.01% to melaminogenica, Propionibacterium acnes, Proteus mirabi 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to lis, P. vulgaris, Pseudomonas aeruginosa, Salmonella 0.4%, or from 0.3% to 0.5% nonionic surfactant), or a com enterica, Serratia marcescens, Staphylococcus aureus, S. bination thereof. In still other embodiments, the magnetic haemolyticus, S. maltophilia, S. saprophyticus, Stenotroph particles include a Surface decorated with 40 ug to 100 ug 30 Omonas maltophilia, S. maltophilia, Streptococcus agalactie, (e.g., 40 ug to 60 ug, 50g to 70 g. 60 ug to 80 ug, or 80 g S. bovis, S. dysgalactie, S. mitis, S. mutans, S. pneumoniae, S. to 100 g) of one or more proteins per milligram of the pyogenes, and S. sanguinis. The pathogen-associated analyte magnetic particles. The liquid sample can include a multiva can be derived from a treatment resistant strain of bacteria, lent binding agent bearing a plurality of analytes conjugated Such as penicillin-resistant, methicillin-resistant, quinolone to a polymeric scaffold. The method for monitoring can 35 resistant, macrollide-resistant, and/or Vancomycin-resistant include any of the magnetic assisted agglomeration methods bacterial strains (e.g., methicillin resistant Staphylococcus described herein. aureus or Vancomycin-resistant enterococci). In particular The invention further features a method of monitoring one embodiments, the second analytes is selected from GRO or more analytes in a liquid sample derived from a patient for alpha, High mobility group-box 1 protein (HMGB-1), IL-1 the diagnosis, management, or treatment of sepsis or SIRS in 40 receptor, IL-1 receptor antagonist, IL-1b, IL-2, IL-4, IL-6, a patient, the method including: (a) combining with the liquid IL-8, IL-10, IL-12, IL-13, IL-18, macrophage inflammatory sample from 1x10° to 1x10" magnetic particles permilliliter protein (MIP-1), macrophage migration inhibitory factor of the liquid sample (e.g., from 1x10° to 1x10, 1x107 to (MIF), osteopontin, RANTES (regulated on activation, nor 1x10, 1x107 to 1x10, 1x10 to 1x10', 1x10 to 1x10'', or mal T-cell expressed and secreted; or CCL5), TNF-C, C-re 1x10' to 1x10" magnetic particles per milliliter), wherein 45 active protein (CRP), CD64, and monocyte chemotactic pro the magnetic particles have a mean diameter of from 150 nm tein 1 (MCP-1). In a particular embodiment, the method to 1200 nm (e.g., from 150 to 250,200 to 350,250 to 450,300 further includes preparing a third assay sample to monitor the to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950,900 concentration of an antiviral agent, antibiotic agent, or anti to 1050, or from 1000 to 1200 nm), and a T, relaxivity per fungal agent circulating in the blood stream of the Subject. In particle of from 1x10 to 1x10' mM's' (e.g., from 1x10 50 certain embodiments, the Subject can be an immunocompro to 1x10, 1x10 to 1x10', 1x10 to 1x10', 1x10 to 1x10'', mised Subject, or a subject at risk of becoming immunocom or from 1x10' to 1x10'’ mM's), and wherein the mag promised. In any of the above methods of monitoring, the netic particles have binding moieties on their surfaces, the monitoring can be intermittent (e.g., periodic), or continuous. binding moieties operative to alter the specific aggregation of In certain embodiments, the magnetic particles are Substan the magnetic particles in the presence of the one or more 55 tially monodisperse; exhibit nonspecific reversibility in the analytes or a multivalent binding agent; (b) placing the liquid absence of the analyte and multivalent binding agent; and/or sample in a device, the device including a Support defining a the magnetic particles further include a Surface decorated well for holding the liquid sample including the magnetic with a blocking agent selected from albumin, fish skingelatin, particles and the one or more analytes, and having an RF coil gamma globulin, lysozyme, casein, peptidase, and an amine disposed about the well, the RF coil configured to detect a 60 bearing moiety (e.g., amino polyethyleneglycol, glycine, eth signal produced by exposing the liquid sample to a bias mag ylenediamine, or amino dextran). In particular embodiments, netic field created using one or more magnets and an RF pulse the liquid sample further includes a buffer, from 0.1% to 3% sequence; (c) exposing the sample to the bias magnetic field (w/w) albumin (e.g., from 0.1% to 0.5%, 0.3% to 0.7%, 0.5% and the RF pulse sequence; (d) following step (c), measuring to 1%, 0.8% to 2%, or from 1.5% to 3% (w/w) albumin), from the signal; (e) on the basis of the result of step (d), monitoring 65 0.01% to 0.5% nonionic surfactant (e.g., from 0.01% to the one or more analytes; and (f) using the result of step (e) to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to diagnose, manage, or treat the sepsis or SIRS. The method can 0.4%, or from 0.3% to 0.5% nonionic surfactant), or a com US 8,409,807 B2 21 22 bination thereof. In still other embodiments, the magnetic 0.0333 Hz, or less (e.g., from 0.000833 Hz to 0.0333 Hz, from particles include a Surface decorated with 40 ug to 100 ug 0.00166 Hz to 0.0333 Hz, or from 0.00333 Hz to 0.0333 Hz). (e.g., 40 ug to 60 ug, 50g to 70 g. 60 ug to 80 ug, or 80 g In other embodiments, the method further includes (iii) fol to 100 ug.) of one or more proteins per milligram of the lowing step (ii), agitating the liquid sample; and (iv) repeating magnetic particles. The liquid sample can include a multiva step (ii). lent binding agent bearing a plurality of analytes conjugated In any of the above methods for assisting specific agglom to a polymeric scaffold. The method for monitoring can eration step (ii) can be repeated from 1 to 100 times (e.g., include any of the magnetic assisted agglomeration methods repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, from 10 to 20 described herein. times, or from 80 to 100 times). In particular embodiments, In a related aspect, the invention features a method for 10 the one or more magnets providing the magnetic field gradi assisting the specific agglomeration of magnetic particles in a ent within the liquid sample have a maximum field strength of liquid sample, the method including: (i) providing a liquid from 0.01 T to 10 T (e.g., from 0.01 T to 0.05T, 0.05 T to 0.1 sample including one or more analytes and the magnetic T, 0.1T to 0.5 T, 0.5 T to 1 T, 1 T to 3 T, or from 3 T to 10 T) particles, wherein the magnetic particles have binding moi and wherein the gradient magnetic field varies from 0.1 eties on their Surfaces, the binding moieties operative to alter 15 mT/mm to 10 T/mm across the liquid sample (e.g., from 0.1 the specific aggregation of the magnetic particles in the pres mT/mm to 0.5 mT/mm, 0.3 mT/mm to 1 mT/mm, 0.5 mT/mm. ence of the one or more analytes or a multivalent binding to 5 mT/mm, 5 mT/mm to 20 mT/mm, 10 mT/mm to 100 agent; (ii) exposing the liquid sample to a magnetic field; (iii) mT/mm, 100 mT/mm to 500 mT/mm, 500 mT/mm to 1 removing the liquid sample from the magnetic field; and (iv) T/mm, or from 1 T/mm to 10 T/mm). In certain embodiments repeating step (ii). of any of the above methods for assisting specific agglomera The invention further features a method for assisting the tion, step (ii) includes applying the magnetic field gradient to specific agglomeration of magnetic particles in a liquid the liquid sample for a period of from 1 second to 5 minutes sample by (i) providing a liquid sample including one or more (e.g., from 1 to 20 seconds, from 20 to 60 seconds, from 30 analytes and the magnetic particles, wherein the magnetic seconds to 2 minutes, from 1 minutes to 3 minutes, or from 2 particles have binding moieties on their Surfaces, the binding 25 minutes to 5 minutes). In particular embodiments, (i) the moieties operative to alter the specific aggregation of the liquid sample includes from 1x10 to 1x10" of the one or magnetic particles in the presence of the one or more analytes more analytes per milliliter of the liquid sample (e.g., from or a multivalent binding agent; (ii) applying a magnetic field 1x10 to 1x10, 1x10 to 1x10, 1x107 to 1x10, 1x10 to gradient to the liquid sample for a time sufficient to cause 1x10', 1x10 to 1x10', or 1x10' to 1x10' analytes per concentration of the magnetic particles in a first portion of the 30 milliliter); (ii) the liquid sample includes from 1x10° to liquid sample, the magnetic field gradient being aligned in a 1x10" of the magnetic particles per milliliter of the liquid first direction relative to the liquid sample; (iii) following step sample (e.g., from 1x10° to 1x10, 1x107 to 1x10, 1x10 to (ii), applying a magnetic field to the liquid sample for a time 1x10, 1x10 to 1x10, 1x10 to 1x10'', or 1x100 to 1x10' Sufficient to cause concentration of the magnetic particles in magnetic particles per milliliter); (iii) the magnetic particles a second portion of the liquid sample, the magnetic field being 35 have a T. relaxivity per particle of from 1x10 to 1x10' aligned in a second direction relative to the liquid sample; and mM's' (e.g., from 1x10" to 1x107, 1x10 to 1x10", 1x107 (iv) optionally repeating steps (ii) and (iii). In certain embodi to 1x10, 1x10 to 1x10, 1x10 to 1x10', 1x10 to 1x10', ments, the angle between the first direction and the second 1x10 to 1x10', or from 1x10' to 1x10 mM.'s"); (iv) the direction relative to the liquid sample is between 0° and 180° magnetic particles have an average diameter of from 150 nm (e.g., from 0° to 10°, 5° to 120°, 20° to 60°, 30° to 80°, 45° to 40 to 1200 nm (e.g., from 150 to 250, 200 to 350,250 to 450,300 90°, 60° to 120°, 80° to 135°, or from 120° to 180°). to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950,900 The invention features a method for assisting the specific to 1050, or from 1000 to 1200 nm); (v) the magnetic particles agglomeration of magnetic particles in a liquid sample by (i) are substantially monodisperse; (vi) the magnetic particles in providing a liquid sample including one or more analytes and the liquid sample exhibit nonspecific reversibility in the the magnetic particles, wherein the magnetic particles have 45 absence of the one or more analytes and multivalent binding binding moieties on their surfaces, the binding moieties agent; (vii) the magnetic particles further include a Surface operative to alter the specific aggregation of the magnetic decorated with a blocking agent selected from albumin, fish particles in the presence of the one or more analytes or a skin gelatin, gamma globulin, lysozyme, casein, peptidase, multivalent binding agent; (ii) applying a magnetic field gra and an amine-bearing moiety (e.g., amino polyethylenegly dient to the liquid sample for a time sufficient to cause con 50 col, glycine, ethylenediamine, or amino dextran); (viii) the centration of the magnetic particles in a first portion of the liquid sample further includes a buffer, from 0.1% to 3% liquid sample; (iii) following step (ii), agitating the liquid (w/w) albumin (e.g., from 0.1% to 0.5%, 0.3% to 0.7%, 0.5% sample; and (iv) repeating step (ii). In certain embodiments, to 1%, 0.8% to 2%, or from 1.5% to 3% (w/w) albumin), from step (iii) includes Vortexing the liquid sample, or mixing the 0.01% to 0.5% nonionic surfactant (e.g., from 0.01% to sample using any method described herein. 55 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to The invention also features a method for assisting the spe 0.4%, or from 0.3% to 0.5% nonionic surfactant), or a com cific agglomeration of magnetic particles in a liquid sample bination thereof, and/or (ix) the magnetic particles include a by (i) providing a liquid sample including one or more ana Surface decorated with 40 ug to 100 Lug (e.g., 40 ug to 60 ug, lytes and the magnetic particles, wherein the magnetic par 50 ug to 70 g. 60 ug to 80 ug, or 80 ug to 100 g.) of one or ticles have binding moieties on their Surfaces, the binding 60 more proteins per milligram of the magnetic particles. moieties operative to alter the specific aggregation of the The invention features a system for the detection of one or magnetic particles in the presence of the one or more analytes more analytes, the system including: (a) a first unit including or a multivalent binding agent; and (ii) exposing the liquid (a1) a permanent magnet defining a magnetic field: (a2) a sample to a gradient magnetic field and rotating the gradient Support defining a well for holding a liquid sample including magnetic field about the sample, or rotating the sample within 65 magnetic particles and the one or more analytes and having an the gradient magnetic field. The sample can be rotated slowly. RF coil disposed about the well, the RF coil configured to In certain embodiments, the sample is rotated at a rate of detect a signal by exposing the liquid sample to a bias homog US 8,409,807 B2 23 24 enous magnetic field created using the permanent magnet and including magnetic particles and the one or more analytes and an RF pulse sequence; and (a3) one or more electrical ele having an RF coil disposed about the well, the RF coil con ments in communication with the RF coil, the electrical ele figured to detect a signal produced by exposing the liquid ments configured to amplify, rectify, transmit, and/or digitize sample to a bias magnetic field created using the permanent the signal; and (b) one or more second units including (b1) a magnet and an RF pulse sequence; and (a3) one or more permanent magnet adjacent a first sample position for holding electrical elements in communication with the RF coil, the a liquid sample and configured to apply a first gradient mag electrical elements configured to amplify, rectify, transmit, netic field to the liquid sample. The one or more second units and/or digitize the signal; and (b) a second unit including a can further include a second permanent magnet adjacent a removable cartridge sized to facilitate insertion into and second sample position for holding a liquid sample and con 10 removal from the system, wherein the removable cartridge is figured to apply a second gradient magnetic field to the liquid a modular cartridge including (i) a reagent module for hold sample, the second magnetic field aligned to apply a gradient ing one or more assay reagents; and (ii) a detection module magnetic field to the sample from a direction different from including a detection chamber for holding a liquid sample the direction of the first field gradient, and a means for moving including the magnetic particles and the one or more analytes, a liquid sample from the first sample position to the second 15 wherein the reagent module and the detection module can be sample position. In certain embodiments, the one or more assembled into the modular cartridge prior to use, and second units is incapable of measuring a signal (e.g., inca wherein the detection chamber is removable from the modu pable of measuring an NMR relaxation rate), and/or lacks an lar cartridge. The modular cartridge can further include an RF coil, or a means for producing an RF pulse. In certain inlet module, wherein the inlet module, the reagent module, embodiments, the angle between the first direction and the and the detection module can be assembled into the modular second direction relative to the liquid sample is between 0° cartridge prior to use, and wherein the inlet module is steril and 180° (e.g., from 0° to 10°, 5° to 120°, 20° to 60°, 30° to izable. In certain embodiments, the system further includes a 80°, 45° to 90°, 60° to 120°, 80° to 135°, or from 120° to system computer with processor for implementing an assay 180°). The system can further include a sample holder for protocol and storing assay data, and wherein the removable holding the liquid sample and configured to move the liquid 25 cartridge further includes (i) a readable label indicating the sample from the first position to the second position. In par analyte to be detected, (ii) a readable label indicating the ticular embodiments, the system includes an array of the one assay protocol to be implemented, (iii) a readable label indi or more second units for assisting the agglomeration of an cating a patient identification number, (iv) a readable label array of samples simultaneously. For example, the array can indicating the position of assay reagents contained in the be configured to rotate one or more liquid from a first position 30 cartridge, or (v) a readable label including instructions for the in which a magnetic field is applied to the side of a sample to programmable processor. The system can include a cartridge a second position in which a magnetic field is applied to the unit, an agitation unit, a centrifuge, or any other System com bottom of a sample. The system can include a cartridge unit, ponent described herein. an agitation unit, a centrifuge, or any other system component The invention features an agitation unit for the automated described herein. For example, the system can further include 35 mixing of a liquid sample in a sample chamber, including a (c) a third unit including a removable cartridge sized to facili motor for providing a rotational driving force to a motor shaft tate insertion into and removal from the system and having a coupled to a drive shaft, the driveshaft having a first end compartment including one or more populations of magnetic coupled to the motor shaft and a second end coupled to a plate particles having binding moieties on their surfaces, wherein bearing a sample holder for holding the sample chamber, the the binding moieties are operative to alter an aggregation of 40 draft shaft including a first axis coaxial to the motor shaft, and the magnetic particles in the presence of the one or more a secondaxis that is offset and parallel to the motor shaft, Such analytes. In particular embodiments, the removable cartridge that the second axis of the driveshaft, the plate, and the sample is a modular cartridge including (i) a reagent module for holder are driven in an orbital path, wherein the motor holding one or more assay reagents; and (ii) a detection mod includes an index mark and/or other position sensing means ule including a detection chamber for holding a liquid sample 45 Such as an optical, magnetic or resistive position encoder for including magnetic particles and one or more analytes, positioning the sample chamber in a predetermined position wherein the reagent module and the detection module can be following the mixing or a sensor which tracks the sample's assembled into the modular cartridge prior to use, and position throughout its path. wherein the detection chamber is removable from the modu The invention features a system for the detection of one or lar cartridge. The modular cartridge can further include an 50 more analytes, the system including: (a) a first unit including inlet module, wherein the inlet module, the reagent module, (a1) a permanent magnet defining a magnetic field: (a2) a and the detection module can be assembled into the modular Support defining a well for holding a liquid sample including cartridge prior to use, and wherein the inlet module is steril magnetic particles and the one or more analytes and having an izable. In another embodiment, the system can further include RF coil disposed about the well, the RF coil configured to a system computer with processor for implementing an assay 55 detect a signal produced by exposing the liquid sample to a protocol and storing assay data, and wherein the removable bias magnetic field created using the permanent magnet and cartridge further includes (i) a readable label indicating the an RF pulse sequence; and (a3) one or more electrical ele analyte to be detected, (ii) a readable label indicating the ments in communication with the RF coil, the electrical ele assay protocol to be implemented, (iii) a readable label indi ments configured to amplify, rectify, transmit, and/or digitize cating a patient identification number, (iv) a readable label 60 the signal; and (b) a second unit for the automated mixing of indicating the position of assay reagents contained in the a liquid sample in a sample chamber, including a motor for cartridge, or (v) a readable label including instructions for the providing a rotational driving force to a motor shaft coupled programmable processor. to a drive shaft, the driveshaft having a first end coupled to the The invention further features a system for the detection of motor shaft and a second end coupled to a plate bearing a one or more analytes, the system including: (a) a first unit 65 sample holder for holding the sample chamber, the draft shaft including (al) a permanent magnet defining a magnetic field; including a first axis coaxial to the motor shaft, and a second (a2) a Support defining a well for holding a liquid sample axis that is offset and parallel to the motor shaft, such that the US 8,409,807 B2 25 26 second axis of the driveshaft, the plate, and the sample holder ity of detection module including a detection chamber for are driven in an orbital path, wherein the motor includes an holding a liquid sample including the magnetic particles and index mark and/or other position sensing means such as an the creatinine, tacrolimus, and Candida, wherein the plurality optical, magnetic or resistive position encoder for positioning of reagent modules includes (i) a first population of magnetic the sample chamberina predetermined position following the 5 particles having a mean diameter of from 150 nm to 699 nm mixing or a sensor which tracks the sample's position (e.g., from 150 to 250, 200 to 350,250 to 450,300 to 500, 450 throughout its path. to 650, or from 500 to 699 nm), a T. relaxivity per particle of In certain embodiments, the system further includes a from 1x10 to 1x10' mM's (e.g., from 1x10 to 1x10, robotic arm for placing the sample chamber in, and removing 1x10 to 1x10, 1x10 to 1x10, 1x10 to 1x10'', or from the sample chamber from, the agitation unit. 10 1x10' to 1x10' mM's'), and creatinine antibodies con The invention further features a system for the detection of jugated to their surface; (ii) a multivalent binding agent bear one or more analytes, the system including: (a) a first unit ing a plurality of creatinine conjugates designed to form including (al) a permanent magnet defining a magnetic field; aggregates with the first population of magnetic particles in (a2) a Support defining a well for holding a liquid sample the absence of creatinine; (iii) a second population of mag including magnetic particles and the one or more analytes and 15 netic particles having a mean diameter of from 150 nm to 699 having an RF coil disposed about the well, the RF coil con nm (e.g., from 150 to 250, 200 to 350,250 to 450,300 to 500, figured to detect a signal produced by exposing the liquid 450 to 650, or from 500 to 699 nm), a T. relaxivity perparticle sample to a bias magnetic field created using the permanent offrom 1x10 to 1x10'’ mM's' (e.g., from 1x10 to 1x10, magnet and an RF pulse sequence; and (a3) one or more 1x10 to 1x10, 1x10 to 1x10, 1x10 to 1x10'', or from electrical elements in communication with the RF coil, the 20 1x10' to 1x10' mM's'), and tacrolimus antibodies con electrical elements configured to amplify, rectify, transmit, jugated to their Surface; (iv) a multivalent binding agent bear and/or digitize the signal; and (b) a centrifuge including a ing a plurality of tacrolimus conjugates designed to form motor for providing a rotational driving force to a drive shaft, aggregates with the second population of magnetic particles the drive shaft having a first end coupled to the motor and a in the absence of tacrolimus; (v) a third population of mag second end coupled to a centrifuge rotor bearing a sample 25 netic particles have a mean diameter of from 700 nm to 1200 holder for holding the sample chamber, wherein the motor nm (e.g., from 700 to 850, 800 to 950, 900 to 1050, or from includes an index mark and/or other position sensing means 1000 to 1200 nm), a T, relaxivity per particle of from 1x10 Such as an optical, magnetic or resistive position encoder for to 1x10 mM.'s (e.g., from 1x10 to 1x10, 1x10 to positioning the sample chamber in a predetermined position 1x10', 1x10 to 1x10', 1x10 to 1x10'', or from 1x10' to following the centrifuging of the sample or a sensor which 30 1x10' mM's'), and having a first probe and a second probe tracks the sample's position throughout its path. conjugated to their Surface selected to form aggregates in the The invention further features a system for the detection of presence of a Candida nucleic acid, the first probe operative one or more analytes, the system including: (a) a disposable to bind to a first segment of the Candida nucleic acid and the sample holder defining a well for holding a liquid sample and second probe operative to bind to a second segment of the having an RF coil contained within the disposable sample 35 Candida nucleic acid. In certain embodiments, the magnetic holder and disposed about the well, the RF coil configured to particles are substantially monodisperse; exhibit nonspecific detect a signal produced by exposing the liquid sample to a reversibility in the absence of the analyte