MOTIONUS 20170234874A1 LAWNMN TANTUM IN ( 19) United States (12 ) Patent Application Publication (10 ) Pub. No. : US 2017/ 0234874 A1 Adams et al. (43 ) Pub. Date : Aug . 17 , 2017 (54 ) INTEGRATED VISUAL MORPHOLOGY AND Related U . S . Application Data CELL PROTEIN EXPRESSION USING (60 ) Provisional application No . 62 / 238 ,605 , filed on Oct . RESONANCE -LIGHT SCATTERING 7 , 2015 . (71 ) Applicant: Clearbridge BioPhotonics Pte Ltd ., Singapore (SG ) Publication Classification (51 ) Int . CI. (72 ) Inventors : Thomas H . Adams, Rancho Santa Fe, GOIN 33 / 569 ( 2006 .01 ) CA (US ) ; Stephen Roman Fait , GOIN 15 / 14 ( 2006 .01 ) Carlsbad , CA (US ) ; Eric Scott A61B 5 /00 ( 2006 .01 ) McCampbell , Carlsbad , CA (US ) ; GOIN 21 /552 ( 2006 .01 ) Michelle Brooke McCampbell , Rancho (52 ) U . S . Cl. Santa Fe, CA (US ); Edward Jablonski, CPC . . . GOIN 33 /56966 (2013 .01 ) ; GOIN 21/ 553 Escondido , CA (US ) ; Robert Earl ( 2013 .01 ) ; GOIN 15 / 1456 ( 2013 .01 ) ; A61B Klem , Rancho Santa Fe, CA (US ) 5 /0082 ( 2013 .01 ); G06T 7/ 0012 ( 2013 .01 ) ( 57 ) ABSTRACT (21 ) Appl . No. : 15 / 286 , 340 The invention relates to detecting cell biomarker signatures and integrated cell biomarker- morphological profiles by detecting resonance - light scattering of functionalized nano (22 ) Filed : Oct. 5 , 2016 particles.

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INTEGRATED VISUAL MORPHOLOGY AND using the resonant light scattering properties of the particles CELL PROTEIN EXPRESSION USING are : ( a ) the nanoparticles can be detected and imaged at RESONANCE -LIGHT SCATTERING magnifications as low as 10x using a simple illuminator, such as a white light illuminator, with dark field illumina RELATED APPLICATIONS tion , (b ) the nanoparticles provide a non -bleaching signal, ( c ) the color of scattered light can be changed by changing [0001 ] This application claims the benefit of U . S. Provi nanoparticle size and / or composition for multicolor multi sional Application Ser . No . 62 /238 , 605 , filed on Oct . 7 , plexing , ( d ) the nanoparticles can be conjugated with bio 2015 , the entire contents of which is hereby incorporated be marker - binding moieties for specific analyte detection to reference in its entirety . create functionalized nanoparticles, ( e ) biological samples contacted with the functionalized nanoparticles are archiv FIELD able , and ( f) the functionalized nanoparticles exhibit a [0002 ] The presently disclosed subject matter relates to greater range of linearity of detection when present on a cell compositions and methods for integrated visualmorphology because the particles do not self- quench . In some embodi and cell protein expression analysis . ments , the methods of this invention are useful in obtaining images of cell - functionalized nanoparticle complexes under INTRODUCTION AND SUMMARY OF THE ambient conditions which do not require use of a darkroom , INVENTION in contrast to fluorescent labeling systems. In some embodi [0003 ] Cellular analysis is an important tool in histopa ments , the samples may be viewed on a microscope in a thology to aid in diagnosing the medical condition of a doctor ' s or pathologist' s office . subject . Two pathological tools clinicians use for cellular 10007 ]. The present disclosure relates in some aspects to analysis is the morphological analysis of cell samples using methods and compositions for detecting cell- functionalized microscopy and cellular biomarker detection using a method nanoparticle binding moiety complexes useful in detecting a such as flow cytometry . Morphological analysis of cells biomarker signature of a cell . In some aspects , the methods samples involves visually identifying features or character and compositions are also useful in detecting the biomarker istics of a cell and associating those features or character morphological profile of an imaged cell . istics with known disease or condition states of a cell . Often , [0008 ] In some aspects of this disclosure , the nanopar morphological analysis is insufficient to diagnose the disease ticles functionalized with biomarker -binding moieties can be state or condition of a cell , and tissues or samples are used for detecting functionalized nanoparticle cell com analyzed for the presence of biomarkers associated with plexes , which are useful, for example , for identifying and known disease or conditions . While biomarkers can be any quantifying biomarkers present on cells . In some embodi molecule that indicates a biological state , they are most often ments , the cells may be imaged to detect morphological peptides or proteins. These peptides or proteins are involved features of the cells complexed with functionalized nano in many roles in the body , including intercellular signaling particles . In some embodiments , the functionalized nano and metabolism . Cell signaling and metabolism refer to the particles can be contacted with the same cells analyzed by a mechanisms behind common disease states and the associ morphological imaging analysis . ated markers may be used for measuring and monitoring [0009 ] In some embodiments , the methods of this inven such observables as disease progression and drug response . tion are useful for improving signal generation , detection [0004 ] Often , biomarkers are designated by their CD limits, dynamic range, automation and / or performance char nomenclature. CD (cluster of differentiation ) molecules are acteristics of the biomarker signature assays . For example , cell surface biomarkers useful for the identification and in some embodiments , the present disclosure relates to characterization of leukocytes. The CD nomenclature was methods for increasing the loading amount of a biomarker developed and is maintained through the HLDA (Human binding moiety onto a cell by using an external force to Leukocyte Differentiation Antigens ) working group . Identi increase the local concentration of the functionalized nano fying various combinations of CD molecules on a cell particles and cells . In some aspects , the biomarker binding surface is often used for the classification of cell types and moiety may be a functionalized nanoparticle , or a biomarker surface molecules which are targets for the immunopheno binding moiety comprising a label other than a nanoparticle . typing of cells. ( Chan , J . et al, Histopathology 12 ( 5 ) : A “ functionalized nanoparticle ” is a nanoparticle presenting 461- 480 ( 1988 ) . a functional group , directly or indirectly . In some aspects , [ 0005 ] Resonance light scattering is a physical phenom the external force may be a centrifugal, electromagnetic , or enon wherein a particle with a diameter less than the magnetic force . wavelength of incident light exhibits a surface plasmon [0010 ] In some aspects of this invention , the method of wave around the particle and said wave becomes coherent to detecting functionalized nanoparticle cell complexes can the circumference of the particle . Particle electrons resonate comprise : in phase with the incident light forming an electromagnetic [0011 ] (a ) providing a sample comprising cells from a dipole that emits energy as scattered light. The wavelength subject ; of the reflected ( scattered ) light is a function of the compo [ 0012 ] (b ) contacting the cells with one or a plurality of sition , shape , and particle size . Often , the composition of the functionalized nanoparticle species , each functional particle is a noble metal, such as gold or silver. Often , the ized nanoparticle species comprising a biomarker- bind size of the particle is below the wavelength of white light ing moiety , wherein an external force is used to accel (below 200 nm ) . Particles under 1000 nm in size are often erate the formation of nanoparticle -cell complexes referred to as " nanoparticles. ” through binding of the nanoparticle species comprising [ 0006 ] The advantages of using particles with a wave a biomarker- binding moiety to its respective bio length less than the wavelength of light for cellular analysis marker ; US 2017 /0234874 A1 Aug . 17 , 2017

[0013 ] (c ) adhering the functionalized nanoparticle -cell refractive index of cells , where the cells are fixed . In some complexes to a substrate ; embodiments , the refractive index of fixed cells is about [0014 ] ( d ) detecting the functionalized nanoparticle cell 1 .52 , or 1 .52 . In some aspects , the index of refraction of the complexes by illuminating the nanoparticle - cell com mountant is from 1 . 51 to 1 .54 . In some aspects , using a plexes with evanescent light and detecting the resonant mountant having an RI within 0 . 1 of the refractive index of light scattering from each observed functionalized fixed cells is useful for reducing the amount of white light nanoparticle cell complex , to obtain a biomarker sig scatter, and obtaining better images of resonance scattering nature of each observed cell ; and from the cell - functionalized nanoparticle complexes . In one [ 0015 ] ( e ) associating the biomarker signature of each aspect, this disclosure relates to a method for detecting substrate -adhered cell to a known disease, condition , or functionalized nanoparticle cell complexes, the method state of a cell exhibiting substantially the same or a comprising: similar biomarker signature to identify the disease , [0025 ] ( a ) providing a sample comprising cells from a condition , or state of the cell from a subject . subject; [0016 ]. In any of the methods disclosed herein , where a [0026 ] ( b ) contacting cells which have been fixed with biomarker or biomarker morphological profile is obtained , one or a plurality of functionalized nanoparticle spe detecting the cell - functionalized nanoparticle complexes on cies , each functionalized nanoparticle species compris the imaged cell optionally includes storing the positional ing a biomarker -binding moiety , and forming nanopar information for each imaged cell . ticle -cell complexes through binding of the [ 0017 ] In some embodiments, the disease , condition , or nanoparticle species comprising a biomarker -binding state of a cell can be identified by forming and detecting moiety to its respective biomarker; complexes between the functionalized nanoparticles and [0027 ] (c ) adhering the nanoparticle -cell complexes to a cells . The method can comprise associating a biomarker substrate , wherein the adhered nanoparticle -cell com signature of each substrate - adhered cell to a known disease , plexes are placed in contact with a mountant , wherein condition , or state of a cell exhibiting substantially the same the refractive index of the mountant is within about 0 . 1 or a similar biomarker signature to identify the disease , of the refractice index of the fixed cells ; condition , or state of the cell from a subject. [ 0028 ] ( d ) detecting the functionalized nanoparticle cell [0018 ] In some embodiments , the biomarker signature of complexes by illuminating the nanoparticle -cell com a cell can be detected in a homogeneous assay , the assay plexes with evanescent light and detecting the resonant comprising the steps : light scattering from each observed functionalized [0019 ] ( a) providing a sample comprising cells from a nanoparticle cell complex , to obtain a biomarker sig subject; nature of each observed cell ; and [0020 ] (b ) contacting the cells with one or a plurality of [0029 ] (e ) associating the biomarker signature of each functionalized nanoparticle species, each functional substrate -adhered cell to a known disease , condition , or ized nanoparticle species comprising a biomarker- bind state of a cell exhibiting substantially the same bio ing moiety , and forming functionalized nanoparticle marker signature to identify the disease , condition , or cell complexes through binding of the nanoparticle state of the cell from a subject. species comprising a biomarker -binding moiety to its In some aspects, the mountant may have a refractive index respective biomarker ; of about 1. 52 . [ 0021 ] (c ) adhering the functionalized nanoparticle - cell [0030 ] In some aspects , the ability to use the compositions complexes to a substrate ; and methods of this disclosure to associate the biomarker [0022 ] (d ) illuminating the functionalized nanoparticle signature of an individual cell , and in some embodiments , cell complexes with evanescent light and detecting the with its morphological image / features greatly enhances the resonant light scattering from each observed com ability to diagnose and monitor abnormal conditions or plexed nanoparticle , to obtain a biomarker signature of disorders . each observed cell , [0031 ] As set forth herein , the inventors have surprisingly where unbound functionalized nanoparticles are not determined that nanoparticles functionalized with bio removed from the field of view . marker -binding moieties can be used for detecting cell [0023 ] Often , unbound species are washed from a target to functionalized nanoparticle complexes and identifying and reduce background noise . In some embodiments wash steps quantifying biomarkers present on imaged cells for example , are omitted , leaving unbound functionalized nanoparticles in when the functionalized nanoparticles are contacted with the the field of view . This embodiment may be used in some same cells analyzed by a morphological imaging analysis . embodiments , for example , for high throughput assays , The ability to use the compositions and methods of this and / or automated assays . The functionalized nanoparticles disclosure to associate the biomarker signature of an indi are specific to the biomarker on the cell , and can substan vidual cell with its morphological image / features greatly tially contact the cell such that little to no signal is observed enhances the ability to diagnose and monitor abnormal for the unbound functionalized nanoparticles . conditions or disorders . 10024 ]. In some aspects of this invention , where biomarker 10032 ] In one aspect , this disclosure relates to composi signatures are detected , the detecting of the resonant light tions and methods for obtaining a biomarker signature for an scattering from each observed complexed -nanoparticle com imaged cell , which is used , in some embodiments, in com prises imaging the cell- functionalized nanoparticle com bination with detected morphological features of the cell plexes in contact with a mountant. In some aspects of this obtained from imaging the cell. In some aspects , composi invention , the mountant can comprise a solution with about tions comprising functionalized nanoparticle species , each the refractive index of the cells . In some aspects of this comprising a specific biomarker -binding moiety , are used to invention , the mountant can be within about 0 . 1 of the detect the biomarker signature of the imaged cell. In some US 2017 /0234874 A1 Aug . 17 , 2017 aspects of this disclosure, combinations of such composi resonant light scattering from each observed com tions are made or used in the methods of this disclosure . The plexed nanoparticle , to obtain a biomarker signature of combinations and kits comprising the combinations can be each observed cell ; and mixtures of such compositions, or may comprise composi [0047 ] ( e ) associating the biomarker signature of an tions segregated before use . imaged cell from the subject with a biomarker signature [ 0033] In one aspect the method comprises the steps of: of a reference cell exhibiting substantially the same [0034 ] (a ) providing a sample comprising cells from a biomarker signature as the imaged cell biomarker sig subject; nature , wherein a diagnostic concordance has been [ 0035 ] (b ) contacting the cells with one or a plurality of established between the reference cell biomarker sig functionalized nanoparticle species, each functional nature and a disease , disorder , condition or state of the ized nanoparticle species comprising a biomarker -bind reference subject. ing moiety , and forming nanoparticle - cell complexes 10048 ]. In some aspects of this disclosure , the sample can through binding of the nanoparticle species comprising be a biological sample . For example , in some embodiments , the sample may be any sample containing cells . In some a biomarker -binding moiety to its respective biomarker embodiments , the sample may be from blood , bone marrow , on the cell; a fine needle aspirate , or tissue . The tissue sample can be [0036 ] ( c ) adhering the functionalized nanoparticle - cell obtained , for example , from a biopsy . In some embodiments complexes to a substrate; the tissue sample may be obtained from a FFPE ( formalin [ 0037 ] ( d ) illuminating the functionalized nanoparticle fixed , paraffin -embedded ) tissue sample . In some aspects of cell complexes with evanescent light and detecting the this disclosure, a biological sample may be processed . For resonant light scattering from each observed com example , the sample may comprise white blood cells . In plexed functionalized nanoparticle, to obtain a bio some embodiments , at least 50 % of the red blood cells are marker signature of each observed cell; removed before contacting the cells with the plurality of [0038 ] ( e ) contacting the substrate -adhered cells with an functionalized nanoparticle species . optical contrast agent; 100491 In some aspects of this disclosure , the cell may be [0039 ] (f ) imaging morphological features of contacted alive , fixed and / or substantially intact . In some embodi cells ; and ments , the detected cells interrogated can be the same type [ 0040 ] ( g ) associating themorphological features of the or different types. When the cells are different types, they contacted cells with the biomarker signature of each may be of different tissue or tumor origin , different stages of substrate - adhered cell to detect the biomarker- morpho cancer progression , metastatic and non -metastatic cancer logical profile of each cell. cells , and may comprise infectious agents or cells , infected [0041 ] In some aspects , biomarker -morphological profiles cells , and uninfected cells , or express different levels , types , can be used to obtain a diagnostic concordance between a variants , mutants , forms, and /or post - translationally modi reference cell biomarker signature and a disease , disorder, fied forms of biomarkers found on normal or reference cells . condition or state of a reference subject. In some embodi In some embodiments , the different cells may exhibit dif ments , a concordance database of biomarker signatures with ferent pathologies, and / or different morphologies from nor diseases , disorders , conditions or states of diagnosed sub mal or reference cells . jects can be obtained . A diagnostic concordance includes an [ 0050 ] In some aspects of this disclosure , the cell is fixed association based on an association between a reference with a fixing agent. The fixing agent may be, for example , biomarker signature and / or a reference biomarker -morpho formaldehyde, glutaraldehyde , or another cross - linking logical profile , and a disease, disorder , condition or state of agent. In other embodiments water - soluble preservatives , for the reference subject . In some embodiments the diagnostic example , methyl or propyl paraben , dimethylolurea , sorbic concordance or association , may be made independent of acid , 2 -pyridinethiol - 1 - oxide , or potassium sorbate may be any treatment decision , for example , using data obtained used . In some embodiments the cell can be permeabilized by from an autopsy and / or based on archived tissue samples surfactants . and patient records . [0051 ] In some aspects of this disclosure , the functional [ 0042 ] This disclosure relates , in some aspects , to a ized nanoparticle cell complexes adhered to a substrate can method for detecting functionalized nanoparticle cell com be placed in contact with a mountant. The volume of the plexes to obtain a biomarker signature , the method com mountant can be from about 2 microliters to about 15 prising : microliters. [ 0043 ] ( a ) providing a sample comprising cells from a [0052 ] In some aspects of this disclosure , the detected subject; biomarker can be present on the cell surface , within the cell , or both on the surface and within the cell . In some embodi [0044 ] ( b ) contacting the cells with one or a plurality of ments the biomarker in the cell may be present in or on one functionalized nanoparticle species, each functional or more cellular features, for example, the cytosol, the ized nanoparticle species comprising a biomarker -bind nucleus, the nuclear membrane, nucleoli , the endoplasmic ing moiety , and forming nanoparticle -cell complexes reticulum , Golgi apparatus , mitochondria , or other cellular through binding of the functionalized nanoparticle spe structure, compartment, or feature . cies comprising a biomarker - binding moiety to its [0053 ] In some aspects of this disclosure , the detected respective biomarker ; biomarker can be a biomolecule identified by the Cluster [0045 ] (c ) adhering the functionalized nanoparticle -cell Determinant antigen (CD ) and or other molecules / antigenic complexes to a substrate; sites . For example , the biomarker can include or exclude any [0046 ] (d ) illuminating the functionalized nanoparticle of the from the following biomarkers : CD1, CD2 , CD3, cell complexes with evanescent light and detecting the CD4, CD5, CD , CD7, CD8, CD9, CD10 , CD11a , CD11b , US 2017 /0234874 A1 Aug . 17 , 2017

CD11c , CD13 , CD14 , CD15 , CD16 , CD19 , CD20 , CD21 , iron stain , Pentachrome stain , Azan stain , Luxol fast blue CD22 , CD23 , CD25 , CD30 , CD33 , CD34 , CD38 , CD41 , stain , Golgi' s method ( reduced silver ) , reduced gold , C43 , CD45 , CD56 , CD57 , CD58 , CD61, CD64 , C71 , chrome alum / haemotoxylin stain , Isamin blue stain , Argen CD79a , CD99 , CD103, CD117 , CD123, CD138 , CD138 , taffin stains , Warthin - Starry silver stain , Nissl stain , Sudan CD163, CD235a , HLA - DR , Kappa , Lambda , Pax - 5 , BCL - 2 , Black and osmium stain , osmium tetroxide stain , hematoxy Ki- 67 , ZAP - 70 , MPO , TDT , and FMC - 7 . In some embodi lin stain , uranyl acetate stain , lead citrate stain , Carmine ments , for example , the biomarker can include or exclude stain , safranin stain , and Ziehl -Neelsen stain . markers expressed by kidney cells , infectious agents , solid [0058 ] In some embodiments , the optical contrast agent tumor cells , circulating tumor cells, or any other cell useful can be a dye or colorant that can include or exclude , for for diagnosis or prognosis. In some embodiments , for example : eosin Y , eosin B , azure B , pyronin G , malachite example , the biomarker can include or exclude biomarkers green , toluidine blue , copper phthalocyanin , alcian blue , expressed on the surface or within kidney cells , infectious auramine- rhodamine , acid fuschin , aniline blue, orange G , agents ( e . g ., bacteria or virus ) , solid tumor cells , or circu acid fuschin , neutral red , Sudan Black B , acridine orange , lating tumor cells . In some embodiments , for example the Oil Red 0 , Congo Red , Fast green FCF , Perls Prussian blue biomarker may include or exclude HER2 , NEU , Prostate stem cell antigen ( PSCA ) , epithelial- specific antigen ( ESA ) , reaction , nuclear fast red , alkaline erythrocin B , and naph epithelial cell adhesion molecule ( EPCAM ), a2B1, VEGFR thalene black . 1, VEGFR -2 , CD133 , or AC133 antigen . [ 0059 ] When the sample is from tissue , the optical contrast [ 0054 ] In some aspects of this disclosure , the biomarker agent can be a H & E (hematoxylin and eosin ) stain . In one binding moiety can be selected from or comprise the fol aspect , the optical contrast agent may be suitable for lowing : an antibody or antibody fragment, nanobody, recep supravital staining . tor fragment, DNA aptamer , DNA /RNA oligonucleotide , [0060 ] In some embodiments , the cells can be contacted RNA aptamer , PNA aptamer , peptide aptamer, LNA with one or a plurality of functionalized nanoparticle species aptamer , carbohydrate , and a lectin . by subjecting the cells and functionalized nanoparticles to an 0055 ] In embodiments where the biomarker binding moi external force to increase the local concentration of the ety comprises an antibody, the antibody can be a monoclonal functionalized nanoparticles and cells . The external force or polyclonal antibody. In some embodiments , the bio can be a gravitational, electric , or magnetic force . The marker binding moiety may comprise an antibody fragment, gravitational force can be generated by centrifugation . The ScFv, or single -domain antibody (nanobody ) . The biomarker magnetic force can be effected by paramagnetic nanopar binding moiety may bind to a protein , protein fragment, ticles . The core of a paramagnetic functionalized nanopar glycosylation moiety or pattern , or a carbohydrate . The ticle comprises a paramagnetic region and the shell of the biomarker binding moiety can include or exclude a bio nanoparticle can include or exclude Ag , Au, Pt, Pd , Rh, Ro , marker binding moiety , e . g . , an antibody or fragment thereof Al, Cu , Ru , Cr , Cd , Zn , Si, Se or mixtures or alloys thereof. or other biomarker binding moiety that binds to , for In some embodiments , charged polymers can be added to the example: CD1, CD2 , CD3 , CD4 , CD5 , CD6 , CD7, CD8 , cells after first providing a sample comprising cells from a CD9, CD10 , CD11a , CD11b , CD11c , CD13 , CD14 , CD15 , subject. These methods may be useful , for example , for CD16 , CD19 , CD20 , CD21 , CD22 , CD23 , CD25 , CD30 , methods of detecting cell -biomarker binding moieties, for CD33 , CD34 , CD38 , CD41, C43 , CD45 , CD56 , CD57 , example , in any embodiment of detecting cell - functional CD58 , CD61 , CD64, C71, CD79a , CD99 , CD103 , CD117 , ized nanoparticle complexes disclosed herein . In some CD123 , CD138 , CD138 , CD163 , CD235a , HLA -DR , embodiments the sample may be re -mixed between two or Kappa , Lambda , Pax - 5 , BCL - 2 , Ki- 67 , ZAP -70 , MPO , TDT , more applications of the external force . As one non - limiting and FMC - 7 . In some embodiments , when the biomarker example , where the force is a centrifugal force , the force can binding moiety is anti -CD45 , the biomarker signature be applied in a forward direction to concentrate the cells and obtained is the white blood cell count. The white blood cell functionalized nanoparticles, and then applied in the reverse count can be measured as a function of the mass or volume direction to resuspend the cells and functionalized nanopar of the sample mass or volume, respectively . ticles . The centrifugal force can also be applied and reversed [0056 ] In some embodiments, the optical contrast agent two or more times . As one non - limiting example where the can be a leuco dye , cell stain , or any dye useful for imaging force is a electric force , the force can be applied by elec for morphological analysis including , for example , any dye trophoresis on a conductive or semiconductive surface , useful for histological, cytological, cytopathological , or his where the functionalized nanoparticles and cells are mixed topathological imaging. In some embodiments , the optical by their different relative electrophoretic mobilities when contrast agent provides visual classification and identifica under a potential bias ( see, Su , H ., et al. , Electrophoresis , 23 tion of cells by differentially staining cells . The leuco dye 1551- 1557 ( 2002 ) and U . S . Patent Application Publication can be red leuco dye , methylene blue , crystal violet , phe No . US 2003 /0119028 ) . The electrical force can also be nolphthalein , thymolphthalein , or methylene green . applied and reversed two or more times . [ 0057 ] In some embodiments , the optical contrast agent 10061] In some embodiments , the imaging of the morpho can include or exclude a cell stain selected from , for logical features of the contacted cells can comprise measur example : Giemsa stain , Wright stain , Wright- Giemsa stain , ing an optical property of the optical contrast agent. The May -Grünwald stain , Mallory trichrome, Periodic acid optical property of the optical contrast agent can include or Schiff reaction stain , Weigert ' s elastic stain , Heidenhain 's exclude , for example : absorbance , scattering, fluorescence , AZAN trichrome stain , Orcein stain , Masson ' s trichrome, photoluminesence, Raman emission , and photoluminescent Alcian blue stain , May -Grünwald -Giemsa , van Gieson stain , lifetime. The optical property of the optical contrast agent Hansel stain , Reticulin Stain , Gram stain , Bielschowsky can be measured under a microscope with either a light field stain , Ferritin stain , Fontana- Masson stain , Hales colloidal illumination or dark field illumination . US 2017 /0234874 A1 Aug . 17 , 2017

[0062 ] In some embodiments , the morphological features midal , cubic , egg - shaped , t- bone -shaped , urchin - or rose identified from the cell can include or exclude , for example : like (with spiky uneven surfaces) or hollow shaped . In some the shape of cellular features, for example , the cell surface embodiments , the nanoparticles may have a round , oval, shape , the cell nucleus shape , the chromatin shape , the triangular , square, egg - shaped , or a t - bone- shaped cross nucleolar shape , the number of cellular features , such as the section . number of nucleoli or mitochondria , the density of staining [0068 ] In some embodiments , the plurality of functional of cellular features , or any combination of any of the ized nanoparticle species can be from 2 to 50 different foregoing, or any other imaged cellular feature or compart species of functionalized nanoparticle species . Each species ment. of functionalized nanoparticle species can be functionalized [0063 ] In some embodiments , the method for detecting the with a different species of biomarker -binding moiety . For biomarker -morphological profile of a cell can further com example , in some aspects of this disclosure, each species of prise : ( h ) diagnosing the subject' s condition based on the functionalized nanoparticle species is functionalized with a biomarker -morphological profile of each cell. In some different biomarker binding moiety . In some aspects of this aspects the subject ' s condition may include or exclude , for disclosure , different biomarker binding moieties used in example , the presence of a hematological cancer, non successive contact of the cell with different pluralities of malignant hematological disorder, solid tumor, kidney dis functionalized nanoparticle species, may be bound to the ease , bladder disease , liver disease , or infectious disease. same functionalized nanoparticle , as disclosed herein . The hematological cancer can include or exclude leukemia , 10069 ] In some embodiments , when the nanoparticle spe lymphoma, or multiple myeloma. The non -malignant hema cies are functionalized with a biomarker binding moiety , tological disorder can be anemia or sickle cell disease. The e .g ., an antibody or antibody fragment or other biomarker solid tumor can include or exclude breast cancer , lung binding moiety that binds to one of the following: CD3 , cancer, prostate cancer, bone cancer, colorectal cancer, or CD22 , CD79a , Kappa , Lambda , Pax - 5 , ZAP - 70 , MPO , or bladder cancer . When the solid tumor is breast cancer , the TdT; the functionalized nanoparticle species can enter the biomarkers can include or exclude , for example , Her2 or cell and bind to its respective intracellular biomarker . The Neu . In some embodiments , the kidney disease can include intracellular biomarker can be in a cellular region which can or exclude acute kidney injury , chronic kidney disease, lupus include or exclude , for example , the cytosol, nucleus , on the nephritis , kidney rejection , or preeclampsia . In some