and multivalent bias magnetic field created using the permanent magnet and binding agent; and/or the magnetic particles further include a an RF pulse sequence, wherein the disposable sample holder Surface decorated with a blocking agent selected from albu includes one or more fusable links; and (b) an MR reader 40 min, fish skin gelatin, gamma globulin, lysozyme, casein, including (b1) a permanent magnet defining a magnetic field; peptidase, and an amine-bearing moiety (e.g., amino polyeth (b2) an RF pulse sequence and detection coil; (b3) one or yleneglycol, glycine, ethylenediamine, or amino dextran). In more electrical elements in communication with the RF coil, particular embodiments, the liquid sample further includes a the electrical elements configured to amplify, rectify, trans buffer, from 0.1% to 3% (w/w) albumin (e.g., from 0.1% to mit, and/or digitize the signal; and (b4) one or more electrical 45 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or from 1.5% elements in communication with the fusable link and config to 3% (w/w) albumin), from 0.01% to 0.5% nonionic surfac ured to apply excess current to the fusable link, causing the tant (e.g., from 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to link to break and rendering the coil inoperable following a 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or from 0.3% to 0.5% predetermined working lifetime. In certain embodiments, the nonionic Surfactant), or a combination thereof. In still other electrical element in communication with the RF coil is 50 embodiments, the magnetic particles include a Surface deco inductively coupled to the RF coil. rated with 40 ug to 100 Lug (e.g., 40 ug to 60 ug, 50 ug to 70 ug, The invention features a system for the detection of crea 60 ug to 80g, or 80 ug to 100 ug.) of one or more proteins per tinine, tacrolimus, and Candida, the system including: (a) a milligram of the magnetic particles. The liquid sample can first unit including (al) a permanent magnet defining a mag include a multivalent binding agent bearing a plurality of netic field: (a2) a Support defining a well for holding a liquid 55 analytes conjugated to a polymeric scaffold. In another sample including magnetic particles and the creatinine, tac embodiment, the liquid sample includes from 1x10° to rolimus, and Candida and having an RF coil disposed about 1x10" of the magnetic particles per milliliter of the liquid the well, the RF coil configured to detect signal produced by sample (e.g., from 1x10° to 1x10, 1x107 to 1x10, 1x107 to exposing the liquid sample to a bias magnetic field created 1x10, 1x10 to 1x10, 1x10 to 1x10'', or 1x100 to 1x10' using the permanent magnet and an RF pulse sequence; and 60 magnetic particles per milliliter). (a3) an electrical element in communication with the RF coil, The invention features a method for measuring the concen the electrical element configured to amplify, rectify, transmit, tration of creatinine in a liquid sample, the method including: and/or digitize the signal; and (b) a second unit including a (a) contacting a solution with (i) magnetic particles to pro removable cartridge sized to facilitate insertion into and duce a liquid sample including from 1x10' to 1x10" mag removal from the system, wherein the removable cartridge is 65 netic particles per milliliter of the liquid sample (e.g., from a modular cartridge including (i) a plurality of reagent mod 1x10 to 1x10, 1x107 to 1x10, 1x107 to 1x10, 1x10 to ules for holding one or more assay reagents; and (ii) a plural 1x10", 1x10 to 1x10', or 1x10' to 1x10" magnetic par US 8,409,807 B2 27 28 ticles per milliliter), wherein the magnetic particles have a -continued mean diameter of from 150 nm to 1200 nm (e.g., from 150 to 250,200 to 350,250 to 450,300 to 500, 450 to 650,500 to 700 nm, 700 to 850, 800 to 950,900 to 1050, or from 1000 to 1200 nm), a T. relaxivity per particle of from 1x10 to 1x10' mM's' (e.g., from 1x10 to 1x10, 1x10 to 1x10", 1x10 to 1x10', 1x10 to 1x10', or from 1x10' to 1x10'’ mM's'), and creatinine antibodies conjugated to their sur face, and (ii) a multivalent binding agent bearing a plurality of creatinine conjugates designed to form aggregates with the 10 magnetic particles in the absence of creatinine; (b) placing the liquid sample in a device, the device including a Support defining a well for holding the liquid sample including the magnetic particles, the multivalent binding agent, and the (B) is a polymeric scaffold covalently attached to each (A), m 15 is an integer from 2 to 10, and n is an integer from 2 to 50. creatinine, and having an RF coil disposed about the well, the The invention features a solution including from 1x10° to RF coil configured to detect a signal produced by exposing 1x10" magnetic particles per milliliter of the solution (e.g., the liquid sample to a bias magnetic field created using one or from 1x10 to 1x10, 1x107 to 1x10, 1x107 to 1x10, 1x10 more magnets and an RF pulse sequence; (c) exposing the to 1x10', 1x10 to 1x10'', or 1x10' to 1x10' magnetic sample to a bias magnetic field and an RF pulse sequence; (d) particles per milliliter), wherein the magnetic particles have a following step (c), measuring the signal; and (e) on the basis mean diameter of from 150 nm to 600 nm (e.g., from 150 to of the result of step (d), determining the concentration of 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, or from creatinine in the liquid sample. In certain embodiments, the 500 to 600 nm), a T. relaxivity per particle of from 1x10 to magnetic particles are substantially monodisperse; exhibit 1x10 mM's' (e.g., from 1x10 to 1x10, 1x10 to 1x10', nonspecific reversibility in the absence of the analyte and 25 1x10 to 1x10, 1x10 to 1x10'', or from 1x109 to 1x10'’ multivalent binding agent, and/or the magnetic particles fur mM's') and a surface bearing creatinine conjugate (A), ther include a Surface decorated with a blocking agent wherein (A) is selected from: selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidase, and an amine-bearing moiety (e.g., amino polyethyleneglycol, glycine, ethylenediamine, 30 or amino dextran). In particular embodiments, the liquid sample further includes a buffer, from 0.1% to 3% (w/w) albumin (e.g., from 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%. 0.8% to 2%, or from 1.5% to 3% (w/w) albumin), from 0.01% to 0.5% nonionic surfactant (e.g., from 0.01% to 0.05%, 35 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or from 0.3% to 0.5% nonionic surfactant), or a combination thereof. In still other embodiments, the magnetic particles include a surface decorated with 40 ug to 100 ug (e.g., 40 ug to 60 ug, 50 ug to 70 ug, 60 ug to 80 ug, or 80 ug to 100 ug.) 40 of one or more proteins per milligram of the magnetic par NY - - - ticles. The liquid sample can include a multivalent binding / / agent bearing a plurality of analytes conjugated to a poly O meric scaffold. The invention features a multivalent binding agent includ 45 ing two or more creatinine moieties covalently linked to a scaffold. In certain embodiments, the multivalent binding agent is a compound of formula (I):

(A)-(B) (I) 50 wherein (A) is

55 and m is an integer from 2 to 10. The invention further features solution including from 1x10° to 1x10" magnetic particles per milliliter of the solu tion (e.g., from 1x10° to 1x10, 1x107 to 1x10, 1x107 to 1x10, 1x10 to 1x10, 1x10 to 1x10'', or 1x100 to 1x10' 60 magnetic particles per milliliter), wherein the magnetic par ticles have a mean diameter of from 150 nm to 600 nm (e.g., from 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to O 650, or from 500 to 600 nm), a T. relaxivity per particle of from 1x10 to 1x10' mM's (e.g., from 1x10 to 1x10, 65 1x10 to 1x10', 1x10 to 1x10', 1x10 to 1x10'', or from 1x10' to 1x10' mM's'), and a surface bearing antibodies having affinity for the creatinine conjugate: US 8,409,807 B2 29 30 metabolites described herein or structurally similar com pounds for which the antibody has affinity covalently linked to a scaffold. In certain embodiments, the multivalent binding (B) agent is a compound of formula (II): (A)-(B) (II) wherein (A) is

HN N HO 10

/ HCO wherein (B) is a polymeric scaffold. The invention further features a method for measuring the 15 concentration of tacrolimus in a liquid sample, the method including: (a) contacting a solution with (i) magnetic particles to produce a liquid sample including from 1x10° to 1x10' magnetic particles per milliliter of the liquid sample (e.g., from 1x10° to 1x10, 1x107 to 1x10, 1x107 to 1x10, 1x10 to 1x10', 1x10 to 1x10'', or 1x10' to 1x10' magnetic particles per milliliter), wherein the magnetic particles have a mean diameter of from 150 nm to 1200 nm (e.g., from 150 to OCH OCH, 250,200 to 350,250 to 450,300 to 500, 450 to 650,500 to 700 25 nm, 700 to 850, 800 to 950,900 to 1050, or from 1000 to 1200 nm), a T, relaxivity per particle of from 1x10 to 1x10' (B) is a polymeric scaffold covalently attached to each (A), mM's (e.g., from 1x10 to 1x10, 1x10 to 1x10", 1x10 and n is an integer from 2 to 50. to 1x10', 1x10 to 1x10', or from 1x10' to 1x10'’ The invention features a solution including from 1x10° to mM's'), and tacrolimus antibodies conjugated to their sur 30 1x10" magnetic particles per milliliter of the solution (e.g., face, and (ii) a multivalent binding agent bearing a plurality of from 1x10 to 1x10, 1x107 to 1x10, 1x107 to 1x10, 1x10 tacrolimus conjugates designed to form aggregates with the to 1x10", 1x10 to 1x10', or 1x10' to 1x10" magnetic magnetic particles in the absence of tacrolimus; (b) placing particles per milliliter), wherein the magnetic particles have a the liquid sample in a device, the device including a Support mean diameter of from 150 nm to 600 nm (e.g., from 150 to defining a well for holding the liquid sample including the 35 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, or from magnetic particles, the multivalent binding agent, and the 500 to 600 nm), a T, relaxivity per particle of from 1x10 to tacrolimus, and having an RF coil disposed about the well, the 1x10 mM's' (e.g., from 1x10 to 1x10, 1x10 to 1x10', RF coil configured to detect a signal produced by exposing 1x10 to 1x10, 1x10 to 1x10'', or from 1x109 to 1x10'’ the liquid sample to a bias magnetic field created using one or mM's'), and a surface bearing antibodies having affinity more magnets and an RF pulse sequence; (c) exposing the 40 for the tacrolimus conjugate: sample to a bias magnetic field and an RF pulse sequence; (d) following step (c), measuring the signal; and (e) on the basis of the result of step (d), determining the concentration of tacrolimus in the liquid sample. In certain embodiments, the magnetic particles are substantially monodisperse; exhibit 45 (B), nonspecific reversibility in the absence of the analyte and multivalent binding agent, and/or the magnetic particles fur ther include a Surface decorated with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidase, and an amine-bearing moiety 50 (e.g., amino polyethyleneglycol, glycine, ethylenediamine, or amino dextran). In particular embodiments, the liquid sample further includes a buffer, from 0.1% to 3% (w/w) albumin (e.g., from 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%. 0.8% to 2%, or from 1.5% to 3% (w/w) albumin), from 0.01% 55 to 0.5% nonionic surfactant (e.g., from 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, oCH, or from 0.3% to 0.5% nonionic surfactant), or a combination OCH thereof. In still other embodiments, the magnetic particles include a surface decorated with 40 ug to 100 ug (e.g., 40 ug 60 wherein (B) is a polymeric scaffold. to 60 ug, 50 ug to 70 ug, 60 ug to 80 ug, or 80 ug to 100 ug.) In an embodiment of any of the above solutions, (i) the of one or more proteins per milligram of the magnetic par magnetic particles are substantially monodisperse; (ii) the ticles. The liquid sample can include a multivalent binding magnetic particles exhibit nonspecific reversibility in plasma; agent bearing a plurality of analytes conjugated to a poly (iii) the magnetic particles further include a Surface decorated meric scaffold. 65 with a blocking agent selected from albumin, fish skingelatin, The invention features a multivalent binding agent includ gamma globulin, lysozyme, casein, peptidase, and an amine ing two or more tacrolimus moieties, including tacrolimus bearing moiety (e.g., amino polyethyleneglycol, glycine, eth US 8,409,807 B2 31 32 ylenediamine, or amino dextran); (iv) the liquid sample fur to 1x10" mM's', and binding moieties on their surfaces ther includes a buffer, from 0.1% to 3% (w/w) albumin (e.g., (e.g., antibodies, conjugated analyte), the binding moieties from 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, operative to alter the specific aggregation of the magnetic or from 1.5% to 3% (w/w) albumin), from 0.01% to 0.5% particles in the presence of the one or more analytes or a nonionic surfactant (e.g., from 0.01% to 0.05%, 0.05% to multivalent binding agent; and (ii) a chamber for holding a 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or from buffer including from 0.1% to 3% (w/w) albumin (e.g., from 0.3% to 0.5% nonionic surfactant), or a combination thereof; 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or from and/or (iv) the magnetic particles include a Surface decorated 1.5% to 3% (w/w) albumin), from 0.01% to 0.5% nonionic with 40 ug to 100 ug (e.g., 40 ug to 60 Lig, 50 ug to 70 ug, 60 surfactant (e.g., from 0.01% to 0.05%, 0.05% to 0.1%, 0.05% ug to 80 ug, or 80 g to 100 ug.) of one or more proteins per 10 to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or from 0.3% to 0.5% milligram of the magnetic particles. The solutions can be used nonionic Surfactant), or a combination thereof. In one in any of the systems or methods described herein. embodiment, the magnetic particles and buffer are together in The invention features a removable cartridge sized to a single chamber within the cartridge. facilitate insertion into and removal from a system of the In any of the systems, kits, cartridges, and methods of the invention, wherein the removable cartridge includes one or 15 invention, the liquid sample can include from 1x10 to 1x10" more chambers for holding a plurality of reagent modules for magnetic particles having a mean diameter of from 100 nm to holding one or more assay reagents, wherein the reagent 350 nm, a T, relaxivity per particle of from 5x10 to 1x10' modules include (i) a chamber for holding from 1x10° to mM'S', and binding moieties on their surfaces (e.g., anti 1x10" magnetic particles (e.g., from 1x10 to 1x10, 1x107 bodies, conjugated analyte), the binding moieties operative to to 1x10, 1x107 to 1x10, 1x10 to 1x10', 1x10 to 1x10'', alter the specific aggregation of the magnetic particles in the or 1x10' to 1x10" magnetic particles) having a mean diam presence of the one or more analytes or a multivalent binding eter of from 100 nm to 699 nm (e.g., from 150 to 250, 200 to agent. 350, 250 to 450, 300 to 500, 450 to 650, or from 500 to 699 In any of the systems, kits, cartridges, and methods of the nm), a T, relaxivity per particle of from 1x10 to 1x10' invention for detection of any analyte in a whole blood mM's' (e.g., from 1x10 to 1x10, 1x10 to 1x10", 1x10 25 sample, the disruption of the red blood cells can be carried out to 1x10, 1x10 to 1x10'', or from 1x10' to 1x10'’ using an erythrocyte lysis agent (i.e., a lysis buffer, or a mM's'), and binding moieties on their surfaces, the binding nonionic detergent). Erythrocyte lysis buffers which can be moieties operative to alter the specific aggregation of the used in the methods of the invention include, without limita magnetic particles in the presence of the one or more analytes tion, isotonic Solutions of ammonium chloride (optionally or a multivalent binding agent; and (ii) a chamber for holding 30 including carbonate buffer and/or EDTA), and hypotonic a buffer. In a related aspect, the invention features a remov Solutions. Alternatively, the erythrocyte lysis agent can be an able cartridge sized to facilitate insertion into and removal aqueous Solution of nonionic detergents (e.g., nonyl phe from a system of the invention, wherein the removable car noxypolyethoxylethanol (NP-40), 4-octylphenol poly tridge comprises one or more chambers for holding a plurality ethoxylate (Triton-X100), Brij-58, or related nonionic surfac of reagent modules for holding one or more assay reagents, 35 tants, and mixtures thereof). The erythrocyte lysis agent wherein the reagent modules include (i) a chamber for hold disrupts at least some of the red blood cells, allowing a large ing from 1x10° to 1x10" magnetic particles (e.g., from fraction of certain components of whole blood (e.g., certain 1x10 to 1x10, 1x107 to 1x10, 1x107 to 1x10, 1x10 to whole blood proteins) to be separated (e.g., as Supernatant 1x10", 1x10 to 1x10', or 1x10' to 1x10" magnetic par following centrifugation) from the white blood cells, yeast ticles) having a mean diameter of from 700 nm to 1200 nm 40 cells, and/or bacteria cells present in the whole blood sample. (e.g., from 700 to 850, 800 to 950,900 to 1050, or from 1000 Following Erythrocyte lysis and centrifugation, the resulting to 1200 nm), a T, relaxivity per particle of from 1x10 to pellet is reconstituted to form an extract. 1x10 mM's' (e.g., from 1x10 to 1x10', 1x10 to The methods, kits, cartridges, and systems of the invention 1x10', or from 1x10' to 1x10' mM's'), and oligonucle can be configured to detect a predetermined panel of patho otide binding moieties on their surfaces, the oligonucleotide 45 gen-associated analytes. For example, the panel can be a binding moieties operative to alter the specific aggregation of candida fungal panel configured to individually detect three the magnetic particles in the presence of the one or more or more of Candida guilliermondii, C. albicans, C. glabrata, analytes; and (ii) a chamber for holding a buffer. The mag C. krusei, C. Lusitaniae, C. parapsilosis, and C. tropicalis. In netic particles can be any described herein, decorated with another embodiment, the panel can be a bacterial panel con any binding moieties described herein, for detecting any ana 50 figured to individually detect three or more of coagulase lyte described herein. In particular embodiments of the negative Staphylococcus, Enterococcus faecalis, E. faecium, removable cartridges, the magnetic particles and buffer are Pseudomonas aeruginosa, Staphylococcus aureus, and together in a single chamber within the cartridge. In still other Escherichia coli. In a particular embodiment, the panel can be embodiments, the buffer includes from 0.1% to 3% (w/w) a viral panel configured to individually detect three or more of albumin, from 0.01% to 0.5% nonionic surfactant, a lysis 55 Cytomegalovirus (CMV), Epstein Barr Virus, BK Virus, agent, or a combination thereof. The removable cartridge can Hepatitis B virus, Hepatitis C virus, Herpes simplex virus further include a chamber including beads for lysing cells; a (HSV), HSV1, HSV2, Respiratory syncytial virus (RSV), chamber including a polymerase; and/or a chamber including Influenza; Influenza A, Influenza A subtype H1, Influenza A a primer. subtype H3, Influenza B, Human Herpes Virus 6, Human The invention features a removable cartridge sized to 60 Herpes Virus 8, Human Metapneumovirus (hMPV), Rhinovi facilitate insertion into and removal from a system of the rus, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, and invention, wherein the removable cartridge includes one or Adenovirus. The panel can be a bacterial panel configured to more chambers for holding a plurality of reagent modules for individually detect three or more of E. coli, CoNS (coagulase holding one or more assay reagents, wherein the reagent negative staph), Pseudomonas aeruginosa, S. aureus, E. modules include (i) a chamber for holding from 1x10 to 65 faecium, E. faecalis, and Klebsiella pneumonia. The panel 1x10" magnetic particles having a mean diameter of from can be a bacterial panel configured to individually detect three 100 nm to 350 nm, a T. relaxivity per particle of from 5x10 or more of A. fumigates, and A. flavum. The panel can be a US 8,409,807 B2 33 34 bacterial panel configured to individually detect three or more polysaccharide, glucose, a lipid, a gas (e.g., oxygen or carbon of Acinetobacter baumannii, Enterobacter aeraogenes, dioxide), an electrolyte (e.g., sodium, potassium, chloride, Enterobacter cloacae, Klebsiella Oxytoca, Proteus mirabilis, bicarbonate, BUN, magnesium, phosphate, calcium, ammo Serratia marcescens, Staphylococcus haemolyticus, nia, lactate), a lipoprotein, cholesterol, a fatty acid, a glyco Stenotrophomonas maltophilia, Streptococcus agalactie, protein, a proteoglycan, a lipopolysaccharide, a cell Surface Streptococcus mitis, Streptococcus pneumonia, and Strepto marker (e.g., CD3, CD4, CD8, IL2R, or CD35), a cytoplas coccus pyogenes. The panel can be a meningitis panel con mic marker (e.g., CD4/CD8 or CD4/viral load), a therapeutic figured to individually detect three or more of Streptococcus agent, a metabolite of a therapeutic agent, a marker for the pneumonia, H. influenza, Neisseria Meningitis, HSV1, detection of a weapon (e.g., a chemical or biological HSV2, Enterovirus, Listeria, E. coli, Group B Streptococcus. 10 weapon), an organism, a pathogen, a pathogen byproduct, a The panel can be configured to individually detect three or parasite (e.g., a protozoan or a helminth), a protist, a fungus more of N. gonnorrhoeae, S. aureus, S. pyogenes, CoNS, and (e.g., yeast or mold), a bacterium, an actinomycete, a cell Borrelia burgdorferi. The panel can be configured to indi (e.g., a whole cell, a tumor cell, a stem cell, a white blood cell, vidually detect three or more of C. Difficile, Toxin A, and a T cell (e.g., displaying CD3, CD4, CD8, IL2R, CD35, or Toxin B. The panel can be a pneumonia panel configured to 15 other surface markers), or another cell identified with one or individually detect three or more of Streptococcus pneumo more specific markers), a virus, a prion, a plant component, a nia, MRSA, Legionella, C. pneumonia, and Mycoplasma plant by-product, algae, an algae by-product, plant growth Pneumonia. The panel can be configured to individually hormone, an insecticide, a man-made toxin, an environmental detect three or more of treatment resistant mutations selected toxin, an oil component, and components derived therefrom. from mecA, VanA. VanB, NDM-1, KPC, and VIM. The panel As used herein, the term “small molecule” refers to a drug, can be configured to individually detect three or more of H. medication, medicament, or other chemically synthesized influenza, N. gonnorrhoeae, H. pylori, Campylobacter, Bru compound that is contemplated for humantherapeutic use. As cella, Legionella, and Stenotrophomonas maltophilia. The used herein, the term “biologic' refers to a substance derived panel can be configured to detect total viral load caused by from a biological source, not synthesized and that is contem CMV, EBV, BKVirus, HIV, HBV, and HCV. The panel can be 25 plated for human therapeutic use. A “biomarker' is a biologi configured to detect fungal load and/or bacterial load. Viral cal Substance that can be used as an indicator of a particular load determination can be using a standard curve and mea disease state or particular physiological state of an organism, Suring the sample against this standard curve or some other generally a biomarker is a protein or other native compound method of quantitation of the pathogen in a sample. The measured in bodily fluid whose concentration reflects the quantitative measuring method may include real-time PCR, 30 presence or severity or staging of a disease state or dysfunc competitive PCR (ratio of two competing signals) or other tion, can be used to monitor therapeutic progress of treatment methods mentioned here. The panel can be configured to of a disease or disorder or dysfunction, or can be used as a detect immune response in a Subject by monitoring PCT, Surrogate measure of clinical outcome or progression. As MCP-1, CRP, GRO-alpha, High mobility group-box 1 protein used herein, the term “metabolic biomarker” refers to a sub (HMBG-1), IL-1 receptor, IL-1 receptor antagonist, IL-1b, 35 stance, molecule, or compound that is synthesized or biologi IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, macroph cally derived that is used to determine the status of a patient or age inflammatory protein (MIP-1), macrophage migration subject’s liver or kidney function. As used herein, the term inhibitory factor (MIF), osteopontin, RANTES (regulated on “genotyping refers to the ability to determine genetic differ activation, normal T-cell expressed and secreted; or CCL5), ences in specific genes that may or may not affect the pheno Th1. Th17, and/or TNF-C. The panel can be configured to 40 type of the specific gene. As used herein, the term "pheno individually detect three or more of Ehrlichea, Mycobacte type' refers to the resultant biological expression, (metabolic rium, Syphillis, Borrelia burgdorferi, Cryptococcus, Histo or physiological) of the protein set by the genotype. As used plasma, and Blastomyces. The panel can be an influenzapanel herein, the term “gene expression profiling refers to the configured to individually detect three or more of Influenza A, ability to determine the rate or amount of the production of a Influenza B, RSV. Parainfluenza, Meta-pneumovirus, Rhi 45 gene product or the activity of gene transcription in a specific novirus, and Adenovirus. tissue, in a temporal or spatial manner. As used herein, the The methods, kits, cartridges, and systems of the invention term “proteomic analysis” refers to a protein pattern or array can be configured to reduce sample to sample variability by to identify key differences in proteins or peptides in normal determining a magnetic resonance signal prior to and after and diseased tissues. Additional exemplary analytes are hybridization. The addition of derivatized nanoparticles to the 50 described herein. The term analyte further includes compo sample prior to methods to enhance clustering may provide a nents of a sample that are a direct product of a biochemical baseline, internal T signal that can either be subtracted or means of amplification of the initial target analyte, such as the used to modify the T signal after analyte-derivatized particle product of a nucleic acid amplification reaction. binding and clustering. This method may also be used to By an "isolated nucleic acid molecule is meant a nucleic determine or manage cartridge to cartridge variability. 