nuclear membrane , or in or on another cellular compartment embodiments , the infectious disease can include or exclude , or structure . In some embodiments , the functionalized nano for example : HIV , hepatitis , sexually transmitted diseases , or particles are small enough to enter the cell without disrupt sepsis . In some embodiments , the hematological cancer can ing the cell membrane . The cells can be treated with a further comprise circulating cancer cells. permeabilizer so as to allow the functionalized nanoparticles [0064 ] In some embodiments, when the subject’ s condi to enter the cell without disrupting the cell membrane . In tion is a cancer, the subject' s condition can be further some embodiments , the biomarker signature can be obtained identified by the lineage of themalignancy , the stage , or state by counting the number or proportion of each of the func of remission . For example , the lineage of the malignancy tionalized nanoparticle species per cell . The number of cells can include or exclude , for example: negative , Myeloid line , having identified normal or abnormalmorphological profiles Lymphoid T cell line , or Lymphoid B cell line . in the sample can be totaled , weighted , or otherwise deter [ 0065 ] In some aspects , the resonant light scattering from mined . each observed complexed nanoparticle can be detected using [0070 ] In some embodiments , the step of (d ) illuminating evanescent or non - evanescent light . In some aspects , the the nanoparticle -cell complexes with evanescent light and non - evanescent light can be transmitted light. The resonant detecting the resonant light scattering from each observed light scattering of the complexed nanoparticle can be complexed nanoparticle , to obtain a biomarker signature of detected when imaging under a dark field illumination . In each observed cell in the method of detecting the biomarker some aspects , an illuminated slide holder can replace the morphological profile of a cell can further comprise : darkfield condenser in the microscope . The illuminated slide [0071 ] (i ) using a software program that counts the holder can use total internal reflection to illuminate the slide number of each of the functionalized nanoparticle holder . The illuminated slide holder can comprise optical species per cell and processes images in each cell in the fibers to deliver light to the edge to the slide . field of view ; [ 006 ] In some embodiments , the detection of the resonant 10072 ] ( ii ) moving the field of view digitally ; light scattering from each observed complexed nanoparticle [ 0073 ] ( iii ) using a software program to count the can be completed in under , for example , 1 second , 500 number of each of the functionalized nanoparticle milliseconds, or 200 milliseconds. species per imaged cell in the next field of view and [0067 ] In some embodiments , the one or a plurality of repeating steps ( ii ) and functionalized nanoparticle species can be comprised from [ 0074 ] (iii ) until the entire substrate area is analyzed ; nanoparticles from 10 to 200 nm in diameter. In some [0075 ] ( iv ) digitally combining all images obtained to embodiments , the nanoparticles can be comprised of, for generate a single image covering the entire substrate example , Ag , Au , Pt, Pd , Rh , Ro , Al, Cu , Ru , Cr, Cd , Zn , Si, area ; and Se or mixtures or alloys thereof. The alloy can be an alloy [0076 ] ( v ) generating from the data obtained for the of gold (Au ) and silver ( Ag) . The alloy can be of copper ( Cu ) entire substrate area the number of each of the func and Gold ( Au ) . The nanoparticles can comprise mixtures of tionalized nanoparticle species per cell, the biomarker the listed metals in discrete shells or layers . When the signature , of each substrate - adhered cell . nanoparticles comprise Si, the nanoparticles can have a Si [0077 ] In some embodiments , the software program also shell , SiO , shell (silica ) or Si core . In some embodiments , stores the positional information for each observed and / or the nanoparticles can be spherical, tubular, cylindrical , pyra imaged cell. US 2017 /0234874 A1 Aug . 17 , 2017

[0078 ] In some embodiments , the field of view is from electrons from the dye . One or more electrons can be about 0 .25 um ? to about 2 . 5 cm2. In some embodiments , the removed from the dye by an oxidation method . The oxida field of view can be from about 100 um ? to about 1000 mm . tion method can be effected by an electrochemical oxidation , In some embodiments , the field of view is a 5 microns by 5 photooxidation , or reaction with an oxidation agent. microns. In some embodiments, the field of view is 100 mm [0095 ] In some embodiments , themethod for detecting the by 100 mm . In some embodiments, the field of view is biomarker -morphological profile of a cell can further com square - shaped . The sides of the square - shaped field of view prise : ( d ) ( 2 ) removing a first plurality of functionalized can be from 0 .25 microns up to 2 . 5 centimeters . The field of nanoparticles; and ( d ) ( 3 ) contacting the cells with a second view can cover one cell, or a plurality of cells. In some plurality of functionalized nanoparticle species . In some embodiments , the field of view can cover the area of the embodiments , removing a first plurality of functionalized entire slide. nanoparticles can be achieved by cleaving a linker between [0079 ] In some embodiments , the step of ( 0 imaging each species of functionalized nanoparticle and each species morphological features of the contacted cells in the method of a functionalized nanoparticle - associated biomarker -bind of detecting the biomarker- morphological profile of a cell ing moiety . In some aspects of this disclosure , different can further comprise : biomarker binding moieties used in successive contact of the [0080 ] (i ) using a software program that processes cell with different pluralities of functionalized nanoparticle images of morphological features of each cell in the species , may be bound to the same functionalized nanopar field of view ; ticle . For example , an anti -CD3 binding moiety may be [0081 ] ( ii ) moving the field of view digitally ; bound to a 10 nm gold particle for use in contacting a first [0082 ] (iii ) using a software program to process images plurality of functionalized nanoparticle species with a cell, ofmorphological features of each cell in the next field and an anti -CD8 antibody may be bound to a 10 nm gold of view and repeating steps (ii ) and ( iii ) until the entire particle for use in a second plurality of functionalized substrate area is analyzed ; nanoparticle species , after the first plurality of functional [0083 ] ( iv ) digitally combining all images obtained to ized nanoparticles has been released . In some aspects of this generate a single image covering the entire substrate disclosure , different pluralities of functionalized nanopar area ; and ticle species may detect biomarkers indicative of two or [0084 ] (v ) generating from the data obtained for the more diseases or conditions . entire substrate area the morphological features of each [0096 ] In some embodiments of this disclosure , the cells substrate - adhered cell to detect the biomarker -morpho may be the same type or different types from each other . In logical profile of each cell . some embodiments , the cells may be associated with dif [ 0085 ) In some embodiments , a method for detecting the ferent conditions . For example , cells may be associated with biomarker -morphological profile of a cell can comprise : one or more of the following conditions : hematological [0086 ] (a ) providing a sample comprising cells from a cancer, non -malignant hematological disorder, solid tumor, subject; bladder disease , liver disease, kidney disease , or infectious [0087 ] (b ) adhering the cells to a substrate ; disease . In some aspects , the cells may be associated with 10088 ] ( c ) contacting the substrate -adhered cells with an two or more types of solid tumors . optical contrast agent; [ 0097 ] In some embodiments , the removal of a first plu [0089 ] ( d ) imaging morphological features of the con rality of functionalized nanoparticles can be achieved by tacted cells ; releasing the first plurality of functionalized nanoparticles [ 0090 ] ( e ) converting the optical contrast agent to a from the biomarker binding moieties . The functionalized colorless form ; nanoparticles can be released by displacing, cleaving, sepa [0091 ] ( f ) contacting the cells with one or a plurality of rating, disconnecting, hydrolyzing , or dissociating the nano functionalized nanoparticle species , each functional particles from the biomarker -binding moieties . In some ized nanoparticle species comprising a biomarker -bind embodiments , the linker between each nanoparticle species ing moiety to form nanoparticle - cell complexes ; in the first plurality of nanoparticles and its respective [0092 ] (g ) illuminating the nanoparticle -cell complexes biomarker binding moiety comprises a first oligonucleotide with evanescent light and detecting the resonant light bound to a first nanoparticle species and a second oligo scattering from each imaged cell nanoparticle complex , nucleotide bound to its respective biomarker binding moiety , to obtain a biomarker signature of each observed cell; where the second oligonucleotide comprises a portion and complementary to at least a portion of the first oligonucle [0093 ] (h ) associating the morphological features of the otide , and hybridization of the first oligonucleotide to the contacted cells with the biomarker signature of each second oligonucleotide forms a linker comprising a double substrate -adhered cell to detect the biomarker -morpho stranded nucleic acid in these oligonucleotide - linker func logical profile of each cell. tionalized nanoparticle species . In some embodiments the [0094 ] In some embodiments , the optical contrast agent first, second and third oligonucleotides may be the same for can be a leuco dye . The leuco dye can be methylene blue , each of the nanoparticle species and respective biomarker methylene green , red leuco dye, crystal violet, phenolphtha binding moiety in the first plurality of nanoparticles . Each lein , or thymolphthalein . The leuco dye can be converted to nanoparticle species can be released from its respective a colorless form by the addition of one or more electrons to biomarker binding moiety by binding of a third oligonucle the dye . Electrons can be added to the dye via a reduction otide to the first oligonucleotide with the hybrid formed by method . The reduction method can be effected by an elec hybridization of the third oligonucleotide and the first oli trochemical reduction , photoreduction , or reaction with a gonucleotide exhibiting a melting temperature higher than reducing agent. In some embodiments, the leuco dye can be the melting temperature of the double - stranded nucleic acid converted to a colored form by the removal of one or more formed by hybridization of the first and second oligonucle US 2017 /0234874 A1 Aug . 17 , 2017 otide . In other embodiments , first, second and third oligo nucleotide , modified polynucleotide , polyribonucleotide, nucleotides associated with each nanoparticle species and its modified polyribonucleotide , peptide , dextran or glycan . respective biomarker binding moiety may be different for The polynucleotide can comprise a DNA restriction enzyme each nanoparticle species and its respective biomarker bind sequence . The modified polynucleotide can comprise a ing moiety . For example , in the first plurality of nanopar di- thiol, diol, abasic , or uracil moiety within the polynucle ticles , the second nanoparticle species may comprise a otide sequence. fourth oligonucleotide , its respective biomarker binding 10104 ] In some embodiments , the linker can comprise a moiety may comprise a fifth oligonucleotide , and the dis peptide that further comprises a protease sequence . The placing oligonucleotide may be a sixth oligonucleotide . protease sequence can be a trypsin or chymotrypsin protease 10098 ] In some aspects of this disclosure , each species of recognition sequence . In some embodiments , the linker can functionalized nanoparticle species can be functionalized comprise a glycan that further comprises an alpha - fucosi with a different DNA oligonucleotide releasing system . In dase recognition site . The alpha - fucosidase recognition site other aspects , all of the functionalized nanoparticle species can be an alpha - 1 , 2 fucoside bond. In some aspects, the in a plurality of functionalized nanoparticle species may be linker can be cleaved with a peptidase , DNAase , and/ or functionalized with the same DNA oligonucleotide releasing RNAse . system . In some aspects all of the functionalized nanopar ticle species used may be functionalized with the same or 10105 ] In some embodiments , the substrate can be glass different DNA oligonucleotide releasing system . silica , clear polymer , gold , or alumina . The substrate can be 10099 ] In some embodiments , one or more iterations of functionalized . The substrate functionalization can be pat interrogating cells and detecting biomarkers can be achieved terned . The substrate functionalization can be a silane - linked by successive contacts with at least a second , third , up to ten cell biomarker , polymer - linked cell biomarker , silane - linked or more plurality of nanoparticle species. In some embodi amine , silane - linked carboxylic acid , polymer - linked amine , ments, the one or more iterations of interrogating biomark polymer - linked carboxylic acid , polyethylene glycol (PEG ) , ers can be one , two, three , four, five , six , seven , eight, nine , gold , silver, alumina , dextran , or glass silica . ten , fifteen , twenty , thirty , forty , fifty , sixty , seventy , or more [0106 ] A combination or kit is described for the detection times . In embodiments where oligonucleotide - linker func of a cellular biomarker signature , the combination compris tionalized nanoparticles are used , each plurality of nanopar ing a plurality of biomarker- binding -moiety - functionalized ticle species and respective biomarker binding moiety may nanoparticles. comprise the same first, second third oligonucleotide for [0107 ] A combination or kit is described for the detection each oligonucleotide- linker functionalized nanoparticle spe of a cellular biomarker signature . In some embodiments , the cies in a given plurality of oligonucleotide - linker function combination can comprise a plurality of biomarker -binding alized nanoparticle species . Alternatively, each oligonucle moiety functionalized nanoparticles where the functional otide - linker functionalized nanoparticle species and its ized nanoparticles can comprise a mixture , or can be seg respective biomarker binding moiety in each plurality of regated . In some embodiments , the functionalized nanopar oligonucleotide -linker functionalized nanoparticle species ticles can further comprise : a nanoparticle functionalized may comprise a unique set of first, second and third oligo with a first oligonucleotide ; a biomarker -binding moiety nucleotides such that each biomarker binding moiety is functionalized with a second oligonucleotide , and the first associated with a unique set of first , second and third oligonucleotide is complementary to a portion of the second oligonucleotides. In this embodiment, from one, to ten or oligonucleotide , and the first and second oligonucleotide more successive rounds of displacement and contact with a form a hybridized duplex . In an alternative embodiment, the new plurality of oligonucleotide - linker functionalized nano functionalized nanoparticles can further comprise a nano particle species can take place. particle functionalized with a first oligonucleotide; a bio [0100 ] In some embodiments , after releasing the second or marker - binding moiety functionalized with a second oligo previous plurality of functionalized nanoparticles from the nucleotide; and a third oligonucleotide , where the first biomarker binding moieties , the following steps are per oligonucleotide is complementary to a portion of the third formed : oligonucleotide , the second oligonucleotide is complemen 0101 ] ( i ) the biomarkers bound on or in the cell by the tary to a separate portion of the third oligonucleotide , and biomarker - bindingmoieties of the functionalized nano the first and second oligonucleotides form a hybridized particles are classified , and duplex to the third oligonucleotide . [0102 ] ( ii ) the cells are contacted with a next plurality of [0108 ] In some aspects , this disclosure relates to a kit for nanoparticles functionalized with different biomarker the detection of a biomarker signature , the kit comprising a binding moieties , where each nanoparticle species of combination comprising a plurality of functionalized nano the next plurality of nanoparticles is functionalized particle species , each comprising a biomarker binding moi with a biomarker binding moiety that binds to a bio ety . In some aspects , a kit may comprise a plurality of marker suspected of being associated with samples or functionalized nanoparticles and an optical contrast agent. conditions, diseases , or disorders that are also associ The optical contrast agent can be the optical contrast agents ated with the first biomarker. described herein . In this aspect, the methods of this disclosure are useful in [0109 ] In some aspects , the kit for the detection of a detecting whether the associated biomarkers are present on biomarker signature , comprising a combination comprising the same or different cell or populations of cells . a plurality of functionalized nanoparticle species , may also [0103 ] In some embodiments , the removing a first plural comprise a mountant having substantially the same refrac ity of functionalized nanoparticles can be achieved by tive index as the cells to be imaged . In some aspects , the cleaving a linker between the nanoparticle and the bio mountant may have a refractive index within 0 . 1 of the marker- binding moiety . The linker can comprise a poly - refractive index of the cells to be imaged . Themountant may US 2017 /0234874 A1 Aug . 17 , 2017 be any of the mountants described herein . As a non - limiting otide - functionalized nanoparticle containing a partial example , the mountant may have a refractive index of 1 .52 . reverse complement sequence to the oligonucleotide [ 0110 ] In some embodiments, the kit may comprises a sequence connected to the antibody. plurality of biomarker -binding moiety functionalized nano [0118 ] FIG . 6 depicts the process for the preparation of the particles where the nanoparticles can comprise mixture , or functionalized nanoparticle with an oligonucleotide , and the can be segregated . In some embodiments , for example , the preparation of a functionalized nanoparticle with a bio functionalized nanoparticles can further comprise a func marker - binding moiety via a displaceable bridging oligo tionalized nanoparticle , where the nanoparticle is releasable nucleotide . from the biomarker binding moiety . In some aspects , a first [0119 ] FIG . 7 is a Brightfield Image of stained , function nanoparticle species is functionalized with a first oligonucle alized nanoparticle labeled cells detection in Bright- Field otide ; a biomarker - binding moiety functionalized with a using 20x objective, Olympus BX60M microscope and second oligonucleotide , and the first oligonucleotide is DP71 color camera . complementary to a portion of the second oligonucleotide , [0120 ] FIGS. 8A and 8B : FIG . 8a is a Dark - field Image of and the first and second oligonucleotide form a hybridized stained , functionalized nanoparticle labeled cells at 20x duplex . In an alternative embodiment , the functionalized objective on Olympus BX60M microscope in Dark - field nanoparticles can further comprise a nanoparticle function utilizing the DarkLite Illuminator light source . FIG . 8b alized with a first oligonucleotide; a biomarker -binding shows an expanded view of two of the selected stained , moiety functionalized with a second oligonucleotide ; and a functionalized nanoparticle labelled cells from FIG . 8a . third oligonucleotide, where the first oligonucleotide is [0121 ] FIG . 9 shows an initial Brightfield image of complementary to a portion of the third oligonucleotide , the Giemsa stained blood smear stained cells imaged for mor second oligonucleotide is complementary to a separate por phology detection in Bright -Field using 20x objective , tion of the third oligonucleotide , and the first and second Olympus BX60M microscope and DP71 color camera . oligonucleotides form a hybridized duplex to the third [0122 ] FIG . 10 shows a Brightfield image of blood cells oligonucleotide. after destain treatment using 20x objective, Olympus 10111 ] A kit is described for the detection of a cellular BX60M microscope and DP71 color camera . biomarker signature . In some embodiments , the kit can 10123 ] FIG . 11 shows a Dark - field image ( 100 ms expo comprise a plurality of functionalized nanoparticles, an sure ) of destained blood smear of the same field imaged for optical contrast agent, and a mountant. residual Giemsa stain (FIG . 4 ) using 20x objective on Olympus BX60M microscope in Dark - field utilizing Dark BRIEF DESCRIPTION OF THE FIGURES Lite Illuminator light source . [ 0112 ]. This patent or application file contains at least one [0124 ] FIG . 12 shows a Dark - field image (40x objective , drawing executed in color. Copies of this patent or patent 200 ms exposure , 400 % zoom ) of CEM cell labeled with application publication with color drawing ( s ) will be pro three colors of nanoparticles . vided by the Office upon request and payment of the [0125 ] FIG . 13 shows a Brightfield image (40x Objective , necessary fee . 0 . 1 ms exposure , 200 % zoom ) of CEM cell stained with [ 0113 ] FIG . 1 depicts the general process for the succes Giemsa and labeled with 4 colors of nanoparticles . sive displacements of functionalized nanoparticles . [0126 ] FIG . 14 shows a Dark - field image (40x Objective , 10114 ) FIG . 2 depicts one embodiment for the displace 100 ms exposure , 200 % zoom ) of CEM cell labeled with 4 ment of a functionalized nanoparticle . colors of nanoparticles. [0115 ] FIG . 3 depicts one embodiment for the displace [0127 ] FIG . 15 shows a Dark - field image (40x Objective , ment of a functionalized nanoparticle using a bridging 100 ms exposure ) of cells contacted with functionalized oligonucleotide . nanoparticles in the absence of a RI- matched mountant. [0116 ] FIG . 4 depicts the process for the preparation of the 10128 ]. FIG . 16 shows Dark - field image of a cell sample functionalized nanoparticle with a biomarker- binding moi where the cells were labeled without applying additional ety . In the figure “ b ” denotes biotin , “ SAV ” denotes strepta force ( Passive Labeling ) . vidin . [0129 ] FIG . 17 shows a Dark - field image of a cell sample [ 0117] FIGS. 5A and 5B : FIG . 5a depicts the process for where the cells were labeled with functionalized nanopar the preparation of the functionalized nanoparticle with an ticles using centrifugation (additional gravitational force ) . oligonucleotide , and the preparation of a functionalized (0130 ] FIG . 18 shows a combined phase contrast and nanoparticle with a biomarker -binding moiety via a dis Dark - field image of a blood smear where cells were labeled placeable oligonucleotide overlap . FIG . 5a , depicts the using centrifugation ( additional gravitational force ). formation of a functionalized nanoparticle by the coupling 0131 FIGS . 19A - J shows a Brightfield image ( FIG . 19A ) of an amine -functionalized oligonucleotide with a carbox of a blood smear where cells were labeled using multiplex ylic acid - functionalized nanoparticle in the presence of EDC labeling and Giemsa staining . FIG . 19B shows a darkfield catalyst. FIG . 5a also depicts the formation of a function image of the same blood smear with the same field of view alized nanoparticle by the coupling of streptavidin to a where cells are labelled using multiplex labeling and Giemsa carboxylic acid -functionalized nanoparticle in the presence staining . FIG . 19D and FIG . 19C show expanded views of of EDC catalyst , followed by the subsequent coupling of a selected labelled cells which were also Giemsa stained and biotin - functionalized oligonucleotide to the streptavidin observed at the same location in the Brightfield image . Au coated nanoparticle . FIG . 5b depicts the formation of a anti -CD - 3 (yellow / lighter ) and Ag anti - CD4 (blue /darker ) functionalized nanoparticle by the reaction of a maleimide functionalized nanoparticles bind to the four lymphocytes in functionalized oligonucleotide with a thiol ( from a cysteine the field . No functionalized nanoparticle binding to neutro amino acid ) on an antibody , followed by hybridization of the phils was detected (FIG . 19F ) , whereas the neutrophils were oligonucleotide - functionalized antibody to an oligonucle observed in the Brightfield image with a Giemsa stain (FIG . US 2017 /0234874 A1 Aug . 17 , 2017

19E ). Au anti -CD - 3 (yellow / lighter ) and Ag anti- CD4 (blue / EDC – 1 - Ethyl- 3 -( 3 - dimethylaminopropyl) carbodiimide darker ) functionalized nanoparticles bound to the four lym - DMSO - dimethylsulfoxide phocytes in the field (FIG . 19H and FIG . 19J) . FIG . 19G and (0139 ] Unless specific definitions are provided , the FIG . 191 show the corresponding Brightfield image of the nomenclature utilized in connection with , and the laboratory Giemsa -stained lymphocytes as those observed in FIG . 19H procedures, techniques and methods described herein are and FIG . 19 ) , respectively . those known in the art to which they pertain . Standard [0132 ] FIGS. 20A - D shows a Brightfield image of a whole chemical symbols and abbreviations are used interchange blood cell suspension where cells were labeled with Au ably with the full names represented by such symbols . Thus , anti -CD3 . FIG . 20A shows the Darkfield image , and FIG . for example , the terms “ hydrogen ” and “ H ” are understood 20B shows the corresponding Brightfield image of the same to have identical meaning . Standard techniques may be used blood smear with the same field of view . Au anti -CD3 for chemical syntheses , chemical analyses , pharmaceutical (yellow / lighter colors ) functionalized nanoparticles bound preparation , formulation , delivery, and treatment of patients . to 13 out of 14 lymphocytes in the field , as observed by Standard techniques may be used for recombinant DNA comparing the labelled cells in FIG . 20A with the Giemsa methodology , oligonucleotide synthesis , tissue culture and stained cells in FIG . 20B . FIG . 20C and FIG . 20D show an the like . Reactions and purification techniques may be expanded view of lymphocytes labelled with functionalized performed e . g ., using kits according to manufacturer ' s anti - CD3 Au nanoparticles. No functionalized nanoparticle specifications, as commonly accomplished in the art or as binding to neutrophils was detected . described herein . [0133 ] FIGS. 21A and 21B show a passively incubated [0140 ] Notwithstanding that the numerical ranges and slide (A ) and a slide electronically enhanced in functional parameters setting forth the broad scope of the invention are ized nanoparticle density ( B ) . approximations, the numerical values set forth in the specific [0134 ] FIG . 22 shows 50 nm (Green /Darker ) and 70 nm examples are reported as precisely as possible . Any numeri ( Yellow /Brighter ) Au Nanoparticles on FFPE Tissue. cal value , however, inherently contains certain errors nec essarily resulting from the standard deviation found in their DETAILED DESCRIPTION respective testing measurements . Moreover , all ranges dis [0135 ] The presently disclosed subject matter is described closed herein are to be understood to encompass any and all more fully hereinafter with reference to the accompanying subranges subsumed therein . For example , a stated range of description and drawings , in which some , but not all “ 1 to 10 ” should be considered to include any and all embodiments of the presently disclosed subject matter are subranges between ( and inclusive of) the minimum value of shown . The presently disclosed subject matter can be 1 and the maximum value of 10 ; that is , all subranges embodied in many different forms and should not be con beginning with a minimum value of 1 or more , e . g . 1 to 6 . 1 , strued as limited to the embodiments set forth herein ; and ending with a maximum value of 10 or less , e . g ., 5 . 5 to instead , these embodiments are provided so that this disclo 10 . Additionally , any reference referred to as being " incor sure will satisfy applicable legal requirements . Like num porated herein " is to be understood as being incorporated in bers refer to like elements throughout. its entirety . Moreover, any listing of multiple values are 10136 ] Many modifications and other embodiments of the understood to include any range between any two of the presently disclosed subject matter set forth herein will be listed values. understood by one skilled in the art to which the presently 10141 ] The terms “ a ” , “ an ” , and “ the” refer to " one or disclosed subject matter pertains having the benefit of the more ” when used in this application , including the claims. teachings presented in the foregoing descriptions and the Thus , for example , reference to " a cell ” includes a plurality associated drawings . Therefore , the presently disclosed sub of such cells , unless the context clearly is to the contrary ject matter is not to be limited to the specific embodiments ( e. g ., a plurality of cells ) , and so forth . disclosed , and those of skill in the art will appreciate that [0142 ] If not otherwise specified , and where applicable , modifications and other embodiments are included within the term " substantially ” when used in association with a the scope of the appended claims. Although specific terms numerical term may refer to 85 , 86 , 87 , 88 , 89 , 90 , 91, 92 , are employed herein , they are used in a generic and descrip 93, 94 , 95 , 96 , 97, 98 , 99, or 99. 5 % . For example , the term tive sense only and not for purposes of limitation . The “ substantially ” reduced may refer to 85 % or greater reduc disclosure utilizes the abbreviations shown below . tion in the recited property . The term “ biomarker signature ” or " biomarker profile ” are not numerical terms for purposes Abbreviations of this definition . [0137 ] DNA - deoxyribonucleic acid Detecting Cell Biomarker Signatures and RNA - ribonucleic acid Biomarker- Morphological Profiles Using Resonance - Light TIRF — total internal reflection fluorescence Scattering PEG - polyethylene glycol [0143 ] In some embodiments , the present disclosure fea tures methods and compositions for detecting cell -nanopar CD Cluster Determination ticle binding moiety complexes useful in detecting a bio [0138 ] ScFv - single chain variable fragment marker signature of a cell. The methods and compositions DPX — dibutyl phthalate -xylene mountant are also useful, in some embodiments , for detecting the RI — refractive index biomarker -morphological profile of an imaged cell . NADH — Nicotinamide adenine dinucleotide , reduced 10144 ] In some embodiments , the nanoparticles function NAD + - Nicotinamide adenine dinucleotide, oxidized alized with biomarker -binding moieties can be used for NADP + nicotinamide adenine dinucleotide phosphate detecting functionalized nanoparticle cell complexes , which DTT — dithioerythritol are useful , for example , for identifying and quantifying US 2017 /0234874 A1 Aug . 