55 acid molecule that is removed from the environment in which The terms "aggregation.” “agglomeration, and “cluster it naturally occurs. For example, a naturally-occurring ing” are used interchangeably in the context of the magnetic nucleic acid molecule present in the genome of cell or as part particles described herein and mean the binding of two or of a gene bank is not isolated, but the same molecule, sepa more magnetic particles to one another, e.g., via a multivalent rated from the remaining part of the genome, as a result of analyte, multimeric form of analyte, antibody, nucleic acid 60 e.g., a cloning event, amplification, or enrichment, is iso molecule, or other binding molecule or entity. In some lated. Typically, an isolated nucleic acid molecule is free instances, magnetic particle agglomeration is reversible. from nucleic acid regions (e.g., coding regions) with which it By “analyte' is meant a substance or a constituent of a is immediately contiguous, at the 5' or 3' ends, in the naturally sample to be analyzed. Exemplary analytes include one or occurring genome. Such isolated nucleic acid molecules can more species of one or more of the following: a protein, a 65 be part of a vector or a composition and still be isolated, as peptide, a polypeptide, an amino acid, a nucleic acid, an Such a vector or composition is not part of its natural envi oligonucleotide, RNA, DNA, an antibody, a carbohydrate, a rOnment. US 8,409,807 B2 35 36 As used herein, “linked' means attached or bound by cova pathogen being detected. Alternatively, the pathogen-associ lent bonds, non-covalent bonds, and/or linked via Van der ated analyte is selected to ascertain a property of the patho Waals forces, hydrogen bonds, and/or other intermolecular gen, Such as resistance to a particular therapy. For example, forces. the pathogen-associated analyte can be a gene, such as a Van The term “magnetic particle' refers to particles including A gene or Van B gene, characteristic of Vancomycin resis materials of high positive magnetic Susceptibility Such as tance in a number of different bacterial species. paramagnetic compounds, Superparamagnetic compounds, By “pulse sequence' or “RF pulse sequence' is meant one and magnetite, gamma ferric oxide, or metallic iron. or more radio frequency pulses to be applied to a sample and As used herein, “nonspecific reversibility” refers to the designed to measure, e.g., certain NMR relaxation rates. Such colloidal stability and robustness of magnetic particles 10 as spin echo sequences. A pulse sequence may also include against non-specific aggregation in a liquid sample and can be the acquisition of a signal following one or more pulses to determined by Subjecting the particles to the intended assay minimize noise and improve accuracy in the resulting signal conditions in the absence of a specific clustering moiety (i.e., value. an analyte or an agglomerator). For example, nonspecific As used herein, the term “signal” refers to an NMR relax reversibility can be determined by measuring the T values of 15 ation rate, frequency shift, Susceptibility measurement, dif a solution of magnetic particles before and after incubation in fusion measurement, or correlation measurements. a uniform magnetic field (defined as <5000 ppm) at 0.45T for As used herein, reference to the "size' of a magnetic par 3 minutes at 37° C. Magnetic particles are deemed to have ticle refers to the average diameter for a mixture of the mag nonspecific reversibility if the difference in T values before netic particles as determined by microscopy, light scattering, and after Subjecting the magnetic particles to the intended or other methods. assay conditions vary by less than 10% (e.g., vary by less than As used herein, the term “substantially monodisperse' 9%, 8%, 6%, 4%, 3%, 2%, or 1%). If the difference is greater refers to a mixture of magnetic particles having a polydisper than 10%, then the particles exhibit irreversibility in the sity in size distribution as determined by the shape of the buffer, diluents, and matrix tested, and manipulation of par distribution curve of particle size in light scattering measure ticle and matrix properties (e.g., coating and buffer formula 25 ments. The FWHM (full width half max) of the particle dis tion) may be required to produce a system in which the tribution curve less than 25% of the peak position is consid particles have nonspecific reversibility. In another example, ered Substantially monodisperse. In addition, only one peak the test can be applied by measuring the T values of a solu should be observed in the light scattering experiments and the tion of magnetic particles before and after incubation in a peak position should be within one standard deviation of a gradient magnetic field 1 Gauss/mm-10000 Gauss/mm. 30 population of known monodisperse particles. As used herein, the term "NMR relaxation rate” refers to a By “T, relaxivity per particle' is meant the average T. measuring any of the following in a sample T, T, T1/T relaxivity per particle in a population of magnetic particles. hybrid, T.T., and T*. The systems and methods of the As used herein, "unfractionated refers to an assay in invention are designed to produce an NMR relaxation rate which none of the components of the sample being tested are characteristic of whether an analyte is present in the liquid 35 removed following the addition of magnetic particles to the sample. In some instances the NMR relaxation rate is char sample and prior to the NMR relaxation measurement. acteristic of the quantity of analyte present in the liquid It is contemplated that units, systems, methods, and pro sample. cesses of the claimed invention encompass variations and As used herein, the term “T/T hybrid refers to any detec adaptations developed using information from the embodi tion method that combines a T and a T measurement. For 40 ments described herein. Throughout the description, where example, the value of a TFT hybrid can be a composite units and systems are described as having, including, or signal obtained through the combination of ratio, or differ including specific components, or where processes and meth ence between two or more differentT and T measurements. ods are described as having, including, or including specific The T/T hybrid can be obtained, for example, by using a steps, it is contemplated that, additionally, there are units and pulse sequence in which T and T are alternatively measured 45 systems of the present invention that consist essentially of, or or acquired in an interleaved fashion. Additionally, the T/T consist of the recited components, and that there are pro hybrid signal can be acquired with a pulse sequence that cesses and methods according to the present invention that measures a relaxation rate that is comprised of both T and T. consist essentially of, or consist of the recited processing relaxation rates or mechanisms. steps. It should be understood that the order of steps or order A "pathogen' means an agent causing disease or illness to 50 for performing certain actions is immaterial, unless otherwise its host, Such as an organism or infectious particle, capable of specified, so long as the invention remains operable. More producing a disease in another organism, and includes but is over, in many instances two or more steps or actions may be not limited to bacteria, viruses, protozoa, prions, yeast and conducted simultaneously. fungi or pathogen by-products. “Pathogen by-products are Other features and advantages of the invention will be those biological Substances arising from the pathogen that 55 apparent from the following detailed description, the draw can be deleterious to the host or stimulate an excessive host ings, and the claims. immune response, for example pathogen antigen/s, metabolic Substances, enzymes, biological Substances, or toxins. BRIEF DESCRIPTION OF THE DRAWINGS By "pathogen-associated analyte' is meant an analyte characteristic of the presence of a pathogen (e.g., a bacterium, 60 FIG. 1A is a schematic diagram of an NMR unit for detec fungus, or virus) in a sample. The pathogen-associated ana tion of a signal response of a sample to an RF pulse sequence, lyte can be a particular Substance derived from a pathogen according to an illustrative embodiment of the invention. (e.g., a protein, nucleic acid, lipid, polysaccharide, or any FIG. 1B depicts a typical coil configuration Surrounding a other material produced by a pathogen) or a mixture derived sample tube for measuring a relaxation signal in a 20 L from a pathogen (e.g., whole cells, or whole viruses). In 65 sample. certain instances, the pathogen-associated analyte is selected FIGS. 2A-2E illustrate micro coil geometries which can be to be characteristic of the genus, species, or specific strain of used in NMR (for excitation and/or detection); designs US 8,409,807 B2 37 38 include, but are not limited to a wound solenoid coil (FIG. ary between the two regimes is ca. 100 nm diameter particles. 2A), a planar coil (FIG. 2B), a MEMS solenoid coil (FIG. When Small magnetic particles form aggregates under 100 2C), a MEMS Helmholz coil (FIG. 2D), and a saddle coil nm in diameter, the result is a decrease in Tupon aggregate (FIG. 2E), according to an illustrative embodiment of the formation. When magnetic particles at or above 100 nm in invention. Three dimensional lithographic coil fabrication of 5 diameterform larger aggregates, the result is an increase in T. well characterized coils used in MR detection is also estab upon aggregate formation. lished and can be used for these applications, Demas et al. FIGS. 5A-5C are drawings depicting different assay for “Electronic characterization of lithographically patterned mats for the assays of the invention. FIG. 5A depicts an microcoils for high sensitivity NMR detection' J Magn agglomerative sandwich immunoassay in which two popula Reson 200:56 (2009). 10 tions of magnetic particles are designed to bind to two differ FIG. 3 is a drawing depicting an aggregation assay of the ent epitopes of an analyte. FIG. 5B depicts a competitive invention. The magnetic particles (dots) are coated with a immunoassay in which analyte in a liquid sample binds to a binding agent (i.e., antibody, oligo, etc.) Such that in the multivalent binding agent (a multivalent antibody), thereby presence of analyte, or multivalent binding agent, aggregates inhibiting aggregation. FIG. 5C depicts a hybridization-me are formed. The dotted circles represent the diffusion sphere 15 diated agglomerative assay in which two populations of par or portion of the total fluid volume that a solution molecule ticles are designed to bind to the first and second portions of may experience via its diffusion during a T measurement a nucleic acid target, respectively. (the exact path traveled by a water molecule is random, and FIG. 6 illustrates a modular cartridge concept in sections this drawing is not to Scale). Aggregation (right hand side) that can be packaged and stored separately. This is done, for depletes portions of the sample from the microscopic mag example, so that the inlet module (shown elevated with netic non-uniformities that disrupt the water's T signal, lead inverted Vacutainer tube attached) can be sterilized while the ing to an increase in T. relaxation. reagent holding module in the middle is not. This allows the FIGS. 4A-4E are a series of graphs depicting the depen component containing reagents to be the only refrigerated dence of transverse relaxivity (R) (FIG. 4A) or T2 (FIGS. component. 4B-4E) on particle diameter and particle aggregation. FIG. 25 FIGS. 7A-7F depict a Vacutainer inlet module. FIG. 7A 4A is a graph depicting the motional averaging regime (light shows it in the inverted position after the user has removed the line, left side); the R (1/T) measured by a CPMG sequence closure from the Vacutainer tube and placed the cartridge onto increases as particle size increases because the refocusing it. FIG.7B shows the molded inpath that the bloodwill follow pulses are ineffective to counteract the dephasing effects of out of the Vacutainer and into the sample loading region once the particles. As the system transitions to the visit limited 30 the cartridge is turned right side up. The foil seal can be the regime (dark line, right side) the refocusing pulses begin to bottom side of the channels, forming an inexpensively become effective and the R decreases as particle size molded part with closed channels. FIG.7C is a cutaway view increases. For homogeneous magnetic fields, the R* in the showing the vent tube which allows air to enter into the vial as motional averaging regime matches the R2 and the R* the blood leaves and fills the sample region. FIGS. 7D-7F reaches a constant value in the visit limited regime. In a 35 depict an inlet module for sample aliquoting designed to homogenous field, when the R* is less than the R of either interface with uncapped vacutainer tubes, and to aliquot two the motional averaging regime or visit limited regime the a sample Volume that can be used to perform, for example, a system is in the static dephasing regime. The empty circle candida assay. The inlet module has two hard plastic parts, represents the R of a solution of 100% dispersed particles that get ultrasonically welded together and foil sealed to form (diameter-15 nm) and the solid circle represents a solution of 40 a network of channels to allow a flow path to form into the first 100% clustered particles (diameter=200 nm). This is an well overflow to the second sample well. A soft vacutainer example of how to interpret these curves for clustering reac seal part is used to for a seal with the vacutainer. It has a port tions. The conditions for this curve are 0.1 mM Fe, A(r)=8.85x for sample flow, and a venting port, to allow the flow to occur. 10, D=2.5x10 m/s, and T-0.25 ms. FIG. 4B is a graph FIG. 8 depicts the sample inlet module with the foil seal depicting the same light and dark curves plotted interms of T 45 removed. On the top, one can see the small air inlet port to the and diameter, on a linear scale. In this figure the black dashed left, the larger sample well in the center and a port which line represents the T measured in a non-uniform magnetic connects them together. This port provides a channel through field where T is always lower than T and doesn't reflect the which air can flow once the foil sealispierced. It also provides particle size. The data points are the same as well. FIG. 4C is an overflow into the body of the module to allow excess blood a graph depicting the monodisperse clustering model and 50 to drain away and not spill over. This effectively meters the showing that T will follow the curve as analyte is added blood sample to the volume contained in the sample well. because the average diameter of the population particles will FIGS. 9A-9C depict a reagent module. FIG.9A depicts the cover all intermediate diameters between the initial and final module of the cartridge that is intended to hold reagents and states. FIG. 4D is a graph depicting the polydisperse model consumables for use during the assay. On the left are sealed and showing that the T will transition between the two points 55 pre-dispensed aliquots of reagents. On the right is a 2.8 ml on this curve when particles form clusters of specific sizes. conical bottomed centrifuge tube that is used for initial cen The response curve will be linear with regard to analyte trifugation of the blood. The other holes can be filled as need addition, but non-linear with regard to volume fraction of with vials, microcentrifuge tubes, and pipette tips. FIG.9B is clusters, because particles transition between State 1 and state a cutaway view of the reagent module showing the holders for 2. The slope of the response curve is directly proportional to 60 the pre-aliquoted reagent tips, including the feature at the the sensitivity of the assay. FIG.4E is a graph showing the two bottom into which the tips are pressed to provide a seal. FIG. regimes for particle aggregation and T affects based on par 9C depicts three representative pipette tips into which ticle size and how clustering assays in the different regimes reagents can be pre-dispensed, and then the backs sealed. The map onto the T. Versus diameter curves (i) for the motional tips are pressed into the sample holder to provide a seal. averaging regime T decreases when particles cluster; and (ii) 65 FIGS. 10A and 10B depict an alternative design of the for the slow motion regime T increases when particles clus modular cartridge, showing a detection module with a ter. Under the conditions shown in these models, the bound recessed well for use in assays that require PCR. Cross US 8,409,807 B2 39 40 contamination from PCR products is controlled in two ways. along the direction of the field produced by the hMAA setup. First, the seals that are on the detection tubes are designed to On the right hand side, the two magnets are represented by seal to a pipette tip as it penetrates. Second, the instrument bars, to depict the formation of a standard dipole field.hMAA provides airflow through the recessed well by means of holes can also be used to evaluate the nonspecific reversibility of a in the well to ensure that any aerosol is carried down and does magnetic particle to assess its utility in an assay of the inven not travel throughout the machine. tion. FIG. 11 depicts a detection module of cartridge showing FIG. 17 depicts a gradient MAA unit configured to apply a detection tubes and one of the holes used to ensure air flow gradient magnetic field to the side and to the bottom of a down and over the tubes during pipetting to help prevent sample. The specific setup has magnets with a surface field of aerosol escape. 10 approximately 0.7 T, while the produced gradient is in the FIG. 12 depicts a bottom view of the detection module, order of 0.25 T/mm. Similar gMAA units, covering a much showing the bottom of the detection tubes and the two holes bigger range of fields and gradients can be used. used to ensure airflow. An optional filter can be inserted here FIGS. 18A-18C depict a gradient MAA unit configured to to capture any liquid aerosol and prevent it from entering the apply a gradient magnetic field to the side and to the bottom machine. This filter could also be a sheet of a hydrophobic 15 ofan array of samples. FIG. 18A depicts the gMAA unit array material like Gore-tex that will allow air but not liquids to of 32 bottom magnets and 40 side magnets (32 functional, 8 escape. used to balance the Stray magnetic fields seen by all sample), FIGS. 13 A-13C depict a detection tube. FIG. 13A is a view each with a field strength of about 0.5 T. used for assisting of the detection tube. The tube itself could be an off the shelf agglomeration in an array of samples simultaneously. FIGS. 200 microliter PCR tube, while the cap is a custom molded 18B-18C depict a top view (FIG. 18B) and side view (FIG. elastomer part that provides a pressure resistant duckbill seal 18C) of a setup for the automation of the an automated gMAA on the inside and a first seal to the pipette tip from the top. The unit wherein a plate gMAA along with a configuration for seal is thus a make-break type of seal, where one seal is made containing an array of samples is cycled between the bottom before the other is broken. FIG. 13B depicts the custom and side magnet positions by a robotic systems, within a molded seal component. Note the circular hole into which the 25 temperature controlled array. The magnets are stationary, pipette tip is inserted and the duckbill seal below, which while the plate holding the sample tubes moves through a provides a second seal that resists pressure developed in the preset trajectory. An exemplary field strength on the Surface tube. FIG. 13C depicts the seal showing the duckbill at bot of individual magnets is 0.4-0.5 T with a gradient in the order tom and the hole at top. of 0.1 T/mm. FIGS. 14A-14C depict a cartridge for performing a multi 30 FIGS. 19A-19B depictatop view (FIG. 19A)and side view plexed assay. FIG. 14A shows a reagent strip for the cartridge. (FIG. 19B) of a homogenous MAA unit configured to apply a The oval holes are the supports for the detection modules, and homogenous magnetic field to an array samples. Field these are constructed separately and then placed into the strengths from 0.2-0.7 T can be used with homogeneity from holes. The detection wells could be custom designed or com 500 to 5000 ppm over the sample tube region. mercially available. FIG. 14B shows the detection module for 35 FIG. 20 is a drawing of a vortexer which includes the the cartridge depicted in FIG. 14A. In this example, the detec following components: (i) a sample Support, (ii) a main plate, tion module contains two detection chambers, but could con (iii) four linkages, (iv) linear rail and carriage system (X2), (v) tain any number of chambers as required by the assay and as a Support for driveshaft and rails, (vi) coupling and driveshaft, the detection system (the MR reader) is designed to accept. (vii) a mounting plate, and (viii) a drive motor. FIG. 14C depicts an alternate footprint for the modular mul 40 FIG. 21 is a drawing of a compact vortexer which includes tiplexed cartridge. This cartridge includes 3 detection mod the following components: (i) a sample Support, (ii) a main ules that are molded as part of the reagent strip, and these plate, (iii) two linkages, (iv) linear rail and carriage system, portions are popped out of the frame and individually pro (v) a support for linear rail, (vi) support for driveshaft, (vii) cessed at other units (i.e., the NMR unit and/or magnetic coupling and driveshaft, (viii) a mounting plate, and (ix) a assisted agglomeration (MAA) unit) within the assay system. 