17 , 2017 10 biomarkers present on cells . In some embodiments , the cells [0156 ] (d ) illuminating the functionalized nanoparticle may also be imaged to detect morphological features of the cell complexes with evanescent light and detecting the cells complexed with functionalized nanoparticles . In some resonant light scattering from each observed com embodiments , the functionalized nanoparticles can be con plexed nanoparticle , to obtain a biomarker signature of tacted with the same cells analyzed by a morphological each observed cell, imaging analysis to obtain a biomarker -morphological pro where unbound functionalized nanoparticles are not file that cell . removed from the field of view . 10145 ] In some embodiments , the methods of this inven [0157 ] Often , unbound species are washed from a target to tion are useful for improving signal generation , detection reduce background noise . In some embodiments wash steps limits, dynamic range, and /or performance characteristics of are omitted , leaving unbound functionalized nanoparticles in the biomarker signature assays . For example , in some the field of view . This embodiment may be used in some embodiments , the present disclosure relates to methods for embodiments , for example , for high throughput assays , increasing the loading amount of a biomarker binding moi and / or automated assays . The functionalized nanoparticles ety onto a cell by using an external force to increase the local concentration of the functionalized nanoparticles and cells. are specific to the biomarker on the cell, and can substan In some embodiments , the biomarker binding moiety may tially contact the cell such that litle to none signal is be a functionalized nanoparticle species . In some aspects , observed for the unbound functionalized nanoparticles. the external force may be a centrifugal, or magnetic force . In [0158 ] In some embodiments , the disease , condition , or some aspects of this invention , the method of detecting state of a cell can be identified by forming and detecting functionalized nanoparticle cell complexes can comprise : complexes between the functionalized nanoparticles and [0146 ] ( a ) providing a sample comprising cells from a cells . The method can comprise associating a biomarker subject; signature of each substrate - adhered cell to a known disease , [0147 ] ( b ) contacting the cells with one or a plurality of condition , or state of a cell exhibiting substantially the same functionalized nanoparticle species, each functional biomarker signature to identify the disease , condition , or ized nanoparticle species comprising a biomarker -bind state of the cell from a subject . ing moiety , wherein an external force is used to accel 101591. In some embodiments of this invention , where erate the formation of nanoparticle - cell complexes biomarker signatures are detected , the detecting of the through binding of the nanoparticle species comprising resonant light scattering from each observed complexed a biomarker -binding moiety to its respective bio nanoparticle comprises imaging the cell- functionalized marker ; nanoparticle complexes in contact with a mountant. In some [0148 ] ( c ) adhering the functionalized nanoparticle -cell embodiments , background and /or interfering white light complexes to a substrate ; scatter can be reduced or substantially eliminated by using [0149 ] (d ) detecting the functionalized nanoparticle cell a mountant comprising a solution with about the refractive complexes by illuminating the nanoparticle- cell com index of the cells . In some embodiments of this invention , plexes with evanescent light and detecting the resonant the mountant can be within about 0 . 1 of the refractive index light scattering from each observed functionalized of cells , where the cells are fixed . In some embodiments , the nanoparticle cell complex , to obtain a biomarker sig refractive index of fixed cells is about 1 .52 , or 1 .52 . In some nature of each observed cell; and embodiments , the index of refraction of the mountant can be [0150 ] ( e ) associating the biomarker signature of each from 1. 51 to 1 . 54 . In some embodiments , using a mountant substrate - adhered cell to a known disease , condition , or having an RI within 0 . 1 of the refractive index of fixed cells state of a cell exhibiting substantially the same bio is useful for reducing the amount of white light scatter , and marker signature to identify the disease , condition , or obtaining better images of resonance scattering from the state of the cell from a subject. cell - functionalized nanoparticle complexes . In one aspect , [0151 ] In some aspects , the phrase substantially the same this disclosure relates to a method for detecting functional or similar biomarker signature , or biomarker- morphological ized nanoparticle cell complexes , the method comprising : profile , may refer to a biomarker signature or profile com [0160 ] ( a ) providing a sample comprising cells from a prising similar levels and / or types of biomarkers and / or subject; biomarker- morphologies for which a concordance has been [ 0161 ] (b ) contacting cells which have been fixed with established or reasonably is expected for the same disease , one or a plurality of functionalized nanoparticle spe condition , state or disorder . cies, each functionalized nanoparticle species compris [ 0152] In some embodiments , the biomarker signature of ing a biomarker- binding moiety , and forming nanopar a cell can be detected in a homogeneous assay , the assay ticle - cell complexes through binding of the comprising the steps of: nanoparticle species comprising a biomarker - binding [0153 ] (a ) providing a sample comprising cells from a moiety to its respective biomarker ; subject ; [0162 ] (c ) adhering the functionalized nanoparticle -cell [0154 ] ( b ) contacting the cells with one or a plurality of complexes to a substrate , wherein the adhered nano functionalized nanoparticle species, each functional particle - cell complexes are placed in contact with a ized nanoparticle species comprising a biomarker -bind mountant, wherein the refractive index of the mountant ing moiety , and forming functionalized nanoparticle is within about 0 . 1 of the refractice index of the fixed cell complexes through binding of the nanoparticle cells ; species comprising a biomarker -binding moiety to its [0163 ] (d ) detecting the functionalized nanoparticle cell respective biomarker ; complexes by illuminating the nanoparticle - cell com [0155 ] (c ) adhering the functionalized nanoparticle -cell plexes with evanescent light and detecting the resonant complexes to a substrate ; light scattering from each observed functionalized US 2017 /0234874 A1 Aug . 17 , 2017

nanoparticle cell complex , to obtain a biomarker sig [0175 ] (g ) associating the morphological features of the nature of each observed cell ; and contacted cells with the biomarker signature of each [0164 ] (e ) associating the biomarker signature of each substrate - adhered cell to detect the biomarker -morpho substrate - adhered cell to a known disease , condition , or logical profile of each cell . state of a cell exhibiting substantially the same bio [0176 ] In addition , it may be desirable to detect the normal marker signature to identify the disease, condition , or or diseased tissue or cells of a patient. The presence or state of the cell from a subject . absence of certain circulating cancer or other cells , for In some embodiments , the mountant may have a refractive example , may be diagnostic for disease . Thus , the endog index of about 1 .52 . enous cells of a human patient are the cells that may be [ 0165 ] In some embodiments , the ability to use the com advantageously detected using the compositions, methods positions and methods of this disclosure to associate the and kits of the present invention . biomarker signature of an individual cell, and in some embodiments , with its morphological image / features greatly Sample Source enhances the ability to diagnose and monitor abnormal conditions or disorders . 101771 The term “ sample ” as used herein refers to an [0166 ] As set forth herein , the inventors have made the aliquot of material, frequently an aqueous solution or an surprising discovery that nanoparticles functionalized with aqueous suspension derived from biological material . In biomarker -binding moieties can be used for detecting cell some embodiments , the sample can be a biological sample . functionalized nanoparticle complexes and identifying and The biological sample can be from a living subject. For quantifying biomarkers present on imaged cells for example , example , in some embodiments, the sample may be any when the functionalized nanoparticles are contacted with the sample containing cells . In some embodiments , the sample same cells analyzed by a morphological imaging analysis . may be from , for example , whole blood , bone marrow , The ability to use the compositions and methods of this serum , plasma , cerebrospinal fluid , sputum , bronchial wash disclosure to associate the biomarker signature of an indi ings, bronchial aspirates, urine , lymph fluids and various vidual cell with its morphological image / features greatly external secretions of the respiratory, intestinal and genito enhances the ability to diagnose and monitor abnormal urinary tracts , tears , saliva , milk , white blood cells , myelo conditions or disorders . mas and the like ; biological fluids such as cell culture [0167 ] In some embodiments , this disclosure relates to supernatants , tissue specimens which may or may not be compositions and methods for obtaining a biomarker signa fixed , and cell specimens which may or may not be fixed , or ture for an imaged cell , which is used , in some embodi a fine needle aspirate . Samples to be assayed for the pres ments , in combination with detected morphological features ence of an analyte by the methods of the present invention of the cell obtained from imaging the cell . In some aspects , include , for example, cells , tissues , homogenates , lysates , compositions comprising functionalized nanoparticle spe extracts, purified or partially purified proteins and other cies, each comprising a specific biomarker -binding moiety , biologicalmolecules and mixtures thereof. As a non - limiting are used to detect the biomarker signature of the imaged cell . example , the tissue sample can be a tissue sample from a In some embodiments of this disclosure , combinations of biopsy , for example , a FFPE (formalin - fixed , paraffin - em such compositions are made or used in the methods of this bedded ) tissue sample , aspirate , or surgically removed tissue disclosure . The combinations can be mixtures of such com sample . The FFPE samples can be sourced from a clinic or positions, or may comprise compositions segregated before laboratory . The samples used in the methods of the present invention will vary based on the assay format and the nature use . of the tissues , cells , extracts or other materials , especially 10168 ] In some embodiments , the method for detecting a biological materials , to be assayed . biomarker -morphological profile of a cell can comprise the [0178 ] In some embodiments , the biological sample may steps of be processed . The processing can be , for example , removal [0169 ] (a ) providing a sample comprising cells from a of selected species in the sample . In some embodiments , the subject; sample may comprise white blood cells . In some embodi [0170 ] ( b ) contacting the cells with one or a plurality of ments , at least 10 % , 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , functionalized nanoparticle species , each functional or 90 % of the red blood cells are removed before contacting ized nanoparticle species comprising a biomarker- bind the cells with the plurality of functionalized nanoparticle ing moiety , and forming nanoparticle -cell complexes species . In some embodiments , at least 50 % of the red blood through binding of the nanoparticle species comprising cells are removed before contacting the cells with the a biomarker - bindingmoiety to its respective biomarker plurality of functionalized nanoparticle species . on the cell ; [0179 ] As used herein , “ subject” refers to any mammal [0171 ] ( c ) adhering the functionalized nanoparticle - cell that can include or exclude humans, domestic and farm complexes to a substrate ; animals, and zoo , or pet animals , such as dogs, horses, cats , [0172 ] ( d ) illuminating the functionalized nanoparticle mouse , rat , llama, sheep , pigs, cows, etc . The preferred cell complexes with evanescent light and detecting the mammal herein is a human , including adults , children , and resonant light scattering from each observed com the elderly . Preferred sports animals are horses and dogs . plexed nanoparticle , to obtain a biomarker signature of Preferred pet animals are dogs and cats . The subject may be , each observed cell ; for example , an aquatic park animal, such as a dolphin , [0173 ] ( e ) contacting the substrate - adhered cells with an whale , seal or walrus . In certain embodiments , the subject, optical contrast agent; individual or patient is a human . Cells for use in the present [0174 ] ( f ) imaging morphological features of the con invention may be obtained from any of the aforementioned tacted cells ; and subjects . US 2017 /0234874 A1 Aug . 17 , 2017

[0180 ] In some embodiments , cells from a subject can primary tumor. A metastasized cancer cell can retain many comprise biomarkers which may be useful to assist in of the characteristics of the original cancer cells . For identifying the cellular state , identity , growth rate , lineage , example , in some embodiments, the methods of this disclo mutations, variants, expression levels , cancer stage or remis sure can detect the origin of metastasizing cancer . The origin sion status, and /or latent or active infection . Such cells may ofmetastasizing cancer can be ascertained by identifying the include or exclude , for example , mammalian cells , immu biomarker signature and / or biomarker -morphological profile nomodulatory cells, lymphocytes, monocytes, polymorphs , of tumor cells in a distant location , and detecting that the T cells , tumor cells, yeast cells , bacterial cell, infectious biomarker signature and / or biomarker -morphological profile agents , parasites, plant cells , transfected cells such as NSO , is the same or similar to the biomarker signature and / or CHO , COS, 293 cells . biomarker -morphological profile from the primary cancer at [0181 ] In some embodiments , the cell may be alive , dead , its original location . In some embodiments , a cancer cell in fixed and /or substantially intact . In some embodiments , the a tissue may exhibit multiple neoplasms. In some embodi cells can be the same type or different types . When the cells ments, the tissue cells can be interrogated separately , such are different types , the cells can be from different tissue or that one or more cancer cell types ( or neoplasm ) can be tumor origin , exhibit a different pathology , express different identified during the detecting a biomarker or biomarker or mutated biomarkers , express different levels of biomark morphological profile of a cell . ers , express biomarkers with different post- translational modifications , or exhibit different morphology . In some Histopathology Methods embodiments , the biomarker -binding moiety is capable of [0186 ] Histopathology imaging often requires cell immo distinguishing between a mutant biomarker and a wild - type bility such that the same region can be analyzed after biomarker. When the cells are from different tumor origin , subjecting the cells in the region with a different imaging the cells can be from a tumor which can include or exclude , modality to correlate the cells from one imaging modality to for example , breast cancer, lung cancer, prostate cancer , the other imaging modality . In some embodiments , the cell bone cancer, colorectal