45 drive motor. FIG. 15 is a scheme depicting one embodiment of the FIGS. 22A and 22B depict portions of a vortexer. FIG.22A cycling gradient magnetic assisted agglomeration (gMAA) is a drawing depicting the bottom portion (i.e., the drive method of the invention. Two magnets are placed in two motor, coupling, and drive shaft) of a Vortexer of the inven positions such that if the sample tube is placed close to the a tion. The motor includes an index mark and/or other position region of strong magnetic field gradient produced by the first 50 sensing means such as an optical, magnetic or resistive posi magnet, the magnetic particles will be drawn towards the tion encoder that allows the motor to find a specific point in its direction of the field gradient produced by the first magnet, rotation. These index marks are used to home the system, and the sample tube is then placed next to the second magnet ensure that the sample can be returned to a known position producing a field gradient, and the magnetic particles are after mixing and allows the Vortexer to be easily accessed by drawn to the direction of the field gradient produced by sec 55 robotic actuators, and thus integrated into an automated sys ond magnet. The cycle can be repeated until the aggregation tem. In lieu of index marks, external home Switches or posi reaction reaches a steady state (as observed by the change in tion tracking sensors could be employed. FIG.22B is a draw the NMR relaxation rate of the sample); a smaller number of ing depicting the guide mechanism of a Vortexer of the cycles can be used as well. A single magnet used to produce invention. The main plate is connected to the offset axis of the a field gradient can also be used, while for cycling the sample 60 drive shaft and is free to rotate. The plate follows the orbital tube can be moved relative to the magnetic field gradient. path around and dictated by the motor shaft. FIG. 16 is a scheme depicting a homogenous magnetic FIGS. 23A-23C are a series of drawings depicting a vor assisted agglomeration (hMAA) setup. On the left hand side, texer utilizing a planetary belt drive. FIG. 23A is an overall the magnetic particles are shown as dots in a partially clus view showing the Vortexer configured for one large tube. tered State. When exposed to a homogeneous magnetic field, 65 FIG. 23B is a section view showing two tube holders for as depicted on the right hand side, clustering of the magnetic small tubes. FIG. 23C is an overall view of vortexer showing particles is promoted as the magnetic particles form chains four tubes and a close up of planetary belt drive mechanism. US 8,409,807 B2 41 42 FIG. 24 is a drawing depicting the components of the 30A and 30B, increasing temperature and increasing dwell creatinine competitive assay of Example 6. A magnetic par time enhance the extent of gMAA assisted aggregation as ticle decorated with creatinine is used in combination with a observed by changes in the observed T. creatinine antibody to form an aggregating system. The crea FIG. 31 is a graph showing that the degree of aggregation tinine present in a liquid sample competes with the magnetic as determined by measuring the T response of the sample is particles for the antibody, leading to a reduction in aggrega increased with increasing the number of gMAA cycles in the tion with increasing creatinine concentration. The change in assay of Example 13. aggregation is observed as a change in the Trelaxation rate of FIG. 32 is a drawing depicting the Candida agglomerative the hydrogen nuclei in the water molecules of the liquid sandwich assay architecture of Example 14. sample. By comparing the observed T. relaxation rate of the 10 FIG.33 is a graph depicting a creatinine inhibition curve liquid sample to a standard curve, the concentration of crea (see Example 7) for using an antibody coated particle and an tinine is determined. amino-dextran-creatinine multivalent binding agent to FIGS. 25A-25C are a series of graphs showing the induce clustering by competing with any target analyte (crea response curve for creatinine competitive assays. FIG. 25A is tinine) present in the sample to cause particle clustering. The a graph showing a standard curve for the creatinine competi 15 binding agent used is a 40 kDa dextran with ~10 creatinines tive assay ofExample 6 correlating the observed T. relaxation per dextran molecule. rate with the concentration of creatinine in the liquid sample. FIG. 34 is a graph depicting the evaluation of Tac-dextran FIG. 25B shows the T response of a creatinine-decorated conjugates for clustering ability (see Example 8) by perform particle with 2 different preparations of antibody. Preparation ing a titration. As observed, that increased molecular weight 1 is pre-production (with aggregated antibody) and Prepara of Tac-dextran results in the improved T signal. tion 2 is production purified (no aggregated antibody FIG. 35 is a graph depicting the evaluation of Tac-dextran present). FIG. 25C shows the T response of a creatinine conjugates for clustering ability (see Example 8) by perform decorated particle with unaggregated antibody, biotinylated ing a titration. As observed, higher substitution improved T. antibody and deliberately multimerized antibody, and con signal. firms the increased clustering ability of multi-valent agglom 25 FIG. 36 is a graph depicting the evaluation of Tac-BSA erating agents. conjugates for clustering ability (see Example 8) by perform FIG.26 is a graph showing the specific clustering achieved, ing a titration similar to that used for the Tac-dextran conju as determined viaT relaxation rates, with various methods of gates. As observed, clustering performance varies with the gMAA as described in Example 10. In FIG. 26 (i) “control is tacrolimus Substitution ratio. gMAA (magnet exposure+Vortex, repeat) in which the rela 30 FIG. 37 is a graph depicting the results of T assays for tive position of the sample and the magnetic field direction are detecting anti-biotin antibody using prepared magnetic par unchanged with each cycle; (ii) “twist is gMAA (magnet ticles in blood and PBS matrices as described in Example 1. exposure--rotation within magnet, repeat) with rotating tube FIG.38 is a graph depicting results of Tassays for detect ca. 90° relative to the gradient magnet with each cycle; (iii) ing anti-biotin antibody using prepared magnetic particles “180° turn” is gMAA (magnet exposure--remove tube from 35 with (open circle) and without (filled circle) a protein block as magnet, rotate, place back in magnet, repeat) with rotating described in Examples 8 and 9. tube ca. 180° relative to the gradient magnet with each cycle: FIG. 39 is a graph depicting results of Tassays for detect and “remove 5 s” is removal of tube from magnet, 5 seconds ing anti-biotin antibody using prepared magnetic particles rest (no rotation), repeat. The results show that the rate at having a BSA block (dark filled diamond, square, triangle) or which a steady state degree of agglomeration, and stable T. 40 an FSG block (light gray X's and circle) as described in reading, is achieved is expedited by cycling between the two Example 2. or more positions over a number of gMAA treatments. Fur FIGS. 40A-40B are schematics of provided particle coat ther, field gradient combinations, cycling field (side or bot 1ngS. tom) to null or side field to bottom, field (side or bottom) to FIGS. 41A-41B depict results of T assays for detecting vortex are also iterations that can be used for gMAA. Expo 45 biotin in a competitive assay format described in Example 4. sure or dwell times (either on the field or away), and number FIG. 41A depicts experimental results in buffer; while FIG. of cycles can be varied to optimize assisted aggregation for a 41B depicts experimental results in lysed blood. specific assay (not shown). FIG. 42 is a sketch of a system of the invention including an FIG. 27 is a graph showing the response curve for the NMR unit, a robotic arm, a hMAA unit, a gMAA unit, two creatinine competitive assay for samples processed with 50 agitation units, a centrifuge, and a plurality of heating blocks. alternating side-bottom magnet gMAA as described in FIGS. 43 A-43D are images depicting various fluid transfer Example 11. units which can be used in the systems of the invention. FIG. 28 is a drawing depicting the tacrolimus competitive FIGS. 44A and 44B are sketches showing how a system of assay architecture of Example 9. the invention can be designed to regulate the temperature of FIG. 29 is a graph showing a standard curve for the tacroli 55 the working space. mus competitive assay of Example 9 correlating the observed FIGS. 45A and 45B are sketches depicting an NMR unit T. relaxation rate observed for a liquid sample with the con having a separate casing for regulation of the temperature at centration of tacrolimus in the liquid sample. the site of the NMR measurement, and useful where tight FIGS. 30A-30B are graphs depicting the degree to which temperature control is needed for precision of the measure gMAA assisted aggregation is dependent upon temperature 60 ment. The temperature control configuration depicted in this and dwell time in the assay of Example 11. FIG. 30A is a figure is one of many different ways to control temperature. graph showing that the degree of aggregation as determined FIG. 46A is a table and 46B is a graph depicting the by measuring the T response of the sample is increased with repeatability of Candida measurements by methods of the increasing dwell time at room temperature. FIG. 30B is a invention over a period of eight days. To determine the repeat graph showing that the degree of aggregation as determined 65 ability of the T2 measurement on C. albicans infected human by measuring the T response of the sample is increased with whole blood, we conducted an eight day study in which the increasing gMAA dwell time at 37° C. As shown in FIGS. same donor spiked and amplified sample was hybridized to US 8,409,807 B2 43 44 the Superparamagnetic particles (n-3) each day and the FIG. 52 is a graph depicting the change in T. signal with resulting T2 values were recorded (see Example 17). The PCR cycling (see Example 18). The results demonstrate that within run precision is shown in FIG. 46A and in general is the methods and systems of the invention can be used to perform real time PCR and provide quantitative information tight with the CVs of all measurands less than 12%. The about the amount of target nucleic acid present in a sample. repeatability observed over the course of eight days is shown FIG. 53 is a series of photographs showing a simple mag in FIG. 46B (Mean T2 values +/- the 95% confidence inter netic separator/PCR block insert. vals measured from the same donor Spiked and amplified FIG. 54 is an image showing the quantity of DNA gener samples over the course of eight days) with the CVs less than ated by amplification of (1) 100 copies of genomic C. albi 10% across the range of Candida concentrations and 6% for cans amplified in the presence of 3' and 5' C. albicans single the negative control. 10 probe nanoparticles; particles were held on the side wall FIG. 47 is a scheme describing the work flow for detection during PCR via magnetic field, (2) 100 copies of genomic C. of a bacterial or fungal pathogen in a whole blood sample (see albicans amplified without nanoparticles, and (3) 100 copies Examples 14 and 17). of genomic C. albicans amplified in the presence of 3' and 5' FIGS. 48A and 48B are graphs depicting results from C. albicans single probe nanoparticles; no magnetic field. 15 FIGS. 55A-55E are schematic views of a sample tube donor Samples. FIG. 48A is a graph depicting the results containing an immobilized portion of magnetizable metal obtained from 16 experiments designed to assess the assays foam (shaded), magnetic particles (circles), and analyte (tri performance in 6 different donor blood samples spiked with a angles). a magnetizable metal foam, e.g., made of nickel, may range of C. albicans cells (see Example 17). Each data point be inserted into a conduit and immobilized by exposure to is the mean+/- the 95% confidence interval (n=48). At the heat, which shrinks the conduitaround the metal foam, result lowest test concentration (10 cells/mL), we failed to detect ing in a tight seal. A sample containing magnetic particles and Candida albicans 37% of the time (6 out of 16 experiments); analytes is then introduced at one end of the conduit (FIG. however at 100 cells/mL Candida albicans was detected 55A). Next, the conduit is exposed to a magnet (FIG. 55B), 100% of the time. This suggests the assay can robustly detect and the magnetic particles are attracted to the metal foam and at C. albicans concentrations greater than or equal to 100 25 become magnetically trapped within its pores, or crevices. cells/mL with no major inhibition of performance introduced The average diameter of the pores in the metal foam is, e.g., between 100-1000 microns. Analyte molecules can be carried through the donor blood samples. FIG. 48B is a graph depict to the metal foam via binding to a magnetic particle, or the ing the results obtained from 7 experiments designed to assess fluid can be forced through the metal foam to reach trapped the assay’s performance in 6 different donor blood samples magnetic particles. While trapped in the metal foam, the spiked with a range of C. krusei cells (see Example 17). Each 30 magnetic particles have enhanced interactions, as they are data point is the mean+/- the 95% confidence interval (n=21). now confined and are closer to other magnetic particles, and We do not detect at 10 cells/mL in any of the experimental clusterformation is enhanced. The metal foam is then demag runs but detect at 100 cells/mL for all experimental runs. This netized (FIG. 55C), i.e., the magnetic field of the metal foam suggests the LOD between 10 and 100 cells/mL. becomes negligible. The magnetic particles and analyte clus FIG. 49 is a dot diagram showing the T2 values measured 35 ter complexes largely remain in the metal foam, as the diffu for five C. albicans clinical isolates spiked into 400 uL whole sion of magnetic particle clusters is relatively low, although blood at concentrations spanning 0 to 1E4 cells/mL. The some natural diffusion of the analyte into and out of the metal plotted results are the mean+/- 1 SD. The data indicates foam occurs (FIG. 55D). Alternatively, the magnetizable despite the scatter of absolute T2 values obtained among the metal foam (hollow cylinder) is free floating in the sample different isolates, at 50 cells/mL all values are above that of 40 tube with the magnetic particles (circles), and analyte (stars). the no Candida control (3 replicate measurements from 20 The magnetization and demagnetization of the free floating independent assays, total of 60 different clustering reactions). metal foam is used to increase the rate of aggregate formation. FIGS. 50A and 50B are ROC plots of T2 results generated FIG. 56A depicts a rotary gMAA configuration. The at 10 cells/mL (FIG.50A) and 50 cells/mL (FIG.50B). The Rotary gMAA can include three configurations for varying area under the curve at 10 cells/mL is 0.72 (95CI=0.56 to 45 magnetic field exposures—side-bottom; side-null and bot 0.88) while at 50 cells/mL the area under the curve is 0.98 tom-null (see Example 21). (95CI=0.95 to 1.001). The area under the curve is often used FIG. 56B is a graph comparing T2 signal as a function of to quantify the diagnostic accuracy; in this case our ability to various rotary gMAA configurations for varying magnetic discriminate between a Candidemic patient with an infection field exposures to a sample at a given agglomerator concen of 10 cells/mL or 50 cells/mL versus a patient with no Can 50 tration. The rotary side-bottom configuration provided the didemia. At 10 cells/mL the area under the curve is 0.72 highest T2 signal at a given agglomerator concentration, fol which means that if the T2 assay was run on a randomly lowed by the comparison side-bottom plate configuration. chosen person with Candidemia at a level of infection of 10 Rotary side-null provides equivalent signal to the plate side cells/mL, there is an 72% chance their T2 value would be bottom; and the bottom-null produces the lowest signal (see higher than a person with no Candidemia. The clinical accu 55 Example 21). racy of the test is much higher at 50 cells/mL with the area FIG.57 is a table depicting the T2MR results for 32 clinical under the curve at 0.98. Again indicating that in a person with specimens indicates fourteen specimens are Candida posi Candidemia at this level of infection, the T2 assay would give tive. The test identifies four specimens containing C. krusei or a value higher than a sample from a patient without Candi C. glabrata, seven specimens containing C. albicans or C. demia 98% of the time. See Example 17. 60 tropicalis, and three containing C. parapsilosis. A solid black FIG. 51 is a graph depicting the sensitivity of the assay line indicates the decision threshold (T2=128 msec) (see using the standard thermocycle (~3 hours turnaround time) Example 22). and a process that combines the annealing/elongation steps (~2 hours, 13 minutes turnaround time). Combining the DETAILED DESCRIPTION annealing and elongation step in the thermocycling reduces 65 the total assay TAT to 2.25 hours without compromising assay The invention features systems, devices, and methods for sensitivity. the rapid detection of analytes or determination of analyte US 8,409,807 B2 45 46 concentration in a sample. The systems and methods of the of aggregated and single (unaggregated) magnetic particles. invention employ magnetic particles, an NMR unit, option The assays of the invention are designed to maximize the ally one or more MAA units, optionally one or more incuba change in Twith aggregation to increase the sensitivity of the tion stations at different temperatures, optionally one or more assay to the presence of analytes, and to differences in analyte Vortexer, optionally one or more centrifuges, optionally a concentration. fluidic manipulation station, optionally a robotic system, and In designing magnetic relaxation Switch (MRSw) biosen optionally one or more modular cartridges. The systems, sors, it is important to consider the relaxation mechanisms of devices, and methods of the invention can be used to assay a the magnetic particles. First, in the case of Superparamagnetic biological sample (e.g., blood, Sweat, tears, urine, saliva, particles the solvent longitudinal and transverse relaxivities semen, serum, plasma, cerebrospinal fluid (CSF), feces, vagi 10 (defined as R=1/T and R=1/T, respectively) are a function nal fluid or tissue, sputum, nasopharyngeal aspirate or Swab, of particle size. Furthermore, R, and R* (where R*=1/T*, lacrimal fluid, mucous, or epithelial Swab (buccal swab). R*-R+AC), where Act) is dephasing due to field inhom tissues, organs, bones, teeth, or tumors, among others). Alter geneities) increase with particle diameter until about 100 nm, natively, the systems, devices, and methods of the invention and then R. decreases with increasing particle size and the are used to monitor an environmental condition (e.g., plant 15 R* reaches a plateau for uniform fields (see FIG. 4A). Super growth hormone, insecticides, man-made or environmental paramagnetic particles are typically divided into categories of toxins, nucleic acid sequences that are important for insect strongly magnetized and weakly magnetized particles, based resistance/susceptibility, algae and algae by-products), as on the relative magnitude of the precession frequency differ part of a bioremediation program, for use in farming plants or ence between nuclei at the surface of the particle and nuclei animals, or to identify environmental hazards. Similarly, the distant from the particle, Act), and the inter-echo delay of the systems, devices, and methods of the invention are used to CPMG detection sequence, T. Act) is essentially a relative detect and monitor biowarfare or biological warfare agents, measure of the effect of the dipolar magnetic field generated Such as ricin, Salmonella typhimurium, botulinum toxin, afla by a Superparamagnetic particle on the resonant frequency of toxin, mycotoxins, Francisella tularesis, Smallpox, anthrax, hydrogen nuclei in adjacent water molecules. When the prod or others. 25 uct Acotl then the particles are classified as strongly mag The magnetic particles can be coated with a binding moiety netized and when AcOT-1 then the particles are classified as (i.e., antibody, oligo, etc.) Such that in the presence of analyte, weakly magnetized. For typical relaxometers, T is no or multivalent binding agent, aggregates are formed. Aggre shorter than tens of microseconds, so Act) must be less than gation depletes portions of the sample from the microscopic 10 for the particles to be within the weakly magnetized magnetic non-uniformities that disrupt the solvents T sig 30 regime. Most Superparamagnetic particles used for MRSw nal, leading to an increase in T. relaxation (see FIG. 3). assays have a surface dephasing Act) of approximately 1x10". The T measurement is a single measure of all spins in the therefore they are classified as strongly magnetized. This ensemble, measurements lasting typically 1-10 seconds, means that the inter-echo delay is always longer than the which allows the solvent to travel hundreds of microns, along amount of dephasing that occurs at the Surface of a particle. distance relative to the microscopic non-uniformities in the 35 Another characteristic of Superparamagnetic particle solu liquid sample. Each solvent molecule samples a Volume in the tions that is used to differentiate physical behavior is the liquid sample and the T signal is an average (net total signal) diffusion time, or travel time, of water (T,) relative to the of all (nuclear spins) on solvent molecules in the sample; in inter-echo time of the pulse sequence, T. Particle solutions other words, the T measurement is a net measurement of the are in the long echo limit when the t, is significantly less than entire environment experienced by a solvent molecule, and is 40 an average measurement of all microscopic non-uniformities that T. T., can be determined by the relationship: in the sample. The observed T. relaxation rate for the solvent molecules in the liquid sample is dominated by the magnetic particles, (1) which in the presence of a magnetic field form high magnetic 45 dipole moments. In the absence of magnetic particles, the observed T. relaxation rates for a liquid sample are typically wheret, is the time it takes a water molecule to diffuse the long (i.e., T (water)=-2000 ms, T. (blood)=~1500 ms). As distance of a particle radius, R, and D the diffusion constant of particle concentration increases, the microscopic non-unifor water, 10 m/s. T. can be thought of as the time it takes a mities in the sample increase and the diffusion of solvent 50 water molecule to pass a hemisphere of a particle, or a flyby through these microscopic non-uniformities leads to an time. When t, is much larger than t, then the particle increase in spin decoherence and a decrease in the T value. system is within the “short echo limit”. Typical CPMG The observed T value depends upon the particle concentra sequences have echo times on the order of hundreds of micro tion in a non-linear fashion, and on the relaxivity per particle seconds to several milliseconds. Therefore, the “short echo parameter. 55 limit” cannot be approached unless the particle diameter In the aggregation assays of the invention, the number of approaches 1000 nm. The most common MRSw biosensors magnetic particles, and if present the number of agglomerant are within the “long echo limit” because the length of the particles, remain constant during the assay. The spatial dis inter-echo delays (T-0.25 ms) is longer than the time it tribution of the particles change when the particles cluster. takes a water molecule to diffuse past the hemisphere of a Aggregation changes the average “experience of a solvent 60 particle (0.2-100 microseconds). molecule because particle localization into clusters is pro As the particle size of a solution of Superparamagnetic moted rather than more even particle distributions. At a high particles at fixed iron concentration is increased there is an degree of aggregation, many solvent molecules do not expe initial increase in R, then a plateau and later decrease (FIG. rience microscopic non-uniformities created by magnetic 4A). The regime on the left hand side of the curve is been particles and the T approaches that of solvent. As the fraction 65 termed the motional averaging regime, the regime in the of aggregated magnetic particles increases in a liquid sample, middle is been termed the static dephasing regime, and the the observed T is the average of the non-uniform Suspension regime on the right is been termed the visit limited, or slow US 8,409,807 B2 47 48 motion regime. The boundaries between the motional aver shown by the light and dark lines in FIGS. 4A and 4B (dark aging and visit limited regimes can be determined by gener line on right side of the curve; light line on left side of the ating plots such as that shown in FIG. 4A, or they can be curve). determined by the relationship between Act) and t. If A modification of Equation 2 can be used to generate a plot Acot,<1, then the system is in the motional averaging regime: 5 that is more intuitive to an assay developer. This plot is in if Acot, 1, then the system is in the visit limited regime (also terms of T and particle diameter with linear units rather than termed the slow motion regime). As the diameter of the par logarithmic units (FIG. 2). As discussed above, magnetic ticles increases in the motional averaging regime the refocus relaxation biosensor assays function due to a transition ing echoes in the CPMG pulse sequence cannot efficiently between dispersed and clustered States. For a given agglom 10 erative assay, the measured T can follow one of two path refocus the magnetization that has been dephased by the ways over the course of an analyte titration. The population of particles, hence the increase in R (or decrease in T). In other dispersed particles can cluster in a uniform manner leading to words, the refocusing pulses cannot compensate for increased an increase in average particle size that is proportional to the dephasing by larger particles. The flat region of the static amount of analyte that has been added. This type of agglom dephasing regime is due to the R being limited by R*. The 15 eration is termed the monodisperse model because it would decreasing R with increasing diameter in the visit limited lead to a monodisperse intermediate population of particles. regime results in the refocusing pulses being able to refocus In this case, T would be expected to decrease as particle size the dephasing caused by the particles. Also apparent in FIG. increases as long as the system is within the motional aver 4A is that the R in the slow motion regime exhibits a depen aging regime. As the system approaches and enters the visit dence on the inter-echo delay of the spin echo sequence. limited regime the T would increase with particle size (FIG. In a homogenous magnetic field, one can determine which 4C). regime applies to a sample by comparing the R2 to the R*, A different type of agglomeration that may occur is one in the two values are identical in the motional averaging or static which the addition of analyte seeds the self-assembly of clus dephasing regime and they are different in the visit limited ters, a process with energetics similar to crystal formation or regime. However, in cases of inhomogeneous fields, such as 25 fractal aggregation. For this model one would expect a pre those present on benchtop and portable MR devices, the T. ferred size for particle clusters that depended on the condi is dominated by the field gradient. In fact, the measured T* tions of the solution. Systems that followed this model would value is not indicative of the particle or particle cluster size exhibit polydisperse intermediate populations; one would state (FIG. 4B). find a mixture of particles with discrete sizes. Given two The shape of the R response as particles agglomerated 30 discrete populations, dispersed particles and clustered par ticles, the system would transition between the T value of the generally matches the expected trend for the increase in aver starting monodisperse population of unclustered particles age particle size. The similarity between the R of particle and the final T value of the fully clustered particles. For both agglomerates and that of spherical particles Suggests that one models, full titration may lead to a monodisperse solution of can equate particle aggregates and spherical shapes. Even 35 clustered particles. Although the exact energetics, kinetics, though this assumption may seem to be in contradiction with and thermodynamics of particle agglomeration will depend the fractal nature of particle agglomerates, the shape of the on characteristics of the assay system such as Valency and particle aggregates observed by the magnetic resonance mea binding affinities, these two models are instructive in under surement is determined by the ensemble of diffusing water standing the dependencies and possible scenarios one may molecules in Solution, which can be approximated by the 40 encounter during MRSw biosensor design. radius of hydration measured by light scattering. There are two regimes for particle clustering and T affects The analytical models for R can be applied to magnetic based on particle size (see FIG. 4D, the boundary is typically relaxation biosensors to aid in the design of biosensor assays. ca. 100 nm diameter particles). For any given assay of a liquid Conveniently, these models accurately predict the depen sample the particle count for 250 nm sized magnetic particles dence of R on parameters that a biosensor designer can 45 can be ca. 1x107 particles, whereas for 30 nm sized magnetic control—iron concentration, temperature, magnetic Suscep particles can be ca. 1x10'. This is because the smaller par tibility, and particle size. Additionally, these analytical mod ticles have a lower relaxivity per particle (for the same type of els allow for predictive modeling of the dependence of T material), resulting in an inherent sensitivity disadvantage. In relaxation on these parameters. Results are not entirely quan a typical assay of the invention, the magnetic particles are titative, but the general trends and response curves predicted 50 selected Such that T increases with an increase in the fraction by these models can be instructive. One useful model is the of aggregated particles. chemical exchange model for strongly magnetized spheres: The assay of the invention can be designed to change the direction of T in the presence of analyte (see FIGS.5A-5C). For example, the assay can be an agglomerative sandwich (479) Vip(Aa).) (2) 55 immunoassay in which two populations of magnetic particles 1/2 - 14/02/12, bind to different epitopes of an analyte (see FIG. 5A); a 1:3 competitive assay in which analyte competes with a multiva Aco CP (3) lent binding agents to inhibit the aggregation of magnetic C. : a + baco CpW particles (see FIG. 5B); or a hybridization-mediated agglom 60 eration in which two populations of magnetic particles bind to where 1/T is the transverse relaxivity, V the volume fraction a first and second portion of an oligonucleotide (see FIG.5C). of iron in Solution, A, the diffusion, or flyby time, Act), the Additional competitive format might include when two par frequency shift at the surface of a particle relative to bulk ticles binding moieties bind without agglomerator (e.g., the solution, t one half the inter-echo delay in a CPMG DNA oligonucleotides are designed so that two nanoparticles sequence, and a and b are derived constants (a 1.34 and 65 have two different oligos and they can anneal together and b=0.99). Equations (2) and (3) can be used to generate a curve when heated the analyte or amplicon or target DNA competes that describes the dependence of R on particle sizes, as or disrupts the np annealing). US 8,409,807 B2 49 50 Other formats for carrying out the assays of the invention useful to produce conjugates include: (i) a high relaxivity, i.e., can be used. Such as: (i) a target sample can be incubated in strong effect on water (or other solvent) relaxation, (ii) a the presence of a magnetic particle that has been decorated functional group to which the binding moiety can be with binding moieties specific to a target analyte and a mul covalently attached, (iii) a low non-specific binding of inter tivalent binding agent, in an inhibition assay the binding of 5 active moieties to the magnetic particle, and/or (iv) stability in the analyte to the magnetic particles blocks agglomeration of Solution, i.e., the magnetic particles remain Suspended in the magnetic particles with the multivalent binding agent; (ii) Solution, not precipitated and/or the nps retain their ability to a target sample can be incubated in the presence of a magnetic be employed in the described method (i.e. the nps have a shelf particle that has been decorated with binding moieties spe life). cific to a target analyte and a multivalent binding agent, in a 10 disaggregation assay the analyte is exposed to a pre-formed The magnetic particles may be linked to the binding moi aggregate of the multivalent binding agent and the magnetic eties via functional groups. In some embodiments, the mag particle and the analyte displaces the multivalent binding netic particles can be associated with a polymer that includes agent to reduce aggregation in the liquid sample; or (iii) a functional groups selected, in part, to enhance the magnetic target sample can be incubated in the presence of a magnetic 15 particles nonspecific reversibility. The polymer can be a syn particle that has been decorated with binding moieties spe thetic polymer, Such as, but not limited to, polyethylene gly cific to a target analyte and the target analyte itself to form a color silane, natural polymers, or derivatives of either syn self-assembling single population of magnetic particles, in an thetic or natural polymers or a combination of these. The inhibition assay or disaggregation assay the presence the polymer may be hydrophilic. In some embodiments, the poly binding of the analyte to the magnetic particles blocks the self mer "coating is not a continuous film around the magnetic agglomeration of the magnetic particles; or (iv) a target metal oxide, but is a “mesh' or "cloud of extended polymer sample can be incubated in the presence of a soluble agglom chains attached to and Surrounding the metal oxide. The poly erating agent and a magnetic particle decorated with the ana mer can include polysaccharides and derivatives, including lyte or analog of the analyte, in an inhibition assay the pres dextran, pullanan, carboxy dextran, carboxmethyl dextran, ence of the analyte binds the soluble agglomerating agent 25 and/or reduced carboxymethyl dextran. The metal oxide can blocking the agglomeration of the particles. be a collection of one or more crystals that contact each other, Where a multivalent binding agent (agglomerant) is or that are individually entrapped or surrounded by the poly employed, multiple analytes are linked to a carrier (e.g., a C. simple synthetic scaffold, or a larger carrier protein or Alternatively, the magnetic particles can be associated with polysaccharide, such as BSA, transferrin, or dextran). 30 non-polymeric functional group compositions. Methods of Magnetic Particles synthesizing stabilized, functionalized magnetic particles The magnetic particles described herein include those without associated polymers are described, for example, in described, e.g., in U.S. Pat. No. 7,564.245 and U.S. Patent Halbreich et al., Biochimie, 80:379 (1998). Application Publication No. 2003-0092029, each of which is The magnetic particles typically include metal oxide mono incorporated herein by reference. The magnetic particles are 35 and polycrystals of about 1-25 nm, e.g., about 3-10 nm, or generally in the form of conjugates, that is, a magnetic par about 5 nm in diameter per crystal. The magnetic particles can ticle with one or more binding moieties (e.g., an oligonucle also include a polymer component in the form of a core and/or otide, nucleic acid, polypeptide, or polysaccharide) linked coating, e.g., about 5 to 20 nm thick or more. The overall size thereto. The binding moiety causes a specific interaction with of the magnetic particles can be, e.g., from 20 to 50 nm, from a target analyte. The binding moiety specifically binds to a 40 50 to 200 nm, from 100 to 300 nm, from 250 to 500 nm, from selected target analyte, for example, a nucleic acid, polypep 400 to 600 nm, from 500 to 750 nm, from 700 to 1,200 nm, tide, or polysaccharide. In some instances, binding causes from 1,000 to 1,500 nm, or from 1,500 to 2,000 nm. aggregation of the conjugates, resulting in a change, e.g., a The magnetic particles may be prepared in a variety of decrease (e.g., in the case of smaller magnetic particles) or an ways. It is preferred that the magnetic particle have functional increase (e.g., in the case of larger magnetic particles) in the 45 groups that link the magnetic particle to the binding moiety. spin-spin relaxation time (T2) of adjacent water protons in an Carboxy functionalized magnetic particles can be made, for aqueous solution (or protons in a non-aqueous solvent). Alter example, according to the method of Gorman (see PCT Pub natively, the analyte binds to a preformed aggregate in a lication No. WO00/61191). In this method, reduced car competitive disaggregation assay (e.g., an aggregate formed boxymethyl (CM) dextran is synthesized from commercial from a multivalent binding agent and magnetic particles), or 50 dextran. The CM-dextran and iron salts are mixed together competes with a multivalent binding agent for binding moi and are then neutralized with ammonium hydroxide. The eties on the magnetic particles in an inhibition assay (i.e., the resulting carboxy functionalized magnetic particles can be formation of aggregates is inhibited in the presence of the used for coupling amino functionalized oligonucleotides. analyte). Carboxy-functionalized magnetic particles can also be made The conjugates have high relaxivity owing to the Super 55 from polysaccharide coated magnetic particles by reaction paramagnetism of their iron, metal oxide, or other ferro or with bromo or chloroacetic acid in strong base to attach ferrimagnetic nanomaterials. Iron, cobalt, and nickel com carboxyl groups. In addition, carboxy-functionalized par pounds and their alloys, rare earth elements such as gado ticles can be made from amino-functionalized magnetic par linium, and certain intermetallics such as gold and Vanadium ticles by converting amino to carboxy groups by the use of are ferromagnets can be used to produce Superparamagnetic 60 reagents such as Succinic anhydride or maleic anhydride. particles. The magnetic particles can be monodisperse (a Magnetic particle size can be controlled by adjusting reac single crystal of a magnetic material, e.g., metal oxide. Such tion conditions, for example, by using low temperature during as Superparamagnetic iron oxide, per magnetic particle) or the neutralization of iron salts with a base as described in U.S. polydisperse (e.g., a plurality of crystals per magnetic par Pat. No. 5.262,176. Uniform particle size materials can also ticle). The magnetic metal oxide can also include cobalt, 65 be made by fractionating the particles using centrifugation, magnesium, zinc, or mixtures of these metals with iron. ultrafiltration, or gel filtration, as described, for example in Important features and elements of magnetic particles that are U.S. Pat. No. 5,492,814. US 8,409,807 B2 51 52 Magnetic particles can also be synthesized according to the the particles may be decorated with antihis antibodies; or the method of Molday (Molday, R. S., and D. MacKenzie, particles may be decorated with his-tagged FAbs. “Immunospecific ferromagnetic iron-dextran reagents for the Low molecular weight materials can be separated from the labeling and magnetic separation of cells. J. Immunol. Meth magnetic particles by ultra-filtration, dialysis, magnetic sepa ods, 52:353 (1982)), and treated with periodate to form alde ration, or other means prior to use. For example, unreacted hyde groups. The aldehyde-containing magnetic particles can binding moieties and linking agents can be separated from the then be reacted with a diamine (e.g., ethylene diamine or magnetic particle conjugates by magnetic separation or size hexanediamine), which will form a Schiff base, followed by exclusion chromatography. In certain instances the magnetic reduction with sodium borohydride or sodium cyanoborohy 10 particles can be fractionated by size to produce mixtures of dride. particles of a particular size range and average diameter. Dextran-coated magnetic particles can be made and cross linked with epichlorohydrin. The addition of ammonia reacts For certain assays requiring high sensitivity, analyte detec with epoxy groups to generate amine groups, see Hogemann, tion using T. relaxation assays can require selecting a proper D., et al., Improvement of MRI probes to allow efficient particle to enable sufficiently sensitive analyte-induced detection of gene expression Bioconjug. Chem., 11:941 15 agglomeration. Higher sensitivities can be achieved using (2000), and Josephson et al., “High-efficiency intracellular particles that contain multiple Superparamagnetic iron oxide magnetic labeling with novel Superparamagnetic-Tat peptide cores (5-15 nm diameter) within a single larger polymer conjugates. Bioconjug. Chem., 10:186 (1999). This material matrix or ferrofluid assembly (100 nm-1200 nm total diam is known as cross-linked iron oxide or “CLIO' and when eter, such as particles having an average diameter of 100 nm, functionalized with amine is referred to as amine-CLIO or 200 nm, 250 nm,300 nm,500 nm, 800 nm, or 1000 nm), or by NH-CLIO. Carboxy-functionalized magnetic particles can using a higher magnetic moment materials or particles with be converted to amino-functionalized magnetic particles by higher density, and/or particles with higher iron content. the use of water-soluble carbodiimides and diamines such as Without being limited by theory, it is postulated these types of ethylene diamine or hexane diamine. 25 particles provided a sensitivity gain of over 100x due to a The magnetic particles can be formed from a ferrofluid much higher number of iron atoms per particle, which is (i.e., a stable colloidal suspension of magnetic particles). For believed to lead to an increase in sensitivity due to the example, the magnetic particle can be a composite of includ decreased number of particles present in the assay Solution ing multiple metal oxide crystals of the order of a few tens of and possibly a higher amount of Superparamagnetic iron nanometers in size and dispersed in a fluid containing a Sur 30 affected by each clustering event. factant, which adsorbs onto the particles and stabilizes them, Relaxivity per particle and particle size is one useful term or by precipitation, in a basic medium, of a solution of metal for selecting an optimal particle for high sensitivity assays. ions. Suitable ferrofluids are sold by the company Liquids Ideally, this term will be as large as possible. Relaxivity per Research Ltd. under the references: WHKS1S9 (A, B or C), particle is a measure of the effect of each particle on the which is a water-based ferrofluid including magnetite 35 (FeO), having particles 10 nm in diameter; WHJS1 (A, B or measured T value. The larger this number, the fewer the C), which is an isoparaffin-based ferrofluid including par number of particles needed to elicit a given T. response. ticles of magnetite (FeO) 10 nm in diameter; and BKS25 Furthermore, lowering the concentration of particles in the dextran, which is a water-based ferrofluid stabilized with reactive Solution can improve the analytical sensitivity of the dextran, including particles of magnetite (Fe-O) 9 nm in 40 assay. Relaxivity per particle can be a more useful parameter diameter. Other suitable ferrofluids for use in the systems and in that the iron density and relaxivity can vary from magnetic methods of the invention are oleic acid-stabilized ferrofluids particle to magnetic particle, depending upon the components available from Ademtech, which include ca. 70% weight used to make the particles (see Table 1). Relaxivity per par O-Fe-O particles (ca. 10 nm in diameter), 15% weight ticle is proportional to the Saturation magnetization of a octane, and 15% weight oleic acid. 45 Superparamagnetic material. The magnetic particles are typically a composite including multiple metal oxide crystals and an organic matrix, and TABLE 1 having a Surface decorated with functional groups (i.e., amine groups or carboxy groups) for the linking binding moieties to Hydroynamic Relaxivity per the Surface of the magnetic particle. For example, the mag 50 Diameter # Metal Atoms Particle netic particles useful in the methods of the invention include (nm) per Particle (mM's) those commercially available from Dynal, Seradyn, Kisker, Miltenyi Biotec, Chemicell, Anvil, Biopal, Estapor, Genovis, 10-30 1.OE-03-1.OE-06 1.OE-6-1.OE+11 Thermo Fisher Scientific, JSR micro, Invitrogen, and 10-50 8.OE--O2-4.OE-04 1.OE-04-4.OE-06 Ademtech, as well as those described in U.S. Pat. Nos. 4,101, 55 10-50 1.OE-04-5.OE-05 1.OE-06-1.OE-08 435; 4,452,773: 5,204,457; 5,262,176; 5,424,419; 6,165,378: SO-100 1.OE-04-1.OE-07 1.OE-06-1.OE-09 6,866,838; 7,001,589; and 7,217,457, each of which is incor 100-200 S.OE-06-5.OE-07 S.OE-08-8.OE-09 porated herein by reference. 200-300 1.OE-07-1.OE-08 3.OE-09-10E-10 Avidin or streptavidin can be attached to magnetic particles 300-500 S.OE-07-1.OE-09 7.OE-09-5.OE-10 for use with a biotinylated binding moiety, Such as an oligo 60 SOO-800 1.OE-08-4.1E-09 1.OE-1O-5.OE-11 nucleotide or polypeptide (see, e.g., Shen et al., “Magneti 800-1000 S.OE-08-SOE-09 S.OE-1O-5.OE-11 cally labeled secretin retains receptor affinity to pancreas 1OOO-12OO 1.OE-09-7.OE-09 1.OE+11-1.OE+12 acinar cells. Bioconjug. Chem., 7:311 (1996)). Similarly, biotin can be attached to a magnetic particle for use with an avidin-labeled binding moiety. Alternatively, the binding 65 The base particle for use in the systems and methods of the moiety is covalently linked to the Surface of the magnetic invention can be any of the commercially available particles particle; the particles may be decorated with IgG molecules; identified in Table 2. US 8,409,807 B2 53 TABLE 2 TABLE 2-continued

Catalogue Diameter Catalogue Diameter No. Source/Description (Lm) No. Source/Description (Lm) Kisker Genovis

MAV-1 Polystyrene, Magnet Particles Avidin coated 1.0-19 AMI-25 Dextran 80-1SO PMSt-O.6 Polystyrene, Magnet Particles Streptavidin O.5-0.69 Thermo Fisher coated PMSt-O.7 Polystyrene, Magnet Particles Streptavidin O.7-0.9 451 S-21 OS Carboxylate-Modified (MG-CM) O coated 10 7815-2104 Neutravidin (MG-NA) O PMSt-1.0 Polystyrene, Magnet Particles Streptavidin 1.0-1.4 5915-2104 Streptavidin (MG-SA) O coated 2415-21 OS Carboxylate-Modified (MG-CM) O PMB-1 Polystyrene, Magnet Particles Biotin covalently 1.0-19 441 S-21 OS Carboxylate-Modified (MG-CM) O coupled o BSA coating JSR micro PMP-2OO Dextran based, No coating, plain 2 PMP-1OOO Dextran based, No coating, plain 15 MB100 Carboxylated .1 PMP-1300 Dextran based, No coating, plain Invitrogen PMP-2500 Dextran based, No coating, plain 2 PMN-1300 Dextran based, NH2-coated 3S4-01 Carboxylated PMN-2SOO Dextran based, NH2-coated 355-OO Tosylactivated PMC-1000 Dextran based, COOH- coated 650-11 Carboxylated PMC-1300 Dextran based, COOH- coated 655-11 Tosylactivated PMC-2SOO Dextran based, COOH- coated 22 Biopal PMAV-1300 Dextran based, Avidin coated PMAV-2SOO Dextran based, Avidin coated 5 M02Q05 Amino activated .5 PMSA-1OOO Dextran based, Streptavidin coated M02Q05 Biotin activated .5 PMSA-1300 Dextran based, Streptavidin coated M02Q05 Strepavidin activated .5 PMSA-2SOO Dextran based, Streptavidin coated 22 PMB-1000 Dextran based, Biotin coated 25 PMB-1300 Dextran based, Biotin coated The magnetic particles for use in the systems and methods PMB-2SOO Dextran based, Biotin coated 2 PMPA-1 OOO Dextran based, Protein A coated of the invention can have a hydrodynamic diameter from 10 PMPA-1300 Dextran based, Protein A coated O. 3 nm to 1200 nm, and containing on average from 8x10-1x PMPA-2SOO Dextran based, Protein A coated 10" metal atoms per particle, and having a relaxivity per PMC-0.1 Dextran based, COOH functionalized 0.1-0.4 30 particle of from 1x10-1x10" mM's'. The magnetic par PMC-04 Dextran based, COOH functionalized O4-O-7 PMC-O.7 Dextran based, COOH functionalized O.7-0.9 ticles used in the systems and methods of the invention can be PMC-10 Dextran based, COOH functionalized 1.0-1.4 any of the designs, composites, or sources described above, PMN-10 Dextran based, NH2 functionalized 1.0-1.4 and can be further modified has described herein for use as a PMC-0.1 Dextran based, COOH functionalized 0.1-0.4 magnetic resonance Switch. Accurate Chemical 35 In addition to relaxivity per particle, several other practical DMO)102O Carboxyl-functionality issues must be address in the selection and design of magnetic DMO 1030 Carboxyl-functionality particles for high analytical sensitivity assays. DMO2O20 Carboxyl-functionality For example, the use of large particles (i.e., 1000 nm or DMO2133 high Carboxyl-functionality greater) may be desired to maximize iron content and the DMO21SO Carboxyl-functionality 40 DMO2220 very hig h Amino-functionality relaxivity per particle. However, we have observed that par DMO2230 very hig h Amino-functionality ticles of this size tend to settle rapidly out of solution. We have DMO22SO Carboxyl-functionality observed that particle settling does not typically interfere DMO2O3O high Carboxyl-functionality with the assay if magnetic particle sizes are kept below 500 DMO2110 high Carboxyl-functionality nm. When use of a particle above 500 nm in the described DMO2120 very hig h Carboxyl-functionality 45 assays or Smaller particles with high density are employed, DMO2130 very hig h Carboxyl-functionality DMO2252 Carboxyl-functionality settling is monitored and effect on T. measurement is deter DMO312O Streptavidin-functionality mined. We have found a magnetic particle size of about 100 DMO3121 Streptavidin-functionality 300 nm particle to be ideal for stability in terms of settling, chemicell even after functionalization (increasing the hydrodynamic 50 diameter to 300 nm by approximately 50 nm), and to afford 1201-S 1 Si—(CH2). COO O.S the high sensitivity enabled by a high relaxivity per particle. 1201-S 1 Si—(CH2). COO 0.75 1201-S 1 Si—(CH2). COO 1.O Particle density certainly plays a role in buoyancy. As such, 1202-S 1 Si—(CH2)3. SOH O.S the relative density of the Solution and particles plays an 1202-S 1 Si-(CH2). SOH 0.75 important role in settling of the particle. Accordingly, a pos 1202-S 1 Si—(CH2)3. SOH 1.O 55 sible Solution to this problem is the use of buoyant magnetic 1205-1 Si—(CH2)3—POH O.S particles (i.e., a hollow particle, or particle containing both a 1205-1 Si-(CH2). POH 0.75 low density matrix and high density metal oxide). Settling 1205-1 Si—(CH2)3—POH 1.O Estapor may affect the T detection, thus, Solution additives may be employed to change the ratio of the particle to solution den M1-130/12 Carboxylated Polys yrene O.7-1.3 60 sity. T. detection can be impacted by settling if there is a M1-18012 Carboxylated Polys yrene O.9-1.3 significant portioning of the Superparamagnetic material M1-18020 Carboxylated Divinylbenzene O.8-1.2 from the measured Volume of liquid. Settling can be assessed M1-050,2O Carboxylated Polys yrene O.S.