cancer, liver cancer, pancreatic can is fixed with a fixing agent. The fixing agent may be , for cer, thyroid cancer, bladder cancer, or other types of cancer. example , formaldehyde , glutaraldehyde , or another cross [0182 ] The terms " cell proliferative disorder ” and “ prolif linking agent. In other embodiments water -soluble preser erative disorder ” , as used herein , refer to disorders that are vatives, for example , methyl or propyl paraben , dimethyl associated with some degree of abnormal cell proliferation . olurea, sorbic acid , 2 - pyridinethiol- 1 - oxide , or potassium In some embodiments , the cell proliferative disorder is sorbate may be used . In some embodiments the cell is cancer . permeabilized by surfactants . [0183 ] The term “ tumor, ” as used herein , refers to all 10187 ] In some embodiments , the functionalized nanopar neoplastic cell growth and proliferation , whether malignant ticle cell complexes adhered to a substrate may be imaged in or benign , and all pre -cancerous and cancerous cells and contact with a mountant. As used herein , a “ mountant” is any tissues . The terms “ cancer, " " cancerous, " " cell proliferative substance in which a specimen is suspended between a slide disorder ,” “ proliferative disorder ,” and “ tumor" are not and a cover glass for microscopic examination . A mountant mutuallymi exclusive as referred to herein . can be used to maintain image fidelity during the course of [0184 ] The terms “ cancer” and “ cancerous, " as used the detection . One of the major causes of image degradation herein , refer to or describe the physiological condition in in microscopy is due to improper matching of the refractive mammals that is typically characterized by unregulated cell index between the immersion medium and mountant growth and/ or proliferation . Some cancers are composed of (Diaspro A , et al ., Appl Opt 2002; 41 (4 ): 685 -690 ). However , rapidly dividing cells while others are composed of cells that if the mountant refractive index is different from the func divide more slowly than normal. Types of cancer examples tionalized nanoparticle cell complex , white light scattering can include or exclude, for example , carcinoma, lymphoma will result . ( e . g ., Hodgkin ' s and non - Hodgkin ' s lymphoma) , blastoma, [ 0188 ] In some embodiments , the mountant can comprise sarcoma, and leukemia . More particular examples of such a solution with a similar refractive index to the refractive cancers can include or exclude , for example , squamous cell index of the cells . The refractive index of a cell may vary by cancer , small -cell lung cancer , non - small cell lung cancer, cell type , and may also vary within the region of the cell . In adenocarcinoma of the lung, squamous carcinoma of the some aspects , the refractive index of cells may vary from 1 . 2 lung , cancer of the peritoneum , hepatocellular cancer, gas to 1 .6 . In some aspects , the refractive index may vary from trointestinal cancer , pancreatic cancer, glioblastoma , cervi 1 . 4 to 1 .5 . In some embodiments , the refractive index of cal cancer , ovarian cancer, liver cancer, bladder cancer , fixed cells can be 1. 52 . In some embodiments, the mountant hepatoma, breast cancer , colon cancer , colorectal cancer, can be within 0 . 1 of the refractive index of the fixed cells . endometrial or uterine carcinoma, salivary gland carcinoma, In some embodiments, the mountant can be within 0 . 09 , kidney cancer, liver cancer , prostate cancer, vulval cancer , 0 . 08 , 0 .07 , 0 .06 , 0 . 05 , 0 .04 , 0 . 03 , 0 . 02 , or 0 .01 of the thyroid cancer, hepatic carcinoma, leukemia and other lym refractive index of the fixed cells . In some embodiments , the phoproliferative disorders , and various types of head and mountant can be within 1 , 2 , 3 , 4 , 5 , 6 , 7 , 89, or 10 % of the neck cancer . refractive index of the reference index of the fixed cells . In [ 0185 ] In some embodiments , local cancer metastases can some embodiments , the mountant can be immersion oil, invade the lymphatic system , leading to distantmestastases . DPX , dissolved polystyrene ( in xylene ), Valap ( an equal Distantmetastases commonly involve the brain , lung , bone mixture of Vaseline , lanolin , and paraffin mixed on a heating and liver . Each cancer has a distinct pattern of metastasis plate 60° C . ) , Gel/ Mount , Fluoromount- G , Fluorsave , Pro ( e . g . prostate cancer may metastasizes to the bone , but rarely long, Vectashield ,MOWIOL , Modified Apathy' s mountant, to the brain ). Metastasis can occur at any time in cancer Permount, or Entellan . The volume of the mountant can be growth , and may occur before or after removal of the from about 2 microliters to about 40 microliters . In some US 2017 /0234874 A1 Aug . 17 , 2017 embodiments , the volume of mountant can be from 5 10º - fold greater than non -specific binding . In some embodi microliters to 15 microliters. In some embodiments , the ments , specific binding may be characterized by affinities volume of mountant can be about 10 microliters . In some greater than 10 % - fold over non -specific binding . Whenever a embodiments , the mountant may further comprise an anti range appears herein , as in “ 1 - 10 or one to ten , the range fade reagent which prevents photodegradation of the optical refers without limitation to each integer or unit of measure contrast agent. In some embodiments , the mountant can in the given range . Thus, by 1 - 10 it is meant each of 1 , 2 , 3 , further comprise spacers or gaskets . In some aspects , the 4 , 5 , 6 , 7 , 8 , 9 , 10 and any subunit in between . spacers or gaskets can be tailored to direct the fluid flow to [0191 ] In some embodiments , the biomarker can be a the cells. In some embodiments , algorithms can may be used biomolecule identified by the Cluster Determinant antigen to minimize the light noise from the white light scattering . (CD ) and or other molecules/ antigenic sites . [0192 ] In some embodiments , the biomarker can include Biomarker- Binding Moieties or exclude, for example , those listed in Table 1 . [0189 ] The term “ biomarker ” as used herein refers to any distinguishing element found on or within a cell . The TABLE 1 distinguishing element can be an antigen or another binding Antigen Name partner recognized by a biomarker binding moiety . The term Other Names CDla R4 , T6 “ antigen ” or “ binding partner ” as used herein refers to any CD16 R1, T6 known or unknown substance that can be recognized by an CD1c M241 , R7 , T6 antibody or other biomarker binding moiety. The term CDid R3G1 “ antigen ” or “ binding partner ” may include , for example , CDle CR2 CD2 T11, LFA - 2 , SRBC- R proteins, peptides, glycoproteins and carbohydrates. In some CD2R T11 - 3 embodiments , the antigen is expressed on the surface of a CD3 gamma, T3g cell . Preferably these antigens include biologically active CD3 delta proteins, such as hormones, cytokines , and their cell surface CD3 epsilon T?? receptors , or bacterial or parasitic cells , agents or antigens , CD4 L3T4 , W3/ 25 , T4 CD5 T1 , Tp67 , Leu- 1 , Ly- 1 membranes or purified components thereof, and viral anti CD6 T12 . TP120 gens or binding partner. Such cells or agents may be those CD7 gp40 , TP41 that naturally express the antigen or binding partner on their CD8a T8 , Leu - 2 CD8b CD8 , Leu2 , Lyt3 surface or a transformed cell expressing the antigen on its CD9 p24 , MRP - 1 surface . In some embodiments, the transformed cell can be CD10 CALLA , NEP, gp100 transfected with an oncogene which is integrated into the CD11a LFA - 1, integrin alphaL cell. In some embodiments , the transformed cells may CD11b Mac - 1 , integrin alphaM CD11c p150 , 95 , CR4, integrin alphaX include or exclude , for example , mammalian cells , immu CD12 p90 - 120 nomodulatory cells , lymphocytes, monocytes, polymorphs, CD13 Aminopeptidase N , APN T cells , tumor cells , yeast cells, bacterial cell , infectious CD14 LPS - R agents , parasites , plant cells , transfected cells such as NSO , CD15 Lewis - x , Lex CD15s Sialyl Lewis X CHO , COS , 293 cells. Transformation of cells such as NSO , CD15u Sulfated Lewis X CHO , COS and 293 cells can be achieved by a method CD16a FcgammaRIIIA which can include or exclude electroporation and nucleo CD16b FcgammaRIIIB fection . In some embodiments , the detected biomarker can CD17 Lactosylceramide , LacCer be present on the cell surface , within the cell , or both on the CD18 Integrin beta2 CD19 B4 surface and within the cell . In some embodiments the CD20 B1, Bp35 biomarker in the cell may be present in or on one or more CD21 C3DR , CR2 , EBV- R cellular features, for example , the cytosol, the nucleus , the CD22 BL - CAM , Siglec - 2 nuclear membrane , nucleoli , the endoplasmic reticulum , CD23 FcepsilonRII CD24 BA - 1 Golgi apparatus or mitochondria . In some embodiments , the CD25 Tac , p55 biomarker can be expressed and transported to the cell CD26 DPP IV surface which is accessible to external biomarker- binding CD27 T14 CD28 Tp44 , T44 moieties . CD29 Integrin betal [0190 ] The terms “ specifically binding ” and “ specific CD31 PECAM - 1 binding ” as used herein mean that an antibody or other CD32 FcgammaRII CD33 p67 , Siglec - 3 molecule , especially a biomarker - binding moiety of the CD34 gp 105 -120 , Hematopoietic progenitor cell antigen 1 invention , binds to a target such as an antigen , ligand or (HPCA1 ) other analyte, with greater affinity than it binds to other CD35 CR1 molecules under the specified conditions of the present CD36 GPIV CD37 gp52 -40 , Leukocyte antigen CD37 , Tetraspanin - 26 , invention . In various embodiments of the invention , “ spe TSPAN26 cifically binding " may mean that an antibody or other CD38 T10 specificity molecule binds to a target analyte molecule with CD39 Ectonucleoside triphosphate diphosphohydrolase 1 at least about a 100- fold greater affinity , preferably at least (ENTPD1 ) , ATP dehydrogenase , NTPdehydrogenase - 1 about a 10 '- fold greater affinity , more preferably at least CD40 Bp50 , MGC9013 , TNFRSF5 , Tumor necrosis about a 10 - fold greater affinity , and most preferably at least factor receptor superfamily member 5 about a 10 % - fold greater affinity than it binds molecules CD41 gpub unrelated to the target molecule . Typically , specific binding CD42a GPIX refers to affinities in the range of about 10º - fold to about US 2017 /0234874 A1 Aug . 17 , 2017 14

TABLE 1 -continued TABLE 1 - continued Antigen Name Other Names Antigen Name Other Names CD42b GPIba CD85a ILT5 , LIR3 , HL9, LILRB3 (Leukocyte CD42c GPIbb immunoglobulin - like receptor, subfamily B ( with CD42d GPV TM and ITIM domains ) , member 3 , LIR - 3 , CD43 Leukosialin , sialophorin MGC138403, PIRB , XXbac - BCX105G6 . 7 H CD44 H -CAM , Pgp - 1 CD85c LILRB5 ( Leukocyte immunoglobulin - like receptor , CD44R CD44v subfamily B ) (with TM and ITIM CD45 LCA , T200 , B220 domains ) , member 5 , LIR8 CD45RA PTPRC CD85d LILRB2 (Leukocyte immunoglobulin - like receptor, CD45RB PTPRC subfamily B (with TM and ITIM CD45RO domains) , member 2 , LILRB2 , ILT4 , LIR2 , MIR10 , CD46 Membrane Cofactor Protein (MCP ) , Trophoblast MIR - 10 leukocyte common antigen , TRA2. 10 CD85e LILRA3 (Leukocyte immunoglobulin - like CD47R receptor, subfamily A (without TM domain ) , CD48 Blast - 1 member 3 , HM31, HM43 , ILT6 , LIR - 4 , CD49a VLA - 1 LIR4 , e3 CD49b VLA - 2 CD85f XXbac - BCX403H19 . 2 , CD85 , CD85F , LIRI , ILT11 , CD49c VLA - 3 LILRB7 (Leukocyte immunoglobulin - like CD49d VLA - 4 receptor, subfamily B (with TM and ITIM domains) , CD49f VLA - 6 member 7 CD50 ICAM - 3 CD85g LILRA4 (Leukocyte immunoglobulin - like receptor, CD51 Vitronectin receptor subfamily A (with TM domain ) , CD52 CAMPATH - 1 member 4 , ILT7 , MGC129597 , MGC129598 CD53 MOX44, TSPAN25 , Tetraspanin - 25 CD85h LILRA2 ( Leukocyte immunoglobulin - like receptor, CD54 ICAM - 1 subfamily A (with TM domain ) , CD55 DAF member 2 , ILT1 , LIR7, LIR - 7 , XXbac -BCX85G21 . 2 , CD56 NCAM ILT- 1 CD57 FINK - 1 , Leu - 7 CD85i LILRA1 (Leukocyte immunoglobulin - like receptor) , CD58 LFA - 3 subfamily A ( with TM domain ), member 1 , LIR - 6 , LIR6 , MGC126563 CD59 Protectin , MAC - inhibitor CD85j LILRB1 ( Leukocyte immunoglobulin - like receptor, CD60a GD3 subfamily B (with TM and ITIM CD60b 9 - O - sialyl GD3 domains) , member 1 , FLJ37515 , ILT2 , LIR - 1 , LIRI, CD60c 7 - O -sialyl GD3 MIR - 7 , MIRT CD61 GPIIIa , Integrin beta - 3 CD85k LILRB4 ( Leukocyte immunoglobulin - like receptor, CD62E E -selectin , ELAM - 1 subfamily B (with TM and ITIM CD62L L -selectin , LECAM - 1 domains ), member 4 , ILT3 , LIR - 5 , HM18 , LIR5 , CD62P P -selectin , PADGEM , GMP - 140 LILRB5 CD63 LIMP, MLA1, gp55 , NGA , LAMP - 3 , ME491 , CD86 B70 , B7 - 2 OMA81H , TSPAN30 , Granulophysin , CD87 UPA - R Melanoma 1 antigen CD88 CSaR CD64 FcgRI, Fc- g Receptor 1 , High affinity CD89 FcalphaR immunoglobulin g Fc Receptor I, FcgRIA CD90 Thy - 1 CD65 Ceramide- dodecasaccharide, VIM2, CD91 LRP1, a2M - R , a2MR , APOER , APR , LRP Fucoganglioside ( Type II) CD92 SLC44A1 , CTL1 , CHTL1, RP11 - 287A8. 1 , p70 CD65s VIM2 CD93 CiQR1 , C1qRP , MXRA4 , C1qR ( P ) , Dj737e23. 1 , CD66a BGP - 1 , NCA - 160 GR11 CD94 KP43 CD66b CD67, CGM6 CD95 CD178 , FASLG , APO - 1 , FAS, TNFRSF6 , CD95L , CD66c NCA APTILG1 , APT1, FAS1, FASTM , CD66d CGM1 ALPS1A , TNFSF6 , FASL CD66e CEA CD96 TACTILE , MGC22596 CD66f PSG , Sp - 1 CD97 TM & LN1, BL -KDD /F12 CD68 Macrosialin , gp110 CD98 SLC3A2, 4F2 , 4F2HC , 4T2HC , MDU1, NACAE , CD69 AIM , EA 1 , MLR3, gp34 / 28 , VEA , CLEC2C , FRP - 1 , RL - 388 BL - AP26 CD99 MIC2, E2 CD70 TNFSF7 , CD27LG , CD27L , Ki- 24 CD99R E2 CD71 TFRC , T9 , Transferrin receptor , TFR , TRFR CD100 SEMAJ, coll - 4 , C9orf164 , FLJ33485 , FLJ34282 , CD72 Ly - 19. 2 , Ly - 32 . 2 , Lyb2 FLJ39737 , FLJ46484 , M - sema - G , CD73 Ecto - 5 '- nuclotidase , NT5E , E5NT, NT5 , NTE , MGC169138 , MGC169141, SEMA4D , SEMAJ eN , ENT CD101 IGSF2 , P126 , V7 , BA27 , BPC # 4 , P126 , V7 - LSB CD74 DHLAG , HLADG , Ia - g , li, invariant chain CD102 ICAM - 2 , Ly60 CD75 ST6GAL1 , MGC48859 , SIAT1 , STOGALL , STON , CD103 HML- 1 , alphab , integrin alphaE CD104 beta4 integrin , TSP1180 , ITGB4 , TSP - 180 ST6 b -Galactosamide a - 2 , 6 - sialyltranferase , CD105 Endoglin , HHT1, ORW , SH - 2 Sialo -masked lactosamine , Carbohydrate of a2 , 6 CD106 VCAM - 1 sialyltransferase CD107a LAMP - 1 CD75s a2, 6 Sialylated lactosamine CD107b LAMP- 2 CD77 Gb3 , Pk blood group CD108 SEMATA , JMH blood group antigen CD79a IGA ( Immunoglobulin - associated a ), MB - 1 CD109 8A3, E123 7D1 , 150kD TGF- b - 1 -binding protein , CD79b IGB ( Immunoglobulin - associated b ) , B29 Platelet- specific Gov antigen CD80 CD28LG , CD28LG1, L AB7, B7 , B7 - 1 , BB1 CD110 MPL , TPO - R CD81 TAPA - 1 CD111 PRR1, Nectin - 1 CD82 4F9, C33 , 144, KAI1 , R2 , ST6, SAR2, GR15 CD112 PRR2, Nectin - 2 CD83 HB15, BL11 CD113 PVRL3, Nectin3 CDw84 LY9B , SLAMF5 , p75 , GR6 , hly9 -b CD114 G -CSFR US 2017 /0234874 A1 Aug . 17 , 2017 15

TABLE 1 -continued TABLE 1 - continued Antigen Name Other Names Antigen Name Other Names CD115 M -CSFR , c - fms CD180 RP - 105 CD116 GM - CSFRalpha CD181 CXCR1 , IL - 8RA CD117 c -kit , SCFR CD182 CXCR2 , IL - SRB CD118 LIFR , gp190 CD183 CXCR3 CD119 IFNgammaR CD184 CXCR4 , fusin CD120a TNFR - I CD185 CXCR5 , BLR1 CD120b TNFR - II CD186 CXCR6 , BONZO CD121a IL - 1R type I CD191 CCR1, MIP - 1alphaR , RANTES - R CD1216 IL -1R , type II CD192 CCR2 , MCP - 1 - R CD122 IL - 2 Rbeta CD193 CCR3 , CKR3 CD123 IL - 3R CD195 CCR5 CD124 IL - 4R CD196 CCR6 , LARC receptor , DRY6 CD125 IL - 5R CD197 CCR7 CD126 IL -6R CD198 CCRS , GPRCY6 , TER1 CD127 IL - 7R CD199 CCR9 , GPR - 9 - 6 CD130 gp130 , ILØST, IL6 - b or CD130 CD200 OX - 2 CD131 CSF2RB , IL3RB , IL5RB , CDw131 CD201 EPC - R CD132 Common gamma chain , IL - 2Rg CD202b Tie2 , Tek CD133 AC133 , prominin - like 1 CD203c NPP3 /PDNP3 , ENpp1 , PD -1b CD134 OX - 40 CD204 Macrophage scavenger- R CD135 Flt3 / Flk2 CD205 DEC - 205 CD136 MSP - R , RON D206 macrophage mannose - R CD137 4 - 1 BB D207 Langerin , C - type Lectin domain family 4 CD138 Syndecan - 1 member K ( CLEC4K ) CD139 CD208 DC -LAMP CD140a PDGFRalpha CD209 DC - SIGN CD140b PDGFRbeta CD210 IL - 10 - R CD141 Thrombomodulin CD212 IL - 12 - R betal CD142 Tissue Factor CD213al IL - 13 - R alphal CD143 ACE D213a2 IL - 13 - R alpha2 CD144 VE - Cadherin , Cadherin - 5 CD217 IL - 17 - R CD145 CD218a IL - 18Ralpha, IL - 1Rip CD146 MUC18 , S -endo CD2186 IL - 18Rbeta , IL18RAP CD147 Neurothelin , basoglin D220 Insulin - R CD148 HPTP - eta CD221 IGF - 1 R CD150 SLAM CD222 IGF - II R , mannose - 6 phosphate - R CD151 PETA - 3 CD223 LAG - 3 CD152 CTLA - 4 CD224 GGT CD153 CD3OL CD225 Leu - 13 CD154 CD40L , gp39 , TRAP CD226 DNAM - 1 , PTA - 1 , TLISA1 CD155 PVR CD227 MUC1, EMA CD156a ADAMS D228 Melanotransferrin CD156b TACE / ADAM 17 CD229 Ly - 9 CD156C ADAM10 CD230 Prion protein CD157 BST- 1 CD231 TALLA - 1 , A15 CD158a p58 . 1 CD232 VESP - R CD1586 p58 . 2 CD233 Band 3 , SLC4A1 CD159a NKG2A CD234 Duffy, DARC CD159c NKG2C CD235a Glycophorin A CD160 BY55 CD235ab Glycophorin A / B CD161 NKR - P1A CD235b Glycophorin B CD162 PSGL - 1 CD236 Glycophorin C /D CD162R PEN - 5 CD236R Glycophorin C CD163 130kD CD238 Kell blood group glycoprotein ( Kel) , Kell blood CD164 MGC - 24 group antigen , Endothelin - 3 - converting CD165 AD2, gp37 enzyme ( ECE3 ) CD166 ALCAM CD239 Basal cell adhesion molecule ( BCAM , B -CAM ) , CD167a DDR1 Lutheran blood group glycoprotein , CD168 RHAMM Lutheran blood group antigen (Lu ) CD169 sialoadhesin , Siglec - 1 D240CE Rh30CE CD170 Siglec - 5 , CD33 - 1 ike2 CD240D Rh30D CD171 LICAM , HSAS , HSAS1, MASA , MIC5, CD241 RhAg , Rh50 N - CAML1, S10 , SPG1, NILE CD242 ICAM - 4 CD172a SIRPgamma CD243 MDR - 1 , p170 , P - gp CD172b SIRPbeta , SIRB1 CD244 2B4 , NAIL , NKR2B4 , Nmrk , SLAMF4 CD172g SIRPgamma, SIRPB2 CD245 p220 / 240 CD173 Blood group H type 2 CD246 ALK , Ki- 1 CD174 Lewis Y , FUT3 , Les , FT3B CD247 CD3 - Z , CD3H , CD3Q , CD3Z , T3Z , TCRZ CD175 Tn CD248 TEM1, Endosialin CD175s Sialyl - Tn CD249 Aminopeptidase A CD176 Thomson Friedrenreich Ag CD252 TNFSF4 , GP34 , OX4OL , TXGP1, CD134L , CD177 NB1 OX -40L , OX40L CD178 FasL , CD95L CD253 TRAIL , Apo -2L , TL2, TNFSF10 CD179a V pre B CD254 TRANCE , RANKL , OPGL CD179b Lambda 5 CD256 APRIL , TALL - 2 US 2017 /0234874 A1 Aug . 17 , 2017 16

TABLE 1 -continued TABLE 1 - continued Antigen Name Other Names Antigen Name Other Names CD257 BLYS , BAFF , TALL - 1 CD344 FZD4, Frizzled homolog 4 CD258 LIGHT, HVEM - L CD349 FZD9 , Frizzled homolog 9 CD261 TRAIL - R1, DR4 CD350 FZD10 , Frizzled homolog 10 CD262 TRAIL -R2 , DR5 CD351 FCAMR , Fc receptor , IgA , IgM , high affinity CD263 TRAIL -R3 , DcR1, LIT CD352 SLAMF6 , Ly108 , NTB - A CD265 RANK , TRANCE - R , ODFR CD353 SLAMF8, BLAME CD266 TWEAK - R , FGF - inducible 14 CD354 TREM1 CD267 TACI, TNFR SF13B CD355 CRTAM , Cytotoxic and regulatory T- cell CD268 BAFFR , TR13C molecule CD269 BCMA , TNFRSF13B CD357 TNFRSF18 , Tumor necrosis factor receptor CD271 NGFR , p75 (NTR ) superfamily , member 18 , GITR CD272 BTLA CD358 TNFRSF21 , Tumor necrosis factor receptor CD273 B7DC , PD -L2 , PDCD1L2 superfamily , member 21, DR6 CD274 B7 -H1 , PD - L1 CD359 P116 CD275 B7 -H2 , ICOSL , B7 - RP1 , GL50 CD360 IL21R CD276 B7 -H3 CD361 EVI2B (ectoptic viral integration site 2B ) CD277 BT3. 1 , butyrophilin SF3 A1 , BTF5 CD362 Syndecan - 2 CD278 ICOS , AILIM CD363 S1PR1, Sphingosine- 1 -phosphate receptor 1, CD279 PD1, SLEB2 EDG - 1 CD280 ENDO180 , UPARAP CD281 TLR1 CD282 TLR2 CD283 TLR3 [0193 ] In some embodiments , the biomarker the bio CD284 TLR4 marker can include or exclude , for example : CD1 , CD2, CD289 TLR9 CD3, CD4, CD5, CD , CD7, CD , CD9, CD10 , CD1la , CD292 BMPR1A , ALK3 CD293 BMPRIB , ALKO CD11b , CD11c , CD13 , CD14 , CD15 , CD16 , CD19 , CD20 , CD294 CRTH2. GPR44 CD21, CD22 , CD23 , CD25 , CD30 , CD33 , CD34 , CD38 , CD295 LeptinR , LEPR CD41 , C43 , CD45 , CD56 , CD57 , CD58 , CD61 , CD64 , C71 , CD296 ART1 , RT6 , ART2 CD79a , CD99 , CD103 , CD117 , CD123 , CD138 , CD138 , CD297 ART4 , dombrock blood group CD298 Na + / K + - ATPase beta3 subunit CD163 , CD235a , HLA - DR , Kappa , Lambda , Pax - 5 , BCL - 2 , CD299 DC - SIGN - related , LSIGN , DC - SIGN2 Ki- 67 , ZAP -70 , MPO , TNT , FMC - 7 , Pro2PSA , ROMA CD300a CMRF35H , IRC1 , IRp60 (HE4 + CA - 125 ), OVA1 (multiple proteins ), HE4 , Fibrin / CD300c CMRF35A , LIR fibrinogen degradation product (DR -70 ) , AFP -L3 % , Circu CD300e CMRF35L CD301 MGL , HML lating Tumor Cells (EpCAM , CD45 , cytokeratins 8, 18 + , CD302 DCL1, BIMLEC 19 + ) , HER2, NEU , Prostate stem cell antigen (PSCA ) , CD303 BDCA2, HECL epithelial- specific antigen ( ESA ) , epithelial cell adhesion CD304 BDCA4 , neuropilin 1 molecule (EPCAM ), a2B1, VEGFR - 1 , VEGFR - 2 , CD133 , CD305 LAIR1 CD306 LAIR2 AC133 antigen , p63 protein , c -Kit , CA19 - 9 , Estrogen recep CD307a FCRL1, IRTA5 tor ( ER ) , Progesterone receptor (PR ), Pro2PSA , HER - 2 /neu , CD307b FCRL2 , IRTA4 CA - 125 , CA15 - 3 , CA27 .29 , Free PSA , Thyroglobulin , CD3070 FCRL3 , IRTA3 Nuclear Mitotic Apparatus protein (NUMA , NMP22 ) , CD307d FCRL4, IRTA1 CD307e FCRL5 , IRTA Alpha - fetoprotein (AFP ) b , ROMA (HE4 + CA - 125 ) , OVAL CD309 VEGFR2, KDR HE4 , DR -70 , p63 protein , c - Kit , CA19 - 9 , Total PSA , alpha CD312 EMR2 Methylacyl -CoA Racemase / AMACR , CA125 /MUC16 , ER CD314 NKG2D , KLR alpha /NR3A1 , ER beta /NR3A2 , Thymidine Kinase 1, AG - 2 , CD315 CD9P1 , SMAP6 , FPRP , PTGFRN BRCA1, BRCA2 , CA15 - 3 /MUC - 1 , Caveolin - 1 , CD117 / c CD316 EWI2, PGRL , CD81P3 , KASP CD317 BST2 , HM1. 24 kit , CEACAM -5 /CD66e , Cytokeratin 14, EGF R / ErbB1 , CD318 CDCP1 , SIMA135 HIN - 1 /SCGB3A1 , Ki- 67 /MKI67 , MKP - 3 , Nestin , NGF CD319 CRACC , SLAMF7 R / TNFRSF16 , NM23 -H1 , PARP , PP4 , Serpin E1/ PAI - 1 , CD320 8D6A , 8D6 CD321 JAMI, F11 receptor 14 - 3 - 3 beta , 14 - 3 - 3 sigma, 14 - 3 - 3 zeta , 15 - PGDH /HPGD , CD322 JAM2, VE - JAM 5T4 , A33 , ABCBS, ABCB6 , ABCG2 , ACE /CD143 , ACLP, CD324 E - Cadherin , Uvomorulin ACP6 , Afadin /AF - 6 , Afamin , AG -2 , AG - 3 , Akt, Aldo -keto CD325 N - Cadherin , NCAD Reductase 1C3 / AKR1C3 , alpha 1B -Glycoprotein , alpha CD326 EP - CAM , Ly74 CD327 SIGLEC6 1 -Microglobulin , AlphaB Crystallin / CRYAB , alpha - Feto CD328 SIGLEC7 , AIRM - 1 protein /AFP , alpha -Methylacyl - CoA Racemase / AMACR , CD329 SIGLEC9 AMFR / gp78 , Annexin A3 , Annexin A8 / ANXA8, APC , Apo CD331 FGFR1, Fms- like tyrosine kinase - 2 , KAL2 , lipoprotein A - I/ ApoA1, Apolipoprotein A - II / ApoA2, Apoli N - SAM CD332 FGFR2, BEK , KGFR poprotein E / ApoE , APRIL / TNFSF13 , ASCL1/Mash1 , CD333 FGFR3, ACH , CEK2 ATBF1/ ZFHX3, Attractin , Aurora A , BAP1, Bcl - 2 , Bcl- 6 , CD334 FGFR4, JTK2, TKF beta 2 -Microglobulin , beta - 1 , 3 - Glucuronyltransferase CD335 NKP46 , Ly - 94 homolog 1 /B3GAT1 , beta -Catenin , beta - III Tubulin , Bikunin , BMI- 1 , CD336 NKp44 , Ly - 95 homolog CD337 NKp30 , Ly117 B - Raf, BRCA1, BRCA2 , Brk , C4 . 4A / LYPD3 , CA15 - 3 / CD338 ABCG2 , BCRP, Bcrpl, MXR MUC - 1, c - Abi, Cadherin -13 , Caldesmon /CALDI , Cal CD339 Jagged - 1 , JAG1, JAGL1, hJ1 ponin 1, Calretinin , Carbonic Anhydrase IX /CA9 , Catalase , CD340 ERB -B2 , Neu , Her - 2 Cathepsin D , Caveolin -1 , Caveolin -2 , CBFB , CCR7, CCR9, CEACAM - 19 , CEACAM - 20 , CEACAM - 4 , CHD1L , Chiti US 2017 /0234874 A1 Aug . 17 , 2017 17 nase 3 - like 1 , Cholecystokinin -B R /CCKBR , Chorionic PIWIL2, PKM2, PLK1, PLRP1, PP4, P -Rex1 , PRMT1 , Gonadotropin alpha Chain (alpha HCG ), Chorionic Gonado Profilin 1 , Progesterone R B /NR3C3 , Progesterone tropin alpha /beta (HCG ) , CKAP4 / p63, Claudin - 18 , Clus R /NR3C3 , Progranulin /PGRN , Prolactin , Prostaglandin E terin , C -Maf , C -Myc , Coactosin - like Protein 1/ CotL1 , Synthase 2/ PTGES2 , PSAP , PSCA , PSMA/ FOLH1 / COMMD1, Cornulin , Cortactin , COX - 2 , CRISP - 3 , CTCF, NAALADase 1 , PSMA1, PSMA2, PSMB7 , PSP94 /MSMB , CTL1/SLC44A1 , CXCL17 / CC - 1 , CXCL8 /IL - 8 , CXCL9/ PTEN , PTEN , PTHIR /PTHR1 , PTK7 / CCK4 , PTP beta / MIG , CXCR4, Cyclin A1, Cyclin A2, Cyclin D2 , Cyclin D3 , zeta /PTPRZ , Rab25 , RARRESI , RARRES3 , Ras , Reg4 , CYLD , Cyr61/ CCN1 , Cytokeratin 14 , Cytokeratin 18 , Ret , RNF2 , RNF43 , S100A1, S100A10 , S100A16 , S100A2, Cytokeratin 19 , DAB2 , DCBLD2 /ESDN , DC - LAMP, Dkk S100A4, S100A6 , S100A7, S100A9, S100B , S100P , 1 , DLL3, DMBT1 , DNMT1, DPPA2 , DPPA4 , E6 , E -Cad SART1 , SCUBE3 , Secretin R , Serpin A9/ Centerin , Serpin herin , ECM - 1 , EGF, EGF R /ErbB1 , ELF3 , ELTD1, EMM E1 /PAI - 1 , Serum Amyloid A1, Serum Amyloid A4 , SEZÓL , PRIN /CD147 , EMP2 , Endoglin /CD105 , Endosialin / CD248 , SEZ6L2/ BSRP - A , Skp2 , SLC16A3 , SLC45A3 /Prostein , Enolase 2 /Neuron - specific Enolase , EpCAM / TROP1 , SLC5A5 , SLC5A8 / SMCT1, SLC7A7 , Smad4 , SMAGP, Eps15 , ER alpha /NR3A1 , ER beta / NR3A2 , ErbB3/ Her3 , SOCS - 1 , SOCS - 2 , SOCS - 6 , SOD2/Mn - SOD , Soggy - 1 / ErbB4 /Her4 , ERCC1, ERK1, ERK5 /BMK1 , Ets - 1 , Exosto DkkL1, SOX11, SOX17 , SOX2, SPARC , SPARC - like sin 1 , EZH2, Ezrin , FABP5 / E - FABP, Fascin , FATP3 , 1 /SPARCL1 , SPINK1, Src , STEAP1 , STEAP2 , STEAP3/ FCRLA , Fetuin A / AHSG , FGF acidic , FGF basic , FGF R3, TSAPÁ , STRO - 1 , STYK1, Survivin , Synaptotagmin - 1 , Syn FGF R4, Fibrinogen , Fibroblast Activation Protein alpha / decan - 1 /CD138 , Syntaxin 4 , Synuclein - gamma, Tankyrase FAP, Follistatin - like 1 / FSTLI, FOLR1, FOLR2 , FOLR3, FOLR4, FosB /GOS3 , FoxM1, FoxO3 , FRAT2 , FXYD5 / 1 , Tau , TCF- 3 /E2A , TCL1A , TCL1B , TEM7/ PLXDC1 , Dysadherin , GABA - A R alpha 1 , GADD153 , GADD45 TEM8/ ANTXR1, Tenascin C , TFF1 , TGF -beta 1 , TGF -beta alpha , Galectin - 3 , Galectin - 3BP /MAC - 2BP , gamma -Gluta 1 , 2 , 3 , TGF - beta 1 / 1 . 2 , TGF - beta 2 / 1 . 2 , TGF - beta RI/ ALK mylcyclotransferase / CRF21, Gasl, Gastrin - releasing Pep 5 , THRSP, Thymidine Kinase 1 , Thymosin beta 10 , Thy tide R /GRPR , Gastrokine 1 , Gelsolin /GSN , GFAP , GLI- 2 , mosin beta 4 , Thyroglobulin , TIMP Assay Kits , TIMP - 1 , Glutathione Peroxidase 3 /GPX3 , Glypican 3 , Golgi Glyco TIMP - 2 , TIMP - 3 , TIMP- 4 , TLE1, TLE2 , TLR2 , TM4SF1/ protein 1 /GLG1 , gp96 /HSP90B1 , GPR10 , GPR110 , GPR18 , L6 , TMEFF2 / Tomoregulin - 2 , TMEM219, TMEM87A , GPR31 , GPR87 , GPRC5A , GPRC6A , GRP78 / HSPA5 , TNF -alpha , TOP2A , TopBP1, t -Plasminogen Activator /tPA , HE4 /WFDC2 , Heparanase /HPSE , Hepsin , Her2 , HGF R / c TRA - 1 -60 ( R ), TRA - 1 -85 / CD147 , TRAF- 4 , Transgelin / TA MET, HIF - 2 alpha /EPAS1 , HIN - 1 /SCGB3A1 , HLA - DR . GLN , Trypsin 2 /PRSS2 , Tryptase alpha/ TPS1 , TSPAN1, HOXB13 , HOXB7 , HSP70 /HSPAIA , HSP90 , Hyaluroni UBE2S , UPAR , u - Plasminogen Activator /Urokinase , Uro dase 1 /HYAL1 , ID1, IgE , IGFBP - 2 , IGFBP - 3 , IGFBP - 4 , tensin - II R , VAP - 1 /AOC3 , VCAM - 1 /CD106 , VEGF, VEGF IGFBP -6 , IGF -I , IGF - I R , IGF - II , IGFL - 3 , IGFLR1, IL - 1 R1/ Flt - 1 , VEGF R2/ KDR / Flk - 1 , VEGF/ P1GF Heterodimer, beta / IL - 1F2 , IL - 17E /IL - 25 , IL - 2 , IL -6 , IMP Dehydrogenase VSIG1, VSIG3, YAP1, ZAG , ZAP70 , ZMIZ1/ Zimp10 , and 1 / IMPDHI, Importin alpha 2 /KPNA2 , ING1, Integrin beta Carcino -embryonic antigen . In some embodiments , the bio 1 / CD29 , Integrin beta 3 /CD61 , IQGAP1, Isocitrate Dehy marker can include or exclude , for example , the proteins drogenase 1 /IDH1 , ITIH4 , ITM2C , Jagged 1, JNK , JunB , listed in the Cancer Atlas (http : // www .proteinatlas . org / JunD , Kallikrein 2 , Kallikrein 6 /Neurosin , KCC2/ search / cancer ) . SLC12A5 ,Ki - 67 /MK167 , KISS1R /GPR54 , KLF10 , KLF17 , LICAM , Lactate Dehydrogenase A /LDHA , Lamin B1 , [0194 ] In some embodiments , the biomarker is selected LEF1, Leptin /OB , LIN - 28A , LIN -28B , Lipocalin - 2 /NGAL , from markers expressed by kidney cells , infectious or para LKB1/ STK11 , LPAR3/ LPA3 / EDG - 7 , LRMP, LRP - 1B , sitic agents , solid tumor cells , circulating tumor cells , or any LRRC3B , LRRC4 , LRRN1/ NLRR - 1 , LRRN3 /NLRR - 3 , other cell useful for diagnosis or prognosis . In some embodi Ly6K , LYPD1, LYPD8, MAP2, Matriptase / ST14 , MCAM / ments , the biomarker is selected from markers expressed on CD146 , M -CSF , MDM2/HDM2 , Melan - A /MART - 1 , Mel the surface or within kidney cells , infectious agents ( e . g . , anocortin - 1 R /MCIR , Melanotransferrin /CD228 , Mela bacteria or virus) , solid tumor cells , or circulating tumor tonin , Mer , Mesothelin , Metadherin , Metastin /KiSSi , cells . In some embodiments , the biomarkers expressed on Methionine Aminopeptidase , Methionine Aminopeptidase the surface or within kidney cells can include or exclude , for 2 /METAP2 , MFAP3L , MGMT, MIA , MIF , MINA , Mind example : KIM - 1 , Albumin , beta - 2 microglobulin , Cystatin Bomb 2 /MIB2 , Mindin , MITF , MKK4, MKP -1 , MKP - 3 , C , Clusterin , Apolipoprotein A - I /ApoA1 , CXCL8 / IL - 8 , MMP- 1 , MMP - 10 , MMP- 13 , MMP - 2 , MMP- 3 , MMP - 8 , ERCC1, Ki- 67 / MKI67 , MMP - 9 , or Trefoil factor- 3 . MMP- 9 , MRP1, MRP4 / ABCC4 , MS4A12 , MSH2, MSP [0195 ] In some embodiments , the biomarker is one or a R /Ron , MSX2 , MUC - 4 , Musashi- 1 , NAC1, Napsin A , plurality of markers for a particular type of cancer. In some NCAM - 1 /CD56 , NCOA3 , NDRGI, NEK2, NELLI , embodiments , the biomarker for breast cancer can include or NELL2 , Nesfatin - 1 /Nucleobindin - 2 , Nestin , NFkB2 , NF - L , exclude her2 - neu , ER , PR , Ki- 67 , and p53 . In some embodi NG2/ MCSP , NGF R / TNFRSF16 , Nicotinamide N -Methyl ments , the biomarker for lung cancer can include or exclude transferase /NNMT , NKX2 . 2 , NKX3 . 1 , NM23 -H1 , NM23 TTF - 1 , Napsin A , CK 5 / 6 , p40 /63 , and Synaptophosmin . In H2 , Notch - 3 , NPDC - 1 , NTS1/ NTSR1 , NTS2 /NTSR2 , some embodiments , the biomarker for prostrate cancer can OGR1, Olig2 , Osteopontin /OPN , Ovastacin , OXGR1/ include or exclude AMACR, PSA , CEA , and p63 . In some GPR80 /P2Y15 , p130Cas, p15INK4b /CDKN2B , p16INK4a / embodiments , the biomarker for colorectal cancer can CDKN2A , p18INK4c /CDKN2C , p21 /CIP1 / CDKN1A , p27 / include or exclude MLH1, MSH2, PMS2 , MSH6 , c -Kit , Kip1 , P2X5/ P2RX5 , p53 , PARP, PAUF /ZG16B , PBEF / p16 , and BRAF V600E . In some embodiments , the bio Visfatin , PDCD4 , PDCD5 , PDGF R alpha , PDGF R beta , marker for tumor infiltrating lymphocytes can include or PDZD2, PEA - 15 , Pepsinogen A5 /PGA5 , Peptidase Inhibitor exclude CD4, CD8 , CD14 , CD20 , CD45RO , FoxP3 , PD - L , 16 /P116 , Peroxiredoxin 2 , PGCP, PI 3 - Kinase p85 alpha , and PD -L1 . In some embodiments , the biomarker for can US 2017 /0234874 A1 Aug . 17 , 2017