-O.7 M1-070.40 Carboxylated Polys yrene O.7-1.3 by diluting the particles to a concentration such that UV-Vis M1-070,60 Carboxylated Polys yrene O.7-1.3 absorbance at 410 nm is between 0.6-0.8 absorbance units M1-02O, SO Carboxylated Polys yrene O.16-0.24 65 and then monitoring the absorbance for 90 minutes. If settling M1-030.40 Carboxylated Polys yrene O.3-O.S occurs, the difference between the initial and final absor bances divided by the initial absorbance will be greater than US 8,409,807 B2 55 56 5%. If 96 settling is above 5% then the particle is typically not ticle which reduce non-specific binding of background pro Suitable for use in assays requiring high analytical sensitivity. teins. There are a variety of agents that one could use to The magnetic particles used in the assays of the invention can achieve the desired effect, and in some cases, it is a combi be, but are not limited to, nonsettling magnetic particles. High nation of agents that is optimal (see Table 3; exemplary par settling represents handling difficulties and may lead to repro 5 ticles, coatings, and binding moieties). ducibility issues. For magnetic particles on the order of 100 nm or larger, the TABLE 3 multiple Superparamagnetic iron oxide crystals that typically include the particle core results in a net dipole moment when Base Particle Coating Binding Moiety 10 in the presence of external magnetic fields, i.e. the dipole NP-COOH: amino Dextran Small molecule monment is a Sufficient force to overcome Brownian motion. Transferrin Nonspecific reversibility is a measure of the colloidal stability Lysozyme and robustness against non-specific aggregation. Nonspecific BSA FSG reversibility is assessed by measuring the T values of a solu BGG tion of particles before and after incubation in a uniform 15 Ovalbumin magnetic field (defined as <5000 ppm). Starting T values are amino PEG Human albumin typically 200 ms for a particle with an iron concentration of Ole Antibody 0.01 mM Fe. If the difference in T values before and after amino PEG incubation in the uniform magnetic field is less than 20 ms, BSA the samples are deemed reversible. Further, 10% is a thresh amino Dextran old allowing starting T, measurements to reflect assay par NP-amino: Ole Small molecule PEG ticle concentration. If the difference is greater than 10%, then NP-SA: Ole biotinylated Ab the particles exhibit irreversibility in the buffer, diluents, and biotinylated amino Antibody matrix tested. The MAA reversibility of the magnetic par PEG 25 NP-SA: biotinylated amino Small molecule ticles can be altered as described herein. For example, colloi PEG dal stability and robustness against non-specific aggregation NP-anti- Ole Antibody can be influenced by the surface characteristics of the par species: ticles, the binding moieties, the assay buffer, the matrix and NP-N: Ole his-tagged antibody the assay processing conditions. Maintenance of colloidal stability and resistance to non-specific biding can be altered 30 by conjugation chemistry, blocking methods, buffer modifi Thus, we have found a protein block may be required to cations, and/or changes in assay processing conditions. achieve assay activity and sensitivity, particularly in proteina We have observed that a very important attribute for robust ceous samples (e.g., plasma samples or whole blood and reproducible assays is the monodispersity in the size samples), that is comparable to results in nonproteinaceous distribution of the magnetic particles used, a distinction 35 buffer samples. Some commonly used protein blockers which observed in polydisperse particles post-coating versus mono may be used in provided preparations include, e.g., bovine disperse particle pre-coating. Polydisperse batches of mag serum albumin (BSA), fish skin gelatin (FSG), bovine gamma netic particles can lack reproducibility and compromise sen globulin (BGG), lysozyme, casein, peptidase, or non-fat dry sitivity. Polydisperse samples can also present problems in milk. In certain embodiments a magnetic particle coating terms of achieving uniform coatings. For certain highly sen 40 includes BSA or FSG. In other embodiments, a combination sitive assays it is desirable that the magnetic particles be of coatings are combinations of those exemplary coatings Substantially monodisperse in size distribution (i.e., having a listed in Table 3. polydispersity index of less than about 0.8-0.9). Alternatively, Furthermore, nonspecific binding can be due to lipids or the assays of the invention can be designed to accommodate other non-proteinaceous molecules in the biological sample. the use of polydisperse magnetic particles. 45 For non-proteinaceous mediated non-specific binding, Given that the assays of the invention require monitoring a changes in pH and buffer ionic strength maybe selected to shift in the clustering states of the agglomeration assays and enhance the particle repulsive forces, but not enough to limit that measuring a change in clustering likely requires a sig the results of the intended interactions. nificant fraction of clustered particles (e.g., thought to be Assay Reagents >1-10%), the total number of particles in an assay should be 50 The assays of the invention can include reagents for reduc minimized to enable the highest sensitivity. However, suffi ing the non-specific binding to the magnetic particles. For cient number of particles must be present to allow utilization example, the assay can include one or more proteins (e.g., of the T detection dynamic range. We have found that the albumin, fish skin gelatin, lysozyme, or transferrin); low highest sensitivity is observed when the number of magnetic molecular weight (<500 Daltons) amines (e.g., amino acids, particles (or molar equivalent) is approximately on the same 55 glycine, ethylamine, or mercaptoethanol amine); and/or order of magnitude of the number (or molar equivalent) of the water soluble non-ionic Surface active agents (e.g., polyeth analyte being detected, and the magnitude of the number (or yleneglycol, Tween(R) 20, Tween R 80, Pluronic R, or molar equivalent) multivalent binding agents employed (i.e., Igepal(R) (see Table 4). in an inhibition assay). For proteinaceous samples it may also be required to 60 TABLE 4 modify the magnetic particle Surface to reduce non-specific Blocking Agents binding of background proteins to the magnetic particles. Non-specific binding of background proteins to particles can PEG BSA Bovine serum albumin induce or impede particle clustering, resulting in false signals HSA—Human serum albumin and/or false lack of signals. For example, in Some instances 65 FSG—Fish skin gelatin the Surface of the magnetic particle can include blocking Lysozyme agents covalently linked to the Surface of the magnetic par US 8,409,807 B2 57 58 TABLE 4-continued certain embodiments, the binding moieties can be designed or selected to serve, when bound to another binding moiety, as Blocking Agents Substrates for a target molecule Such as enzyme in Solution. Transferrin Binding moieties include, for example, oligonucleotide Glycine or other Small amine containing 5 binding moieties (DNA, RNA, or substituted or derivatized molecules nucleotide Substitutes), polypeptide binding moieties, anti Ethylamine Mercaptoethanolamine body binding moieties, aptamers, and polysaccharide binding Tween 20 moieties. Tween 80 Oligonucleotide Binding Moieties Pluronic 10 In certain embodiments, the binding moieties are oligo Igepal Triton X-100 nucleotides, attached/linked to the magnetic particles using Other surfactants/detergents any of a variety of chemistries, by a single, e.g., covalent, bond, e.g., at the 3' or 5' end to a functional group on the magnetic particle. Such binding moieties can be used in the The surfactant may be selected from a wide variety of is systems, devices, and methods of the invention to detect soluble non-ionic Surface active agents including Surfactants mutations (e.g., SNPs, translocations, large deletions, Small that are generally commercially available under the IGEPAL deletions, insertions, Substitutions) or to monitor gene trade name from GAF Company. The IGEPAL liquid non expression (e.g., the presence of expression, or changes in the ionic Surfactants are polyethylene glycol p-isooctylphenyl level of gene expression, monitoring RNA transcription), or ether compounds and are available in various molecular 20 CHP analysis characteristic of the presence of a pathogen, weight designations, for example, IGEPAL CA720, IGEPAL disease state, or the progression of disease. CA630, and IGEPAL CA890. Other suitable non-ionic Sur An oligonucleotide binding moiety can be constructed factants include those available under the trade name using chemical synthesis. A double-stranded DNA binding TETRONIC 909 from BASF Wyandotte Corporation. This moiety can be constructed by enzymatic ligation reactions material is a tetra-functional block copolymer Surfactant ter- 25 using procedures known in the art. For example, a nucleic minating in primary hydroxyl groups. Suitable non-ionic Sur acid (e.g., an oligonucleotide) can be chemically synthesized factants are also available under the VISTAALPHONIC trade using naturally occurring nucleotides or variously modified name from Vista Chemical Company and Such materials are nucleotides designed to increase the biological stability of the ethoxylates that are non-ionic biodegradables derived from molecules or to increase the physical stability of the duplex linear primary alcohol blends of various molecular weights. 30 formed between the complementary strands, e.g., phospho The Surfactant may also be selected from poloxamers, such as rothioate derivatives and acridine substituted nucleotides can polyoxyethylene-polyoxypropylene block copolymers, such be used. The nucleic acid also can be produced biologically as those available under the trade names Symperonic PE series using an expression vector into which a nucleic acid has been (ICI), PluronicR) series (BASF), Supronic, Monolan, Plura subcloned. care, and Plurodac, polysorbate surfactants, such as Tween R 35 One method uses at least two populations of oligonucle 20 (PEG-20 sorbitan monolaurate), and glycols such as eth otide magnetic particles, each with strong effects on water (or ylene glycol and propylene glycol. other solvent) relaxation. As the oligonucleotide-magnetic Such non-ionic Surfactants may be selected to provide an particle conjugates react with a target oligonucleotide, they appropriate amount of detergency foranassay without having form aggregates (e.g., clusters of magnetic particles). Upon a deleterious effect on assay reactions. In particular, Surfac- 40 prolonged standing, e.g., overnight at room temperature, the tants may be included in a reaction mixture for the purpose of aggregates form large clusters (micron-sized clusters). Using Suppressing non-specific interactions among various ingredi the methods of the invention, the formation of large clusters ents of the aggregation assays of the invention. The non-ionic can be accomplished more quickly by employing multiple Surfactants are typically added to the liquid sample prior in an cycles of magnetic assisted agglomeration. Magnetic reso amount from 0.01% (w/w) to 5% (w/w). 45 nance is used to determine the relaxation properties of the The non-ionic Surfactants may be used in combination with solvent, which are altered when the mixture of magnetic one or more proteins (e.g., albumin, fish skin gelatin, oligonucleotide magnetic particles reacts with a target lysozyme, or transferrin) also added to the liquid sample prior nucleic acid to form aggregates. in an amount from 0.01% (w/w) to 5% (w/w). Certain embodiments employ a mixture of at least two Furthermore, the assays, methods, and cartridge units of 50 types of magnetic metal oxide magnetic particles, each with a the invention can include additional suitable buffer compo specific sequence of oligonucleotide, and each with more nents (e.g., Tris base, selected to provide a pH of about 7.8 to than one copy of the oligonucleotide attached, e.g., 8.2 in the reaction milieu); and chelating agents to Scavenge covalently, per magnetic particle. For example, the assay cations (e.g., EDTA disodium, ethylene diamine tetraacetic protocol may involve preparing a mixture of populations of acid (EDTA), citric acid, tartaric acid, glucuronic acid, sac- 55 oligonucleotide-magnetic particle conjugates and reacting charic acid or suitable salts thereof). the mixture with a target nucleic acid. Alternatively, oligo Binding Moieties nucleotide-magnetic particle conjugates can be reacted with In general, a binding moiety is a molecule, synthetic or the target in a sequential fashion. Certain embodiments fea natural, that specifically binds or otherwise links to, e.g., ture the use of magnetic resonance to detect the reaction of the covalently or non-covalently binds to or hybridizes with, a 60 oligonucleotide-magnetic particle conjugates with the target target molecule, or with another binding moiety (or, in certain nucleic acid. When a target is present, the dispersed conju embodiments, with an aggregation inducing molecule). For gates self-assemble to form Small aggregates. example, the binding moiety can be an antibody directed For example, oligonucleotide binding moieties can be toward an antigen or any protein-protein interaction. Alterna linked to the metal oxide through covalent attachment to a tively, the binding moiety can be a polysaccharide that binds 65 functionalized polymer or to non-polymeric Surface-func to a corresponding target or a synthetic oligonucleotide that tionalized metal oxides. In the latter method, the magnetic hybridizes to a specific complementary nucleic acid target. In particles can be synthesized according to the method of US 8,409,807 B2 59 60 Albrecht et al., Biochimie, 80:379 (1998). Dimercapto-suc binding moiety-magnetic particle is used to measure the pres cinic acid is coupled to the iron oxide and provides a carboxyl ence of analytes in a test media reacting with the binding functional group. moiety. In certain embodiments, oligonucleotides are attached to Additionally, a polypeptide binding moiety can be used in magnetic particles via a functionalized polymer associated a universal reagent configuration, where the target of the with the metal oxide. In some embodiments, the polymer is binding moiety (e.g., Small molecule, ligand, or binding part hydrophilic. In certain embodiments, the conjugates are made ner) is pre-attached to the target analyte to create a labeled using oligonucleotides that have terminal amino, Sulfhydryl, analyte that, in the presence of the polypeptide decorated orphosphate groups, and Superparamagnetic iron oxide mag particles, induces clustering. 10 Examples of protein hormones which can be utilized as netic particles bearing amino or carboxy groups on a hydro binding moieties include, without limitation, platelet-derived philic polymer. There are several methods for synthesizing growth factor (PDGF), which binds the PDGF receptor; insu carboxy and amino derivatized-magnetic particles. lin-like growth factor-I and -II (Igf), which binds the Igf In one embodiment, oligonucleotides are attached to a receptor; nerve growth factor (NGF), which binds the NGF particle via ligand-protein binding interaction, Such as biotin 15 receptor; fibroblast growth factor (FGF), which binds the streptavidin, where the ligand is covalently attached to the FGF receptor (e.g., aFGF and bFGF); epidermal growth fac oligonucleotide and the protein to the particle, or vice versa. tor (EGF), which binds the EGF receptor; transforming This approach can allow for more rapid reagent preparation. growth factor (TGF, e.g., TGFC. and TGF-B), which bind the Otherforms of oligonucleotides may be used. For example, TGF receptor, erythropoietin, which binds the erythropoitin aptamers are single-stranded RNA or DNA oligonucleotides receptor; growth hormone (e.g., human growth hormone), 15 to 60 base in length that in solution form intramolecular which binds the growth hormone receptor; and proinsulin, interactions that fold the linear nucleic acid molecule into a insulin, A-chain insulin, and B-chain insulin, which all bind three dimensional complex that then can bind with high affin to the insulin receptor. ity to specific molecular targets; often with equilibrium con Receptor binding moieties are useful for detecting and stants in the range of 1 pM to 1 nM which is similar to some 25 imaging receptor clustering on the Surface of a cell. Useful monoclonal antibodies-antigen interactions. Aptamers can ectodomains include those of the Notch protein, Delta pro specifically bind to other nucleic acid molecules, proteins, tein, integrins, cadherins, and other cell adhesion molecules. Small organic compounds, Small molecules, and cells (organ Antibody Binding Moieties isms or pathogens). Other polypeptide binding moieties include immunoglo Polypeptide Binding Moieties 30 bulin binding moieties that include at least one immunoglo In certain embodiments, the binding moiety is a polypep bulin domain, and typically at least two such domains. An tide (i.e., a protein, polypeptide, or peptide), attached, using “immunoglobulin domain” refers to a domain of an antibody any of a variety of chemistries, by a single covalent bond in molecule, e.g., a variable or constant domain. An "immuno Such a manner so as to not affect the biological activity of the globulin Superfamily domain refers to a domain that has a polypeptide. In one embodiment, attachment is done through 35 three-dimensional structure related to an immunoglobulin the thiol group of single reactive cysteine residue so placed domain, but is from a non-immunoglobulin molecule. Immu that its modification does not affect the biological activity of noglobulin domains and immunoglobulin Superfamily the polypeptide. In this regard the use of linear polypeptides, domains typically include two B-sheets formed of about with cysteine at the C-terminal or N-terminal end, provides a seven B-strands, and a conserved disulfide bond (see, e.g., single thiol in a manner similar to which alkanethiol Supplies 40 Williams and Barclay Ann. Rev Immunol. 6:381 (1988)). a thiol group at the 3' or 5' end of an oligonucleotide. Similar Proteins that include domains of the Ig Superfamily domains bifunctional conjugation reagents, such as SPDP and reacting include T cell receptors, CD4, platelet derived growth factor with the amino group of the magnetic particle and thiol group receptor (PDGFR), and intercellular adhesion molecule of the polypeptide, can be used with anythiol bearing binding (ICAM). moiety. The types of polypeptides used as binding moieties 45 One type of immunoglobulin binding moiety is an anti can be antibodies, antibody fragments, and natural and Syn body. The term “antibody, as used herein, refers to a full thetic polypeptide sequences. The peptide binding moieties length, two-chain immunoglobulin molecule and an antigen have a binding partner, that is, a molecule to which they binding portion and fragments thereof, including synthetic selectively bind. variants. A typical antibody includes two heavy (H) chain Use of peptides as binding moieties offers several advan 50 variable regions (abbreviated herein as VH), and two light (L) tages. For example, polypeptides can be engineered to have chain variable regions (abbreviated herein as VL). The VH uniquely reactive residues, distal from the residues required and VL regions can be further subdivided into regions of for biological activity, for attachment to the magnetic particle. hyperVariability, termed "complementarity determining The reactive residue can be a cysteine thiol, an N-terminal regions' (CDR), interspersed with regions that are more con amino group, a C-terminal carboxyl group or a carboxyl 55 served, termed “framework regions’ (FR). The extent of the group of aspartate or glutamate, etc. A single reactive residue framework region and CDR's has been precisely defined (see, on the peptide is used to insure a unique site of attachment. Kabat, E. A., et al. (1991) Sequences of Proteins of Immuno These design principles can be followed with chemically logical Interest, Fifth Edition, U.S. Department of Health and synthesized peptides or biologically produced polypeptides. Human Services, NIH Publication No. 91-3242, and Chothia The binding moieties can also contain amino acid 60 et al., J. Mol. Biol., 196:901 (1987)). Each VH and VL is sequences from naturally occurring (wild-type) polypeptides composed of three CDR's and four FRs, arranged from or proteins. For example, the natural polypeptide may be a amino-terminus to carboxy-terminus in the following order: hormone, (e.g., a cytokine, a growth factor), a serum protein, FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. a viral protein (e.g., hemagglutinin), an extracellular matrix An antibody can also include a constant region as part of a protein, a lectin, or an ectodomain of a cell Surface protein. 65 light or heavy chain. Light chains can include a kappa or Another example is a ligand binding protein, such as Strepta lambda constant region gene at the COOH-terminus (termed vidin or avidin that bind biotin. In general, the resulting CL). Heavy chains can include, for example, a gamma con US 8,409,807 B2 61 62 stant region (IgG1, IgG2, IgG3, IgG4; encoding about 330 Another feature of the methods includes identification of amino acids). A gamma constant region can include, e.g., specific cell types, for hematological or histopatholgical CH1, CH2, and CH3. The term “full-length antibody” refers investigations for example CD4/CD3 cell counts and circu to a protein that includes one polypeptide that includes VL lating tumor cells using any of the binding moieties described and CL, and a second polypeptide that includes VH, CH1, above. CH2, and CH3. Multivalent Binding Agents The term “antigen-binding fragment of an antibody, as The assays of the invention can include a multivalent bind used herein, refers to one or more fragments of a full-length ing agent (i) bearing multiple analytes are linked to a carrier antibody that retain the ability to specifically bind to a target. (e.g., a simple synthetic scaffold, or a larger carrier protein or Examples of antigen-binding fragments include, but are not 10 polysaccharide, such as BSA, transferrin, or dextran), or bear limited to: (i) an Fab fragment, a monovalent fragment con ing multiple epitopes forbinding to, for example, two or more sisting of the VL, VH, CL and CH1 domains; (ii) an F(ab') populations of magnetic particles to form an aggregate. fragment, a bivalent fragment including two Fab fragments Where a multivalent binding agent is employed, multiple linked by a disulfide bridge at the hinge region; (iii) an Fd analytes can be linked to a carrier (e.g., a simple synthetic fragment consisting of the VH and CH1 domains; (iv) an Fv 15 scaffold, or a larger carrier protein or polysaccharide, Such as fragment consisting of the VL and VH domains of a single BSA, transferrin, or dextran). Alternatively, the multivalent arm of an antibody, (v) a dAb fragment (Ward et al., Nature binding agent can be a nucleic acid designed to bind to two or 341:544 (1989)), which consists of a VH domain; and (vi) an more populations of magnetic particles. Such multivalent isolated complementarity determining region (CDR). Fur binding agents act as agglomerants and the assay architecture thermore, although the two domains of the Fv fragment, VL is characterized by a competition between the analyte being and VH, are coded for by separate genes, they can be joined, detected and the multivalent binding agent (e.g., in an inhi using recombinant methods, by a synthetic linker that enables bition assay, competition assay, or disaggregation assay). them to be made as a single protein chain in which the VL and The functional group present in the analyte can be used to VH regions pair to form monovalent molecules (known as form a covalent bond with the carrier. Alternatively, the ana single chain Fv (sclv); see e.g., Bird et al., Science 242:423 25 lyte can be derivatized to provide a linker (i.e., a spacer (1988); and Huston et al., Proc. Natl. Acad. Sci. USA, separating the analyte from the carrier in the conjugate) ter 85:5879 (1988)). Such single chain antibodies are also minating in a functional group (i.e., an alcohol, an amine, a encompassed within the term “antigen-binding fragment.” carboxyl group, a Sulfhydryl group, or a phosphate group), A single domain antibody (SdAb, nanobody) is an antibody which is used to form the covalent linkage with the carrier. fragment consisting of a single monomeric variable antibody 30 The covalent linking of an analyte and a carrier may be domain, and may also be used in the systems and methods of effected using a linker which contains reactive moieties the invention. Like a whole antibody, sdAbs are able to bind capable of reaction with such functional groups present in the selectively to a specific antigen. With a molecular weight of analyte and the carrier. For example, a hydroxyl group of the only 12-15 kDa, single domain antibodies are much smaller analyte may react with a carboxyl group of the linker, or an than common antibodies (150-160 kDa) which are composed 35 activated derivative thereof, resulting in the formation of an of two heavy protein chains and two light chains, and even ester linking the two. smaller than Fab fragments (~50kDa, one light chain and half Examples of moieties capable of reaction with sulfhydryl a heavy chain) and single-chain variable fragments (~25kDa, groups include C-haloacetyl compounds of the type two variable domains, one from a light and one from a heavy XCHCO— (where X=Br, C1 or I), which show particular chain). 40 reactivity for sulfhydryl groups, but which can also be used to Polysaccharide Binding Moieties modify imidazolyl, thioether, phenol, and amino groups as In certain embodiments, the binding moiety is a polysac described by Gurd, Methods Enzymol. 11:532 (1967). N-Ma charide, linked, for example, using any of a variety of chem leimide derivatives are also considered selective towards Sulf istries, by a single bond, e.g., a covalent bond, at one of the hydryl groups, but may additionally be useful in coupling to two ends, to a functional group on the magnetic particle. The 45 amino groups under certain conditions. Reagents such as polysaccharides can be synthetic or natural. Mono-, di-, tri 2-iminothiolane (Traut et al., Biochemistry 12:3266 (1973)), and polysaccharides can be used as the binding moiety. These which introduce athiol group through conversion of an amino include, e.g., glycosides, N-glycosylamines, O-acyl deriva group, may be considered as Sulfhydryl reagents if linking tives, O-methyl derivatives, osaZones, Sugar alcohols, Sugar occurs through the formation of disulphide bridges. acids, Sugar phosphates when used with appropriate attach 50 Examples of reactive moieties capable of reaction with ment chemistry to the magnetic particle. amino groups include, for example, alkylating and acylating A method of accomplishing linking is to couple avidin to a agents. Representative alkylating agents include: (i) C.-halo magnetic particle and react the avidin-magnetic particle with acetyl compounds, which show specificity towards amino commercially available biotinylated polysaccharides, to yield groups in the absence of reactive thiol groups and are of the polysaccharide-magnetic particle conjugates. For example, 55 type XCH-CO— (where X=Cl, Br or I), for example, as sialyl Lewis based polysaccharides are commercially avail described by Wong, Biochemistry 24:5337 (1979); (ii) N-ma able as biotinylated reagents and will react with avidin-CLIO leimide derivatives, which may react with amino groups (see Syntesome, Gesellschaft fur medizinische Biochemie either through a Michael type reaction orthrough acylation by mbH.). The sialyl Lewis Xtetrasaccharide (Sle') is recognized addition to the ring carbonyl group, for example, as described by proteins known as Selectins, which are present on the 60 by Smyth et al., J. Am. Chem. Soc. 82:4600 (1960) and Surfaces of leukocytes and function as part of the inflamma Biochem. J. 91:589 (1964); (iii) aryl halides such as reactive tory cascade for the recruitment of leukocytes. nitrohaloaromatic compounds; (iv) alkyl halides, as Still other targeting moieties include a non-proteinaceous described, for example, by McKenzie et al., J. Protein Chem. element, e.g., a glycosyl modification (such as a Lewis anti 7:581 (1988); (v) aldehydes and ketones capable of Schiffs gen) or another non-proteinaceous organic molecule. 65 base formation with amino groups, the adducts formed usu Another method is covalent coupling of the protein to the ally being stabilized through reduction to give a stable amine; magnetic particle. (vi) epoxide derivatives such as epichlorohydrin and bisox US 8,409,807 B2 63 64 iranes, which may react with amino, Sulfhydryl, or phenolic a spacer permits bifunctional linkers to react with specific hydroxyl groups; (vii) chlorine-containing derivatives of functional groups within the analyte and the carrier, resulting S-triazines, which are very reactive towards nucleophiles Such in a covalent linkage between the two. The reactive moieties as amino, Sufhydryl, and hydroxyl groups; (viii) aziridines in a linker may be the same (homobifunctional linker) or based on S-triazine compounds detailed above, e.g., as different (heterobifunctional linker, or, where several dis described by Ross, J. Adv. Cancer Res. 2:1 (1954), which similar reactive moieties are present, heteromultifunctional react with nucleophiles such as amino groups by ring open linker), providing a diversity of potential reagents that may ing; (ix) Squaric acid diethyl esters as described by Tietze, bring about covalent attachment between the analyte and the Chem. Ber. 124:1215 (1991); and (x) C.-haloalkyl ethers, carrier. which are more reactive alkylating agents than normal alkyl 10 Spacer elements in the linker typically consist of linear or halides because of the activation caused by the ether oxygen branched chains and may include a Co alkyl, a heteroalkyl atom, as described by Benneche et al., Eur. J. Med. Chem. of 1 to 10 atoms, a Coalkene, a C2-lo alkyne, Cso aryl, a 28:463 (1993). cyclic system of 3 to 10 atoms, or —(CH2CH2O) Representative amino-reactive acylating agents include: (i) CHCH , in which n is 1 to 4. isocyanates and isothiocyanates, particularly aromatic 15 Typically, a multivalent binding agent will include 2, 3, 4, derivatives, which form stable urea and thiourea derivatives 5,6,7,8, 15,50, or 100 (e.g., from 3 to 100, from 3 to 30, from respectively; (ii) sulfonyl chlorides, which have been 4 to 25, or from 6 to 20) conjugated analytes. The multivalent described by Herzig et al., Biopolymers 2:349 (1964); (iii) binding agents are typically from 10 kDa to 200 kDa in size acid halides; (iv) active esters such as nitrophenylesters or and can be prepared as described in the Examples. N-hydroxysuccinimidyl esters; (V) acid anhydrides such as Analytes mixed, symmetrical, or N-carboxyanhydrides; (vi) other use Embodiments of the invention include devices, systems, ful reagents for amide bond formation, for example, as and/or methods for detecting and/or measuring the concen described by M. Bodansky, Principles of Peptide Synthesis, tration of one or more analytes in a sample (e.g., a protein, a Springer-Verlag, 1984; (vii) acylazides, e.g. wherein the peptide, an enzyme, a polypeptide, an amino acid, a nucleic azide group is generated from a preformed hydrazide deriva 25 acid, an oligonucleotide, a therapeutic agent, a metabolite of tive using sodium nitrite, as described by Wetz et al., Anal. a therapeutic agent, RNA, DNA, circulating DNA (e.g., from Biochem. 58:347 (1974); and (viii) imidoesters, which form a cell, tumor, pathogen, or fetus), an antibody, an organism, a stable amidines on reaction with amino groups, for example, virus, bacteria, a carbohydrate, a polysaccharide, glucose, a as described by Hunter and Ludwig, J. Am. Chem. Soc. lipid, a gas (e.g., oxygen and/or carbon dioxide), an electro 84:3491 (1962). Aldehydes and ketones may be reacted with 30 lyte (e.g., sodium, potassium, chloride, bicarbonate, BUN. amines to form Schiffs bases, which may advantageously be magnesium, phosphate, calcium, ammonia, and/or lactate), stabilized through reductive amination. Alkoxylamino moi general chemistry molecules (creatinine, glucose), a lipopro eties readily react with ketones and aldehydes to produce tein, cholesterol, a fatty acid, a glycoprotein, a proteoglycan, stable alkoxamines, for example, as described by Webb et al., and/or a lipopolysaccharide). The analytes may include iden Bioconjugate Chem. 1:96 (1990). 35 tification of cells or specific cell types. The analyte(s) may Examples of reactive moieties capable of reaction with include one or more biologically active Substances and/or carboxyl groups include diazo compounds such as diaZoac metabolite(s), marker(s), and/or other indicator(s) of biologi etate esters and diazoacetamides, which react with high speci cally active Substances. A biologically active Substance may ficity to generate ester groups, for example, as described by be described as a single entity or a combination of entities. Herriot, Adv. Protein Chem. 3:169 (1947). Carboxyl modify 40 The term “biologically active substance' includes without ing reagents such as carbodiimides, which react through limitation, medications; vitamins; mineral Supplements; Sub O-acylurea formation followed by amide bond formation, stances used for the treatment, prevention, diagnosis, cure or may also be employed. mitigation of disease or illness; or Substances which affect the It will be appreciated that functional groups in the analyte structure or function of the body; or pro-drugs, which become and/or the carrier may, if desired, be converted to other func 45 biologically active or more active after they have been placed tional groups prior to reaction, for example, to confer addi in a predetermined physiological environment; or biologi tional reactivity or selectivity. Examples of methods useful cally toxic agents such as those used in biowarfare including for this purpose include conversion of amines to carboxyls organisms such as anthrax, ebola, Salmonella typhimurium, using reagents such as dicarboxylic anhydrides; conversion Marburg virus, plague, cholera, Francisella tulariesis (tula of amines to thiols using reagents such as N-acetylhomocys 50 remia), brucellosis, Q fever, Bolivian hemorrhagic fever, teine thiolactone, S-acetylmercaptosuccinic anhydride, Coccidioides mycosis, glanders, Melioidosis, Shigella, 2-iminothiolane, or thiol-containing Succinimidyl deriva Rocky Mountain spotted fever, typhus, Psittacosis, yellow tives; conversion of thiols to carboxyls using reagents such as fever, Japanese Bencephalitis, Rift Valley fever, and small C-haloacetates; conversion of thiols to amines using reagents poX; naturally-occurring toxins that can be used as weapons Such as ethylenimine or 2-bromoethylamine; conversion of 55 include ricin, aflatoxin, SEB, botulinum toxin, saxitoxin, and carboxyls to amines using reagents such as carbodiimides many mycotoxins. Analytes may also include organisms such followed by diamines; and conversion of alcohols to thiols as Candida albicans, Candida glabrata, Candida krusei, using reagents such as tosyl chloride followed by transesteri Candida parapsilosis, Candida tropicalis, Coagulase nega fication with thioacetate and hydrolysis to the thiol with tive Staphalococcus, Enterococcus faecalis, Enterococcus Sodium acetate. 60 faecium, Escherichia coli, Klebsiella pneumonia, Pseudomo So-called Zero-length linkers, involving direct covalent nas aeruginosa, Staphylococcus aureus, Acinetobacter bau joining of a reactive chemical group of the analyte with a mannii, Aspergillus filmigates, Bacteroides fragilis, Bacteroi reactive chemical group of the carrier without introducing des fragilis, blaSHV, Burkholderia cepacia, Campylobacter additional linking material may, if desired, be used in accor jejuni/coli, Candida guilliermondii, Candida lusitaniae, dance with the invention. Most commonly, however, the 65 Clostridium pefringens, Enterobacter aeraogenesl, Entero linker will include two or more reactive moieties, as described bacter cloacae, Enterobacteriaceae spp., Haemophilus influ above, connected by a spacer element. The presence of Such enza, Kingella kingae, Klebsiella Oxytoca, Listeria monocy US 8,409,807 B2 65 66 togenes, Mec A gene-bearing bacteria (MRSA), Morganella losporin antibiotics, macrollide antibiotics, miscellaneous morgana, Neisseria meningitides, Neisseria spp., non-men B-lactam antibiotics, penicillin antibiotics, quinolone antibi ingitidis, Prevotella buccae, Prevotella intermedia, Prevo otics, Sulfonamide antibiotics, tetracycline antibiotics, anti tella melaminogenica, Propionibacterium acnes, Proteus mycobacterials, antituberculosis antimycobacterials, anti mirabilis, Proteus vulgaris, Salmonella enteric, Serratia protozoals, antimalarial antiprotozoals, antiviral agents, marcescens, Staphylococcus haemolyticus, Staphylococcus antiretroviral agents, Scabicides, and urinary anti-infectives; maltophilia, Staphylococcus saprophyticus, Stenotrophomo antineoplastic agents, such as alkylating agents, nitrogen nas maltophilia, Stenotrophomonas maltophilia, Streptococ mustard alkylating agents, nitrosourea alkylating agents, cus agalactie, Streptococcus bovis, Streptococcus dysgalac antimetabolites, purine analog antimetabolites, pyrimidine tie, Streptococcus initis, Streptococcus initita F2S, 10 analog antimetabolites, hormonal antineoplastics, natural Streptococcus pneumonia, Streptococcus pyogenes, Strepto antineoplastics, antibiotic natural antineoplastics, and Vinca coccus sanguinis, Van Agene, Van B gene. Analytes may also alkaloid natural antineoplastics; autonomic agents, such as include viral organisms such as dsDNA viruses (e.g., aden anticholinergics, antimuscarinic anticholinergics, ergot alka oviruses, herpes viruses, poxviruses); ssDNA viruses loids, parasympathomimetics, cholinergic agonist parasym (+)sense DNA (e.g., parvoviruses); dsRNA viruses (e.g., 15 pathomimetics, cholinesterase inhibitor parasympathomi reoviruses); (+)ssRNA viruses (+)sense RNA (e.g., picor metics, sympatholytics, alpha-blocker sympatholytics, beta naviruses, togaviruses): (-)ssRNA viruses (-)sense RNA blocker sympatholytics, sympathomimetics, and adrenergic (e.g., orthomyxoviruses, rhabdoviruses); ssRNA-RT viruses agonist sympathomimetics; cardiovascular agents, such as (+)sense RNA with DNA intermediate in life-cycle (e.g., antianginals, beta-blocker antianginals, calcium-channel retroviruses); and dsDNA-RT viruses (e.g., hepadnaviruses). blocker antianginals, nitrate antianginals, antiarrhythmics, Opportunistic infections which can be detected using the cardiac glycoside antiarrhythmics, class I antiarrhythmics, systems and methods of the invention include, without limi class II antiarrhythmics, class III antiarrhythmics, class IV tation, fungal, viral, bacterial, protozoan infections, such as: antiarrhythmics, antihypertensive agents, alpha-blocker anti 1) fungal infections, such as those by Candida spp. (drug hypertensives, angiotensin-converting enzyme inhibitor resistant and non-resistant Strains), C. albicans, C. krusei, C. 25 (ACE inhibitor) antihypertensives, beta-blocker antihyper glabrata, and Aspergillus fumigates; 2) gram negative infec tensives, calcium-channel blocker antihypertensives, central tions, such as those by E. coli, Stenotrophomonas malto acting adrenergic antihypertensives, diuretic antihyperten philia, Klebsiella pneumonia/oxytoca, and Pseudomonas sive agents, peripheral vasodilator antihypertensives, aeruginosa; and 3) gram positive infections, such as those by antilipemics, bile acid sequestrant antilipemics, HMG-COA Staphylococcus spp., S. aureus, S. pneumonia, Enterococcus 30 reductase inhibitor antilipemics, inotropes, cardiac glycoside spp. (Efaecalis and E. faecium). The infection can be by inotropes, and thrombolytic agents; dermatological agents, coagulase negative staphylococcus, Corynebacterium spp., such as antihistamines, anti-inflammatory agents, corticoster Fusobacterium spp., Morganella morganii, Pneumocystis oid anti-inflammatory agents, antipruritics/local anesthetics, jirovecii (previously known as Pneumocystis carinii), F. topical anti-infectives, antifungal topical anti-infectives, anti hominis, S. pyogenes, Pseudomonas aeruginosa, polyomavi 35 viral topical anti-infectives, and topical antineoplastics; elec ruS JC polyomavirus (the virus that causes progressive mul trolytic and renal agents, such as acidifying agents, alkalin tifocal leukoencephalopathy), Acinetobacter baumanni, izing agents, diuretics, carbonic anhydrase inhibitor Toxoplasma gondii, cytomegalovirus, Aspergillus spp., diuretics, loop diuretics, osmotic diuretics, potassium-spar Kaposi’s Sarcoma, Cryptosporidium spp., Cryptococcus ing diuretics, thiazide diuretics, electrolyte replacements, and neoformans, and Histoplasma capsulatum. 40 uricoSuric agents; enzymes, such as pancreatic enzymes and Non-limiting examples of broad categories of analytes thrombolytic enzymes; gastrointestinal agents, such as which can be detected using the devices, systems, and meth antidiarrheals, antiemetics, gastrointestinal anti-inflamma ods of the invention include, without limitation, the following tory agents, salicylate gastrointestinal anti-inflammatory therapeutic categories: anabolic agents, antacids, anti-asth agents, antacid anti-ulcer agents, gastric acid-pump inhibitor matic agents, anti-cholesterolemic and anti-lipid agents, anti 45 anti-ulcer agents, gastric mucosal anti-ulcer agents, coagulants, anti-convulsants, anti-diarrheals, anti-emetics, H-blocker anti-ulcer agents, cholelitholytic agents, diges anti-infective agents, anti-inflammatory agents, anti-manic tants, emetics, laxatives and stool softeners, and prokinetic agents, anti-nauseants, anti-neoplastic agents, anti-obesity agents; general anesthetics, such as inhalation anesthetics, agents, anti-pyretic and analgesic agents, anti-spasmodic halogenated inhalation anesthetics, intravenous anesthetics, agents, anti-thrombotic agents, anti-uricemic agents, anti 50 barbiturate intravenous anesthetics, benzodiazepine intrave anginal agents, antihistamines, anti-tussives, appetite Sup nous anesthetics, and opiate agonist intravenous anesthetics; pressants, biologicals, cerebral dilators, coronary dilators, hematological agents, such as antianemia agents, hematopoi decongestants, diuretics, diagnostic agents, erythropoietic etic antianemia agents, coagulation agents, anticoagulants, agents, expectorants, gastrointestinal sedatives, hyperglyce hemostatic coagulation agents, platelet inhibitor coagulation mic agents, hypnotics, hypoglycemic agents, ion exchange 55 agents, thrombolytic enzyme coagulation agents, and plasma resins, laxatives, mineral Supplements, mucolytic agents, Volume expanders; hormones and hormone modifiers, such as neuromuscular drugs, peripheral vasodilators, psychotropics, abortifacients, adrenal agents, corticosteroid adrenal agents, sedatives, stimulants, thyroid and anti-thyroid agents, uterine androgens, anti-androgens, antidiabetic agents, Sulfonylurea relaxants, vitamins, and prodrugs. antidiabetic agents, antihypoglycemic agents, oral contracep More specifically, non-limiting examples of analytes 60 tives, progestin contraceptives, estrogens, fertility agents, which can be detected using the devices, systems, and meth oxytocics, parathyroid agents, pituitary hormones, ods of the invention include, without limitation, the following progestins, antithyroid agents, thyroid hormones, and toco therapeutic categories: analgesics, such as nonsteroidal anti lytics; immunobiologic agents, such as immunoglobulins, inflammatory drugs, opiate agonists and Salicylates; antihis immunosuppressives, toxoids, and vaccines; local anesthet tamines, such as H-blockers and H-blockers; anti-infective 65 ics. Such as amide local anesthetics and ester local anesthet agents, such as anthelmintics, antianaerobics, antibiotics, ics; musculoskeletal agents, such as anti-gout anti-inflamma aminoglycoside antibiotics, antifungal antibiotics, cepha tory agents, corticosteroid anti-inflammatory agents, gold US 8,409,807 B2 67 68 compound anti-inflammatory agents, immuno-Suppressive neoplastic agents, such as fluorouracil (5-FU) and gemcitab anti-inflammatory agents, nonsteroidal anti-inflammatory ine; hormonal antineoplastics, such as goserelin, leuprolide, drugs (NSAIDs), Salicylate anti-inflammatory agents, skel and tamoxifen; natural antineoplastics, such as aldesleukin, etal muscle relaxants, neuromuscular blocker skeletal muscle interleukin-2, docetaxel, etoposide (VP-16), interferon alfa, relaxants, and reverse neuromuscular blocker skeletal muscle paclitaxel, and tretinoin (ATRA); antibiotic natural antine relaxants; neurological agents, such as anticonvulsants, bar oplastics, such as bleomycin, dactinomycin, daunorubicin, biturate anticonvulsants, benzodiazepine anticonvulsants, doxorubicin, and mitomycin; Vinca alkaloid natural antine anti-migraine agents, anti-parkinsonian agents, anti-Vertigo oplastics, such as vinblastine and Vincristine; autonomic agents, opiate agonists, and opiate antagonists; ophthalmic agents, such as nicotine; anticholinergic autonomic agents, agents, such as anti-glaucoma agents, beta-blocker anti-glau 10 Such as benztropine and trihexyphenidyl; antimuscarinic coma agents, miotic anti-glaucoma agents, mydriatics, adr anticholinergic autonomic agents, such as atropine and oxy energic agonist mydriatics, antimuscarinic mydriatics, oph butynin; ergot alkaloid autonomic agents, such as bromocrip thalmic anesthetics, ophthalmic anti-infectives, ophthalmic tine; cholinergic agonist parasympathomimetics, such as aminoglycoside anti-infectives, ophthalmic macrollide anti pilocarpine; cholinesterase inhibitor parasympathomimetics, infectives, ophthalmic quinolone anti-infectives, ophthalmic 15 Such as pyridostigmine; alpha-blocker sympatholytics, such Sulfonamide anti-infectives, ophthalmic tetracycline anti-in as praZosin; 9-blocker sympatholytics, such as atenolol, adr fectives, ophthalmic anti-inflammatory agents, ophthalmic energic agonist sympathomimetics, such as albuterol and corticosteroid anti-inflammatory agents, and ophthalmic dobutamine; cardiovascular agents, such as aspirin (ASA) nonsteroidal anti-inflammatory drugs (NSAIDs); psychotro (enteric coated ASA); i-blocker antianginals. Such as atenolol pic agents, such as antidepressants, heterocyclic antidepres and propranolol, calcium-channel blocker antianginals. Such sants, monoamine oxidase inhibitors (MAOIs), selective as nifedipine and Verapamil; nitrate antianginals, such as serotonin re-uptake inhibitors (SSRIs), tricyclic antidepres isosorbide dinitrate (ISDN); cardiac glycoside antiarrhyth sants, antimanics, antipsychotics, phenothiazine antipsychot mics. Such as digoxin; class I antiarrhythmics, such as ics, anxiolytics, sedatives, and hypnotics, barbiturate seda lidocaine, mexiletine, phenyloin, procainamide, and quini tives and hypnotics, benzodiazepine anxiolytics, sedatives, 25 dine; class II antiarrhythmics, such as atenolol, metoprolol. and hypnotics, and psychoStimulants; respiratory agents, propranolol, and timolol; class III antiarrhythmics, such as Such as antitussives, bronchodilators, adrenergic agonist amiodarone; class IV antiarrhythmics, such as diltiazem and bronchodilators, antimuscarinic bronchodilators, expecto Verapamil: alpha-blocker antihypertensives, such as pra rants, mucolytic agents, respiratory anti-inflammatory Zosin; angiotensin-converting enzyme inhibitor (ACE inhibi agents, and respiratory corticosteroid anti-inflammatory 30 tor) antihypertensives, such as captopril and enalapril; beta agents; toxicology agents. Such as antidotes, heavy metal blocker antihypertensives, such as atenolol, metoprolol. antagonists/chelating agents. Substance abuse agents, deter nadolol, and propanolol; calcium-channel blocker antihyper rent Substance abuse agents, and withdrawal Substance abuse tensive agents, such as diltiazem and nifedipine; central-act agents; minerals; and vitamins, such as vitamin A, vitamin B, ing adrenergic antihypertensives, such as clonidine and meth vitamin C, vitamin D, vitamin E, and vitamin K. 35 yldopa; diuretic antihypertensive agents, such as amiloride, Examples of classes of biologically active Substances from furosemide, hydrochlorothiazide (HCTZ), and spironolac the above categories which can be detected using the devices, tone; peripheral vasodilator antihypertensives, such as systems, and methods of the invention include, without limi hydralazine and minoxidil; antilipemics, such as gemfibrozil tation, nonsteroidal anti-inflammatory drugs (NSAIDs) anal and probucol; bile acid sequestrant antilipemics, such as gesics, such as diclofenac, ibuprofen, ketoprofen, and 40 cholestyramine; HMG-CoA reductase inhibitor antilipemics, naproxen; opiate agonist analgesics, such as codeine, fenta Such as lovastatin and pravastatin: inotropes, such as ami nyl, hydromorphone, and morphine; salicylate analgesics, none, dobutamine, and dopamine; cardiac glycoside ino such as aspirin (ASA) (enteric coated ASA); H-blocker anti tropes, such as digoxin; thrombolytic agents, such as histamines, such as clemastine and terfenadine; H-blocker alteplase (TPA), anistreplase, streptokinase, and urokinase; antihistamines, such as cimetidine, famotidine, nizadine, and 45 dermatological agents, such as colchicine, isotretinoin, meth ranitidine; anti-infective agents, such as mupirocin, anti otrexate, minoxidil, tretinoin (ATRA); dermatological corti anaerobic anti-infectives, such as chloramphenicol and clin costeroid anti-inflammatory agents, such as betamethasone damycin; antifungal antibiotic anti-infectives, such as and dexamethasone; antifungal topical anti-infectives, such amphotericinb, clotrimazole, fluconazole, and ketoconazole; as amphotericin B, clotrimazole, miconazole, and nystatin: macrollide antibiotic anti-infectives, such as azithromycin and 50 antiviral topical anti-infectives, such as acyclovir, topical erythromycin; miscellaneous beta-lactam antibiotic anti-in antineoplastics, such as fluorouracil (5-FU); electrolytic and fectives, such as aztreonam and imipenem; penicillin antibi renal agents, such as lactulose; loop diuretics, such as furo otic anti-infectives, such as nafcillin, oxacillin, penicillin G, semide; potassium-sparing diuretics, such as triamterene; thi and penicillin V: quinolone antibiotic anti-infectives, such as azide diuretics, such as hydrochlorothiazide (HCTZ); urico ciprofloxacin and norfloxacin, tetracycline antibiotic anti 55 Suric agents, such as probenecid; enzymes Such as RNase and infectives, such as doxycycline, minocycline, and tetracy DNase; thrombolytic enzymes, such as alteplase, anistre cline; antituberculosis antimycobacterial anti-infectives Such plase, streptokinase and urokinase; antiemetics, such as as isoniazid (INH), and rifampin; antiprotozoal anti-infec prochlorperazine; salicylate gastrointestinal anti-inflamma tives. Such as atovaquone and dapsone; antimalarial antipro tory agents, such as Sulfasalazine; gastric acid-pump inhibitor toZoal anti-infectives, such as chloroquine and 60 anti-ulcer agents, such as omeprazole; H-blocker anti-ulcer pyrimethamine; anti-retroviral anti-infectives, such as agents, such as cimetidine, famotidine, nizatidine, and raniti ritonavir and Zidovudine; antiviral anti-infective agents. Such dine; digestants, such as pancrelipase; prokinetic agents, such as acyclovir, ganciclovir, interferon alfa, and rimantadine; as erythromycin; opiate agonist intravenous anesthetics Such alkylating antineoplastic agents, such as carboplatin and cis as fentanyl; hematopoietic antianemia agents, such as eryth platin: nitrosourea alkylating antineoplastic agents, such as 65 ropoietin, filgrastim (G-CSF), and sargramostim (GM-CSF); carmustine (BCNU); antimetabolite antineoplastic agents, coagulation agents, such as antihemophilic factors 1-10 Such as methotrexate; pyrimidine analog antimetabolite anti (AHF 1-10); anticoagulants, such as warfarin; thrombolytic US 8,409,807 B2 69 70 enzyme coagulation agents, such as alteplase, anistreplase, (r-IGF-1); recombinant interferon beta-1a; lenograstim streptokinase and urokinase; hormones and hormone modi (G-CSF); olanzapine; recombinant thyroid stimulating hor fiers. Such as bromocriptine; abortifacients, such as methotr mone (r-TSH); topotecan; acyclovir Sodium; aldesleukin; exate; antidiabetic agents, such as insulin; oral contracep atenolol, bleomycin Sulfate, human calcitonin; Salmon calci tives, such as estrogen and progestin; progestin tonin; carboplatin: carmustine; dactinomycin, daunorubicin contraceptives, such as levonorgestrel and norgestrel; estro HCl; docetaxel: doxorubicin HCl; epoetin alfa: etoposide gens such as conjugated estrogens, diethylstilbestrol (DES). (VP-16); fluorouracil (5-FU); ganciclovir sodium; gentami estrogen (estradiol, estrone, and estropipate); fertility agents, cin Sulfate; interferon alfa: leuprolide acetate; meperidine Such as clomiphene, human chorionic gonadatropin (HCG), HCl; methadone HCl; methotrexate sodium; paclitaxel; ran and menotropins; parathyroid agents such as calcitonin; pitu 10 itidine HCl; vinblastin sulfate; and zidovudine (AZT). itary hormones, such as desmopressin, goserelin, oxytocin, Further specific examples of biologically active substances and vasopressin (ADH); progestins, such as medroxyproges from the above categories which can be detected using the terone, norethindrone, and progesterone; thyroid hormones, devices, systems, and methods of the invention include, with Such as levothyroxine; immunobiologic agents, such as inter out limitation, antineoplastics Such as androgen inhibitors, feron beta-1b and interferon gamma-1b; immunoglobulins, 15 antimetabolites, cytotoxic agents, and immunomodulators; such as immune globulin IM, IMIG, IGIM and immune anti-tussives Such as dextromethorphan, dextromethorphan globulin IV, IVIG, IGIV: amide local anesthetics, such as hydrobromide, noscapine, carbetapentane citrate, and chlor lidocaine; ester local anesthetics, such as benzocaine and phedianol hydrochloride; antihistamines Such as chlorphe procaine; musculoskeletal corticosteroid anti-inflammatory niramine maleate, phenindamine tartrate, pyrilamine male agents, such as beclomethasone, betamethasone, cortisone, ate, doxylamine Succinate, and phenyltoloxamine citrate; dexamethasone, hydrocortisone, and prednisone; musculosk decongestants such as phenylephrine hydrochloride, phenyl eletal anti-inflammatory immunosuppressives, such as aza propanolamine hydrochloride, pseudoephedrine hydrochlo thioprine, cyclophosphamide, and methotrexate; musculosk ride, and ephedrine; various alkaloids Such as codeine phos eletal nonsteroidal anti-inflammatory drugs (NSAIDs), such phate, codeine Sulfate and morphine; mineral Supplements as diclofenac, ibuprofen, ketoprofen, ketorlac, and naproxen; 25 Such as potassium chloride, Zinc chloride, calcium carbon skeletal muscle relaxants, such as baclofen, cyclobenzaprine, ates, magnesium oxide, and other alkali metal and alkaline and diazepam; reverse neuromuscular blocker skeletal earth metal salts; ion exchange resins such as muscle relaxants, such as pyridostigmine; neurological cholestryramine; anti-arrhythmics such as N-acetylprocaina agents, such as nimodipine, riluzole, tacrine and ticlopidine; mide; antipyretics and analgesics Such as acetaminophen, anticonvulsants, such as carbamazepine, gabapentin, lamot 30 aspirin and ibuprofen; appetite Suppressants such as phenyl rigine, phenyloin, and valproic acid; barbiturate anticonvul propanolamine hydrochloride or caffeine; expectorants such sants, such as phenobarbital and primidone; benzodiazepine as guaifenesin; antacids such as aluminum hydroxide and anticonvulsants, such as clonazepam, diazepam, and magnesium hydroxide; biologicals such as peptides, lorazepam, anti-parkisonian agents, such as bromocriptine, polypeptides, proteins and amino acids, hormones, interfer levodopa, carbidopa, and pergolide; anti-vertigo agents. Such 35 ons or cytokines, and other bioactive peptidic compounds, as meclizine; opiate agonists, such as codeine, fentanyl. Such as interleukins 1-18 including mutants and analogues, hydromorphone, methadone, and morphine; opiate antago RNase, DNase, luteinizing hormone releasing hormone nists, such as naloxone; beta-blocker anti-glaucoma agents, (LHRH) and analogues, gonadotropin releasing hormone Such as timolol, miotic anti-glaucoma agents, such as pilo (GnRH), transforming growth factor-beta (TGF-beta), fibro carpine, ophthalmic aminoglycoside antiinfectives, such as 40 blast growth factor (FGF), tumor necrosis factor-alpha &beta gentamicin, neomycin, and tobramycin; ophthalmic qui (TNF-alpha & beta), nerve growth factor (NGF), growth hor nolone anti-infectives, such as ciprofloxacin, norfloxacin, and mone releasing factor (GHRF), epidermal growth factor ofloxacin, ophthalmic corticosteroid anti-inflammatory (EGF), fibroblast growth factor homologous factor (FGFHF), agents, such as dexamethasone and prednisolone; ophthalmic hepatocyte growth factor (HGF), insulin growth factor (IGF), nonsteroidal anti-inflammatory drugs (NSAIDs). Such as 45 invasion inhibiting factor-2 (IIF-2), bone morphogenetic pro diclofenac; antipsychotics, such as clozapine, haloperidol. teins 1-7 (BMP1-7), somatostatin, thymosin-O-1, T-globulin, and risperidone; benzodiazepine anxiolytics, sedatives and superoxide dismutase (SOD), complement factors, hCH, hypnotics, such as clonazepam, diazepam, lorazepam, tPA, calcitonin, ANF, EPO and insulin; and anti-infective oxazepam, and prazepam, psychostimulants, such as meth agents such as antifungals, anti-virals, antiseptics and antibi ylphenidate and pemoline; antitussives. Such as codeine; 50 otics. bronchodilators, such as theophylline; adrenergic agonist Biologically active Substances which can be detected using bronchodilators, such as albuterol; respiratory corticosteroid the devices, systems, and methods of the invention also anti-inflammatory agents, such as dexamethasone; antidotes, include radiosensitizers, such as metoclopramide, sensamide Such as flumazenil and naloxone; heavy metal antagonists/ or neusensamide (manufactured by Oxigene); profiromycin chelating agents. Such as penicillamine; deterrent Substance 55 (made by Vion); RSR13 (made by Allos); Thymitaq (made by abuse agents, such as disulfuram, naltrexone, and nicotine; Agouron), etanidazole or lobenguane (manufactured by withdrawal Substance abuse agents, such as bromocriptine; Nycomed); gadolinium texaphrin (made by Pharmacyclics); minerals, such as iron, calcium, and magnesium; Vitamin B BuDR/Broxine (made by NeoPharm); IPdR (made by compounds, such as cyanocobalamin (vitamin B) and nia Sparta); CR2412 (made by Cell Therapeutic); L1X (made by cin (vitamin B); vitamin C compounds, such as ascorbic 60 Terrapin); or the like. acid; and vitamin D compounds, such as calcitriol; recombi Biologically active Substances which can be detected using nant beta-glucan: bovine immunoglobulin concentrate; the devices, systems, and methods of the invention include, bovine Superoxide dismutase; the formulation including fluo without limitation, medications for the gastrointestinal tract rouracil, epinephrine, and bovine collagen; recombinanthiru or digestive system, for example, antacids, reflux Suppres din (r-Hir), HIV-1 immunogen; human anti-TAC antibody; 65 sants, antiflatulents, antidoopaminergics, proton pump recombinant human growth hormone (r-hGH); recombinant inhibitors, H-receptor antagonists, cytoprotectants, prostag human hemoglobin (r-Hb); recombinant human mecasermin landin analogues, laxatives, antispasmodics, antidiarrheals, US 8,409,807 B2 71 72 bile acid sequestrants, and opioids; medications for the car tions, electrolytes, vitamins, anti-obesity drugs, anabolic diovascular system, for example, beta-receptor blockers, cal drugs, haematopoietic drugs, and food product drugs; medi cium channel blockers, diuretics, cardiac glycosides, antiar cations for neoplastic disorders, for example, cytotoxic drugs, rhythmics, nitrate, antianginals, Vascoconstrictors, sex hormones, aromatase inhibitors, somatostatin inhibitors, vasodilators, peripheral activators, ACE inhibitors, angio recombinant interleukins, G-CSF, and erythropoietin: medi tensin receptor blockers, alpha blockers, anticoagulants, hep cations for diagnostics, for example, contrast agents; and arin, HSGAGs, antiplatelet drugs, fibrinolytics, anti-hemo medications for cancer (anti-cancer agents). philic factors, haemostatic drugs, hypolipaemic agents, and Examples of pain medications (e.g., analgesics) which can statins; medications for the central nervous system, for be detected using the devices, systems, and methods of the example, hypnotics, anaesthetics, antipsychotics, antidepres 10 invention include opioids such as buprenorphine, butorpha sants, anti-emetics, anticonvulsants, andepileptics, anxiolyt nol, dextropropoxyphene, dihydrocodeine, fentanyl, diamor ics, barbiturates, movement disorder drugs, stimulants, ben phine (heroin), hydromorphone, morphine, nalbuphine, oxy Zodiazepine, cyclopyrrolone, dopamine antagonists, codone, oxymorphone, pentazocine, pethidine (meperidine), antihistamine, cholinergics, anticholinergics, emetics, can and tramadol; salicylic acid and derivatives such as acetyl nabinoids, 5-HT antagonists; medications for pain and/or 15 salicylic acid (aspirin), diflunisal, and ethenZamide; pyrazo consciousness, for example, NSAIDs, opioids and orphans lones such as aminophenaZone, metamizole, and phenaZone; Such as paracetamol, tricyclic antidepressants, and anticon anilides such as paracetamol (acetaminophen), phenacetin: Vulsants; for musculo-skeletal disorders, for example, and others such as Ziconotide and tetradyrocannabinol. NSAIDs, muscle relaxants, and neuromuscular drug anticho Examples of blood pressure medications (e.g., antihyper linersterase; medications for the eye, for example, adrenergic tensives and diuretics) which can be detected using the neurone blockers, astringents, ocular lubricants, topical anes devices, systems, and methods of the invention include antia thetics, sympathomimetics, parasympatholytics, mydriatics, drenergic agents such as clonidine, doxazocin, guanethidine, cycloplegics, antibiotics, topical antibiotics, Sulfa drugs, ami guanfacine, mecamylamine, methyldopa, moxonidinie, pra noglycosides, fluoroquinolones, anti-virals, anti-fungals, Zosin, rescinnamine, and reserpine; vasodilators such as dia imidazoles, polyenes, NSAIDs, corticosteroids, mast cell 25 Zoxide, hydralazine, minoxidil, and nitroprusside; low ceil inhibitors, adrenergic agonists, beta-blockers, carbonic anhy ing diuretics such as bendroflumethiazide, chlorothiazide, drase inhibitors/hyperosmotics, cholinergics, miotics, para chlortalidone, hydrochlorothiazide, indapamide, quineth sympathomimetics, prostaglandin, agonists/prostaglandin aZone, mersalyl, metolaZone, and theobromine; high ceiling inhibitors, nitroglycerin; medications for the ear, nose and diuretics such as bumetanide, furosemide, and torasemide; oropharynx, for example, sympathomimetics, antihista 30 potassium-sparing diuretics such as amiloride, eplerenone, mines, anticholinergics, NSAIDs, steroids, antiseptics, local spironolactone, and triamterene; and other antihypertensives anesthetics, antifungals, cerumenolytics; medications for the such as bosentan and ketanserin. respiratory system, for example, bronchodilators, NSAIDs, Examples of anti-thrombotics (e.g., thrombolytics, antico anti-allergies, antitussives, mucolytics, decongestants, corti agulants, and antiplatelet drugs) which can be detected using costeroids, beta-receptor antagonists, anticholinergics, Ste 35 the devices, systems, and methods of the invention include roids; medications for endocrine problems, for example, Vitamin Kantagonists such as acenocoumarol, clorindione, androgen, antiandrogen, gonadotropin, corticosteroids, dicumarol, diphenadione, ethyl biscoumacetate, phenproc growth hormone, insulin, antidiabetics, thyroid hormones, oumon, phenindione, tioclomarol, and warfarin; heparin antithyroid drugs, calcitonin, diphosphonate, and vasopressin group (platelet aggregation inhibitors) such as antithrombin analogues; medications for the reproductive system or uri 40 III, bemiparin, dalteparin, danaparoid, enoxaparin, heparin, nary system, for example, antifungals, alkalising agents, qui nadroparin, parnaparin, reviparin, Sulodexide, and tinzaparin; nolones, antibiotics, cholinergics, anticholinergics, anticho other platelet aggregation inhibitors such as abciximab, ace linesterase, antispasmodics, 5-alpha reductase inhibitor, tylsalicylic acid (aspirin), aloxiprin, beraprost, ditazole, car selective alpha-1 blockers, and sildenafil; medications for basalate calcium, cloricromen, clopidogrel, dipyridamole, contraception, for example, oral contraceptives, spermicides, 45 epoprostenol, eptifibatide, indobufen, iloprost, picotamide, and depot contraceptives; medications for obstetrics and prasugrel, ticlopidine, tirofiban, treprostinil, and triflusal; gynecology, for example, NSAIDs, anticholinergics, haemo enzymes Such as alteplase, ancrod, anistreplase, brinase, static drugs, antifibrinolytics, hormone replacement therapy, drotrecoginalfa, fibrinolysin, procein C, reteplase, Saruplase, bone regulator, beta-receptor agonists, follicle stimulating streptokinase, tenecteplase, and urokinase; direct thrombin hormone, luteinising hormone, LHRH gamolenic acid, gona 50 inhibitors such as argatroban, bivalirudin, desirudin, lepiru dotropin release inhibitor, progestogen, dopamine agonist, din, melagatran, and Ximelagatran; other antithrombotics oestrogen, prostaglandin, gonadorelin, clomiphene, tammox Such as dabigatran, defibrotide, dermatan Sulfate, fonda ifen, and diethylstilbestrol; medications for the skin, for parinux, and rivaroXaban; and others such as citrate, EDTA, example, emollients, anti-pruritics, antifungals, disinfec and oxalate. tants, scabicide, pediculicide, tar products, vitamin A deriva 55 Examples of anticonvulsants which can be detected using tives, vitamin D analogue, keratolytics, abrasives, systemic the devices, systems, and methods of the invention include antibiotics, topical antibiotics, hormones, desloughing barbiturates such as barbexaclone, metharbital, methylphe agents, exudate absorbents, fibrinolytics, proteolytics, Sun nobarbital, phenobarbital, and primidone; hydantoins such as screens, antiperspirants, and corticosteroids; medications for ethotoin, fosphenyloin, mephenyloin, and phenyloin, oxazo infections and infestations, for example, antibiotics, antifun 60 lidinediones such as ethadione, paramethadione, and tri gals, antileprotics, antituberculous drugs, antimalarials, methadione; succinimides such as ethoSuximide, mesuXim anthelmintics, amoebicide, antivirals, antiprotozoals, and ide, and phensuximide; benzodiazepines Such as clobazam, antiserum; medications for the immune system, for example, clonazepam, cloraZepate, diazepam, lorazepam, midazolam, vaccines, immunoglobulin, immunosuppressants, interferon, and nitrazepam, carboxamides such as carbamazepine, monoclonal antibodies; medications for allergic disorders, 65 oXcarbazepine, rufinamide; fatty acid derivatives such as Val for example, anti-allergies, antihistamines, and NSAIDs.; promide and Valnoctamide; carboxylic acids Such as Valproic medications for nutrition, for example, tonics, iron prepara acid, tiagabine; GABA analogs such as gabapentin, pregaba US 8,409,807 B2 73 74 lin, progabide, and givabatrin; monosaccharides such as topi Other biologically active substances which can be detected ramate; aromatic allylic alcohols such as stiripentol; ureas using the devices, systems, and methods of the invention Such as phenacemide and pheneturide; carbamates such as include those mentioned in Basic and Clinical Pharmacology emylcamate, felbamate, and meprobamate; pyrrolidines Such (LANGE Basic Science), Katzung and Katzung, ISBN as brivaracetam, levetiracetam, nefiracetam, and seletrac 007 1410929, McGraw-Hill Medical, 9 edition (2003). etam; Sulfa drugs such as acetazolamide, ethoXZolamide, Sul Medical Conditions tiame, and Zonisamide; propionates Such as beclamide; alde Embodiments of the invention may be used in the moni hydes such as paraldehyde; and bromides such as potassium toring of one or more analytes in the diagnosis, management, bromide. and/or treatment of any of a wide range of medical conditions. Examples of anti-cancer agents which can be detected 10 Various categories of medical conditions include, for using the devices, systems, and methods of the invention example, disorders of pain; of alterations in body temperature include acivicin, aclarubicin; acodazole hydrochloride; acro (e.g., fever); of nervous system dysfunction (e.g., Syncope, nine; adriamycin; adoZelesin; aldesleukin; altretamine; myalgias, movement disorders, numbness, sensory loss, ambomycin; ametantrone acetate; aminoglutethimide; amsa delirium, dementia, memory loss, or sleep disorders); of the crine; anastrozole; anthramycin; asparaginase; asperlin; aza 15 eyes, ears, nose, and throat; of circulatory and/or respiratory citidine; azetepa; azotomycin; batimastat; benzodepa; functions (e.g., dyspinea, pulmonary edema, cough, hemop bicalutamide; bisantrene hydrochloride; bisnafide dimesy tysis, hypertension, myocardial infarctions, hypoxia, cyano late; bizelesin, bleomycin Sulfate; brequinar sodium; bropir sis, cardiovascular collapse, congestive heart failure, edema, imine; buSulfan, cactinomycin, calusterone; caracemide; car or shock); of gastrointestinal function (e.g., dysphagia, diar betimer, carboplatin: carmustine; carubicin hydrochloride; rhea, constipation, GI bleeding, jaundice, ascites, indiges carzelesin, cedefingol: chlorambucil; cirolemycin; cisplatin: tion, nausea, vomiting); of renal and urinary tract function cladribine; crisinatol mesylate; cyclophosphamide; cytara (e.g., acidosis, alkalosis, fluid and electrolyte imbalances, bine; dacarbazine; dactinomycin; daunorubicin hydrochlo azotemia, or urinary abnormalities); of sexual function and ride; decitabine; dexormaplatin; deZaguanine; deZaguanine reproduction (e.g., erectile dysfunction, menstrual distur mesylate; diaziquone; docetaxel, doxorubicin, doxorubicin 25 bances, hirsutism, virilization, infertility, pregnancy associ hydrochloride; droloxifene; droloxifene citrate; dromo ated disorders and standard measurements); of the skin (e.g., stanolone propionate; duaZomycin; ediatrexate; eflornithine eczema, psoriasis, acne, rosacea, cutaneous infection, immu hydrochloride; elsamitrucin; enloplatin; enpromate; epipro nological skin diseases, or photosensitivity); of the blood pidine; epirubicin hydrochloride; erbulozole; esorubicin (e.g., hematology); of genes (e.g., genetic disorders); of drug hydrochloride; estramustine; estramustine phosphate 30 response (e.g., adverse drug responses); and of nutrition (e.g., Sodium; etanidazole; etoposide; etoposide phosphate: eto obesity, eating disorders, or nutritional assessment). Other prine: fadrozole hydrochloride: fazarabine; fenretinide: medical fields with which embodiments of the invention find floxuridine; fludarabine phosphate; fluorouracil; fluorocitab utility include oncology (e.g., neoplasms, malignancies, ine; fosquidone; fostriecin Sodium; gemcitabine; gemcitab angiogenesis, paraneoplasic Syndromes, or oncologic emer ine hydrochloride; hydroxyurea; idarubicin hydrochloride: 35 gencies); hematology (e.g., anemia, hemoglobinopathies, ifosfamide; ilmofosine; interferon alfa-2a: interferon alfa-2b: megalooblastic anemias, hemolytic anemias, aplastic ane interferon alfa-n1; interferon alfa-n3; interferon beta-I a: mia, myelodysplasia, bone marrow failure, polycythemia interferon gamma-Ib, iproplatin; irinotecan hydrochloride; Vera, myloproliferative diseases, acute myeloid leukemia, lanreotide acetate; letrozole; leuprolide acetate; liarozole chronic myeloid leukemia, lymphoid malignancies, plasma hydrochloride; lometrexol Sodium, lomustine; losoxantrone 40 cell disorders, transfusion biology, or transplants); hemosta hydrochloride; masoprocol; maytansine; mechlorethamine sis (e.g., disorders of coagulation and thrombosis, or disor hydrochloride; megestrol acetate; melengestrol acetate; mel ders of the platelet and vessel wall); and infectious diseases phalan; menogaril; mercaptopurine; methotrexate; methotr (e.g., sepsis, septic shock, fever of unknown origin, endocar exate Sodium; metoprine; meturedepa; mitindomide; mito didtis, bites, burns, osteomyelitis, abscesses, food poisoning, carcin, mitocromin, mitogillin, mitomalcin, mitomycin; 45 pelvic inflammatory disease, bacterial (e.g., gram positive, mitosper, mitotane; mitoxantrone hydrochloride; mycophe gram negative, miscellaneous (nocardia, actimoyces, mixed), nolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran: mycobacterial, Spirochetal, rickettsia, or mycoplasma); paclitaxel; pegaspargase; peliomycin; pentamustine; peplo chlamydia; viral (DNA, RNA), fungal and algal infections: mycin Sulfate; perfosfamide; pipobroman; piposulfan, piroX protozoal and helminthic infections; endocrine diseases; antrone hydrochloride; plicamycin; plomestane; porfimer 50 nutritional diseases; and metabolic diseases. Sodium; porfiromycin; prednimustine; procarbazine hydro Other medical conditions and/or fields with which embodi chloride; puromycin; puromycin hydrochloride; pyrazofurin; ments of the invention find utility include those mentioned in riboprine; rogletimide; Safingol: Safingol hydrochloride; Harrison's Principles of Internal Medicine, Kasper et al., Semustine; simtraZene; sparfosate Sodium; sparsomycin; ISBN 0071402357, McGraw-Hill Professional, 16' edition spirogermanium hydrochloride; spiromustine; spiroplatin: 55 (2004), as well as those mentioned in Robbins Basic Pathol streptonigrin: Streptozocin, Sulofenur; talisomycin; tecogalan ogy, Kumar, Cotran, and Robbins, eds. ISBN 1416025340, Sodium, tegafur, teloxantrone hydrochloride; temoporfin; Elsevier, 7" edition (2005). teniposide; teroxirone; testolactone; thiamiprine; thiogua Medical tests (e.g., blood tests, urine tests, and/or other nine; thiotepa; tiazofurin; tirapazamine; topotecan hydro human or animal tissue tests) that may be performed using chloride; toremifene citrate; trestolone acetate; triciribine 60 various embodiments of the invention described herein phosphate; trimetrexate; trimetrexate glucuronate; triptore include, for example, general chemistry tests (e.g., analytes lin; tubulozole hydrochloride; Uracil mustard; redepa; vap include albumin, blood urea nitrogen, calcium, creatinine, reotide; verteporfin; vinblastine sulfate; Vincristine sulfate; magnesium, phosphorus, total protein, and/or uric acid); elec vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate trolyte tests (e.g., analytes include Sodium, potassium, chlo sulfate; Vinleurosine sulfate; Vinorelbine tartrate; Vinrosidine 65 ride, and/or carbon dioxide); diabetes tests (e.g., analytes Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin: Zinostatin: include glucose, hemoglobin A1C, and/or microalbumin); and Zorubicin hydrochloride. lipids tests (e.g., analytes include apolipoprotein A1, apoli