cers of the urinary system ( bladder, kidney , urethra ) can antibody fragment may comprise an antigen binding arm include or exclude CK7, p63 , CK20 , p53 , Ki- 67 , PSA , linked to an Fc sequence capable of conferring in vivo Vimentin , and PAX8. stability to the fragment. [ 0196 ] In some embodiments , the biomarker is cell -spe [0200 ] “ Polyclonal Antibodies ” or “ PAbs, ” are heteroge cific . The cells can be in a healthy state (normal ) or diseased neous populations of antibody molecules derived from the state (abnormal ) . Monocytes and macrophages can exhibit a sera of animals immunized with an antigen , or an antigenic biomarker that includes or excludes the CD14 and CD16 functional derivative thereof. For the production of poly biomarkers. Lymphocyte B cells can exhibit a biomarker clonal antibodies, host animals such as rabbits , mice and that includes or excludes the CD20 biomarker. Lymphocyte goats , may be immunized by injection with an antigen or NK cells can exhibit a biomarker that includes or excludes antigen - conjugate , optionally supplemented with adjuvants . the CD56 biomarker. Lymphocytes T cells can exhibit a Polyclonal antibodies may be unpurified , purified or par biomarker that includes or excludes the CD3 biomarker . T tially purified from other species in an antiserum . The Reg cells can exhibit a biomarker that includes or excludes techniques for the preparation and purification of polyclonal the CD4 , CD25 , and FoxP3 biomarkers . Cytotoxic T cells antibodies are described in various general and more specific can exhibit a biomarker that includes or excludes the CD8 references , including but not limited to Kabat & Mayer , biomarker . Helper T cells can exhibit a biomarker that Experimental Immunochemistry , 2d ed . , ( Thomas , Spring includes or excludes the CD4 biomarker. Naïve T cells can field , I11. ( 1961) ); Harlow & Lane , Antibodies : A Laboratory exhibit a biomarker that includes or excludes the CD45RA Manual (Cold Spring Harbor Laboratory Press , Cold Spring biomarker . Memory T cells can exhibit a biomarker that Harbor, N . Y . ( 1988 ) ) ; and Weir, Handbook of Experimental includes or excludes the CD45RO biomarker. Tth cells can Immunology , 5th ed . (Blackwell Science , Cambridge, Mass . exhibit a biomarker that includes or excludes the CXR5 ( 1996 ) . biomarker . Th17 cells can exhibit a biomarker that includes [0201 ] “ Monoclonal antibodies ," or " MAbs, ” are homo or excludes the CCR6 biomarker . Th2 cells can exhibit a geneous populations of antibodies to a particular antigen and biomarker that includes or excludes the CCR4 biomarker. may be obtained by any technique that provides for the Th1 cells can exhibit a biomarker that includes or excludes production of antibody molecules, such as by continuous the CXCR3 biomarker. Tumor cells can exhibit a biomarker culture of cell lines. These techniques include , but are not limited to the hybridoma technique of Köhler and Milstein , that includes or excludes the PanCK biomarker. Nature , 256 : 495 - 7 ( 1975 ) ; and U . S . Pat. No . 4 ,376 , 110 ), the [0197 ] The term “ biomarker -binding moiety ” as used human B - cell hybridoma technique (Kosbor , et al. , Immu herein is a moiety that can specifically bind to a biomarker . nology Today, 4 :72 ( 1983 ); Cote , et al. , Proc. Natl . Acad . In some embodiments , the biomarker -binding moiety can Sci . USA , 80 : 2026 - 30 ( 1983 ) ) , and the EBV - hybridoma include or exclude , for example , an antibody or antibody technique (Cole , et al. , in Monoclonal Antibodies And Can fragment, nanobody , receptor fragment, DNA aptamer , cer Therapy , Alan R . Liss , Inc . , New York , pp . 77 - 96 DNA /RNA oligonucleotide, RNA aptamer , PNA aptamer , (1985 ) ) . Such antibodies may be of any immunoglobulin peptide aptamer , LNA aptamer , carbohydrate , or a lectin . class including IgG , IgM , IgE , IgA , IgD and any subclass [0198 ] The term “ antibody ” as used herein is a protein that thereof. Thehybridoma producing theMAb of this invention can specifically bind to an antigen . In some embodiments , an may be cultivated in vitro or in vivo . Production of high antibody can include or exclude , for example , any recom titers of MAbs in vivo makes this a presently preferred binant or naturally occurring immunoglobulin molecule method of production . such as a member of the IgG class e .g . IgG1 and also any 0202 ] Techniques developed for the production of “ chi antigen binding immunoglobulin fragment, such as Fv , Fab meric antibodies " (Morrison , et al . , Proc. Natl . Acad . Sci. , and F (ab ' ) 2 fragments , antibody fragment, ScFv (single 81: 6851 - 6855 ( 1984 ) ; Takeda , et al. , Nature , 314 : 452 -54 chain variable fragment, a fusion protein of the variable (1985 ) ) by splicing the genes from a mouse antibody mol regions of the heavy ( V ) and light chains ( V ) of immu ecule of appropriate antigen specificity together with genes noglobulins , connected with a short linker peptide of ten to from a human antibody molecule of appropriate biological about 25 amino acids ) , or single - domain antibody ( nano activity can be used . A chimeric antibody can be a molecule body ) , and any derivatives thereof. In embodiments where in which different portions are derived from different animal the biomarker binding moiety comprises an antibody, the species , such as those having a variable region derived from antibody can be a monoclonal or polyclonal antibody . a murine MAb and a human immunoglobulin constant [0199 ] The term “ antibody fragments ” as used herein , region . refers to a portion of an intact antibody, wherein the portion [0203 ] Alternatively , techniques described for the produc retains at least one, and as many as most or all , of the tion of single chain antibodies ( U . S . Pat. No . 4 , 946 , 778 ; functions normally associated with that portion when pres Bird , Science 242 : 423 - 26 ( 1988 ) ; Huston , et al. , Proc . Natl . ent in an intact antibody. In some embodiments , an antibody Acad . Sci . USA , 85 :5879 - 83 ( 1988 ) ; and Ward , et al. , Nature , fragment comprises an antigen binding site of the intact 334 : 544 -46 ( 1989 ) ) can be adapted to produce gene - single antibody and thus retains the ability to bind antigen . In some chain antibodies suitable for use in the present invention . embodiments , an antibody fragment , for example one that Single chain antibodies are typically formed by linking the comprises the Fc region , retains at least one of the biological heavy and light chain fragments of the Fv region via an functions normally associated with the Fc region when amino acid bridge, resulting in a single chain polypeptide . present in an intact antibody, such as FcRn binding , antibody [0204 ] Antibody fragments that recognize specific half - life modulation , ADCC function and complement bind - epitopes may be generated by known techniques . For ing . In some embodiments , an antibody fragment is a example , such fragments include but are not limited to : the monovalent antibody that has an in vivo half- life substan - F ( ab ') 2 fragments that can be produced by pepsin digestion tially similar to an intact antibody. For example , such an of the antibody molecule and the Fab fragments that can be US 2017 /0234874 A1 Aug . 17 , 2017 19 generated by reducing the disulfide bridges of the F ( ab ') 2 protein , c -Kit , CA19 - 9, Estrogen receptor ( ER ), Progester fragments . Alternatively , Fab expression libraries may be one receptor (PR ), HER - 2 /neu , CA - 125 , CA15 - 3 , CA27 .29 , constructed (Huse , et al. , Science, 246 : 1275 - 81 ( 1989 )) to Free PSA , Thyroglobulin , Nuclear Mitotic Apparatus pro allow rapid and easy identification of monoclonal Fab tein (NUMA , NMP22 ) , Alpha - fetoprotein (AFP ) b , Total fragments with the desired specificity . PSA , and Carcino - embryonic antigen , or any of the bio [0205 ] The monoclonal antibodies herein specifically markers described herein . In some embodiments , when the include " chimeric ” antibodies in which a portion of the biomarker- binding moiety is anti - CD45 , the biomarker sig heavy and /or light chain is identical with or homologous to nature obtained is the white blood cell count . corresponding sequences in antibodies derived from a par ticular species or belonging to a particular antibody class or Optical Contrast Agents subclass , while the remainder of the chain ( s ) is identical [ 0208 ] In some embodiments, the contacting the cells with with or homologous to corresponding sequences in antibod an optical contrast agent can comprise adding a dye or ies derived from another species or belonging to another colorant to the cells . In some embodiments , the optical antibody class or subclass, as well as fragments of such contrast agent can be a leuco dye , cell stain , or any dye antibodies, so long as they exhibit the desired biological useful for imaging for morphological analysis including , for activity . example , any dye useful for histological, cytological, cyto 10206 ]. The term “ humanized ” forms of non - human ( e . g ., pathological, or histopathological imaging . In some embodi murine ) antibodies are chimeric antibodies that comprise ments , the optical contrast agent provides visual classifica minimal sequence derived from non -human immunoglobu tion and identification of cells by differentially staining cells . lin . In some embodiments , a humanized antibody is a human The histopathological imaging can be imaging method used immunoglobulin ( recipient antibody ) in which residues from in a treatment or diagnostic clinic . The leuco dye can be red a hypervariable region of the recipient are replaced by leuco dye , methylene blue, crystal violet, phenolphthalein , residues from a hypervariable region of a non -human spe thymolphthalein , or methylene green . cies (donor antibody ) such as mouse , rat, rabbit or nonhu 102091 In some embodiments , the optical contrast agent man primate having the desired specificity , affinity , and /or can be a cell stain selected from : Giemsa stain , Wright stain , capacity . In some instances, framework region (FR ) residues Wright -Giemsa stain , May -Grünwald stain , Mallory of the human immunoglobulin are replaced by correspond trichrome, Periodic acid - Schiff reaction stain , Weigert ' s ing non -human residues . Furthermore , humanized antibod elastic stain , Heidenhain 's AZAN trichrome stain , Orcein ies may comprise residues that are not found in the recipient stain , Masson ' s trichrome, Alcian blue stain , May -Grün antibody or in the donor antibody . These modifications are wald -Giemsa , van Gieson stain , Hansel stain , Reticulin made to further refine antibody performance . In general, the Stain , Gram stain , Bielschowsky stain , Ferritin stain , Fon humanized antibody will comprise substantially all of at tana -Masson stain , Hales colloidal iron stain , Pentachrome least one , and typically two, variable domains, in which all stain , Azan stain , Luxol fast blue stain , Golgi ' s method or substantially all of the hypervariable loops correspond to (reduced silver ), reduced gold , Chrome alum /haemotoxylin those of a non -human immunoglobulin and all or substan stain , Isamin blue stain , Argentaffin stains, Warthin - Starry tially all of the FRs are those of a human immunoglobulin silver stain , Nissl stain , Sudan Black and osmium stain , sequence . The humanized antibody optionally will also osmium tetroxide stain , hematoxylin stain , Uranyl acetate comprise at least a portion of an immunoglobulin constant stain , lead citrate stain , Carmine stain , safranin stain , and region (Fc ) , typically that of a human immunoglobulin . For Ziehl- Neelsen stain . further details , see Jones et al ., Nature 321 :522 - 525 ( 1986 ) ; [ 0210 ] In some embodiments , the optical contrast agent Riechmann et al. , Nature 332 :323 -329 ( 1988 ) ; and Presta , can be a dye or colorant selected from : eosin Y , eosin B , Curr. Op. Struct . Biol. 2 : 593 -596 ( 1992 ). azure B , pyronin G , malachite green , toluidine blue , copper [0207 ] In some embodiments , the biomarker binding moi phthalocyanin , alcian blue , auramine- rhodamine , acid ety may bind to a peptide , protein , protein fragment, glyco fuschin , aniline blue , orange G , acid fuschin , neutral red , sylation moiety or pattern , or a carbohydrate . The biomarker Sudan Black B , acridine orange , Oil Red 0 , Congo Red , Fast binding moiety can be selected from a biomarker binding green FCF, Perls Prussian blue reaction , nuclear fast red , moiety , e . g . , an antibody or fragment thereof or other alkaline erythrocin B , and naphthalene black . biomarker binding moiety that binds to any of the biomark [ 0211] In some embodiments , the cells labelled with the ers in Table 1 . In some embodiments , the biomarker binding optical contrast agent can be selectively decolored . The moiety can be selected from a biomarker binding moiety that selective decloration can comprise removal of the stain , binds to , for example : CD1, CD2 , CD3 , CD4 , CD5 , CD6 , conversion of the stain or dye to a colorless form , or CD7, CD , CD9, CD10 , CD11a , CD11b , CD11c , CD13 , degradation of the dye. The stain can be removed by CD14 , CD15 , CD16 , CD19 , CD20 , CD21, CD22 , CD23 , washing . The washing can be done in the presence of a CD25, CD30 , CD33 , CD34 , CD38, CD41, C43 , CD45 , different pH than during contacting with the stain such that CD56 , CD57 , CD58 , CD61, CD64, C71, CD79a , CD99 , the overall charge of the stained proteins changes thereby CD103 , CD117 , CD123 , CD138 , CD138 , CD163 , CD235a , affecting removal of the stain . The washing can be done with HLA -DR , Kappa , Lambda , Pax - 5 , BCL - 2 , Ki -67 , ZAP -70 , a solvent system which solubilizes the stain at the altered MPO , TNT, FMC - 7 , Pro2PSA , ROMA (HE4 + CA - 125 ) , pH . In some embodiments , optical contrast agent can be a OVA1 (multiple proteins ) , HE4, Fibrin / fibrinogen degrada leuco dye . In some embodiments , the leuco dye can be tion product (DR - 70 ) , AFP -L3 % , Circulating Tumor Cells converted to a colorless form by the addition of one or more (EPCAM , CD45 , cytokeratins 8 , 18 + , 19 + ) , HER2, NEU , electrons to the dye . Electrons can be added to the dye via Prostate stem cell antigen ( PSCA ), epithelial- specific anti a reduction method . The reduction method can be effected gen ( ESA ) , epithelial cell adhesion molecule (EpCAM ) , by an electrochemical reduction , photoreduction , or reaction a2B1, VEGFR -1 , VEGFR - 2 , CD133 , AC133 antigen , p63 with a reducing agent. The reducing agent can by sodium US 2017 /0234874 A1 Aug . 17 , 2017 20 cyanoborohydride sodium borohydride, NADH ( formed in cancer cells can exhibit mitoses. In a malignant cancer cell , situ or separately added ) , ascorbic acid (and salts thereof, for the number ofmitoses can increase , with an atypical mitosis example sodium ascorbate , potassium ascorbate , ammonium forming with defects in the mitotic spindle appear, which ascorbate , etc . ) or dithiothreitol (DTT ) . In some embodi results in triple or quadruple asters ( cellular structures ments , the leuco dye can be converted to a colored form by shaped like a star, comprised of microtubules , formed the removal of one or more electrons from the dye . One or around each centrosome during mitosis ) and dissymmetrical more electrons can be removed from the dye by an oxidation structures and atypical forms of chromosomes. In some method . The oxidation method can be effected by an elec embodiments , a cancerous cell may exhibit nuclear changes trochemical oxidation , photooxidation , or reaction with an that can explain the presence of different cell clones and oxidation agent . In some embodiments , the oxidation agent genetic anomalies associated with these changes . can be NAD + , NADP + , pyruvate , acetaldehyde , cystine , [0216 ] The cytoplasm of a cancerous cell can also change , alpha -ketoglurate , ibquinone , 2 cytochrome c , 2 cytochrome with the appearance of new structures appear or disappear c , 2 cytochrome a3 , or oxygen . ance of normal structures . In some embodiments , the new [0212 ] When the sample is from tissue , the optical contrast structures in cancer cells can be cytoplasmic inclusions. agent can be a H & E (hematoxylin and eosin ) stain . In one Cytoplasmic inclusions can include Auer rods, clumps of aspect, the optical contrast agent may be suitable for stainable cellular granular material that form elongated supravital staining . In one aspect, the optical contrast agent needles seen in the cytoplasm of leukemic blasts (partially may be suitable for vital staining . differentiated cells ). In some forms of neoplasms, apoptosis Cell Morphology from Imaging the Optical Contrast Agent occurs, with the presence of apoptotic bodies in the cyto [0213 ] Histopathological analysis often involves imaging plasm . of a sample contacted with an optical contrast agent . In some [0217 ] Malignant cancer cells have a small cytoplasmic aspects , cellular morphological features can be identified by amount, frequently with vacuoles . the visual characteristics of a cell , including or excluding , [0218 ]. In cancerous cells , the granular endoplasmic reticu for example, the size, shape , or the presence and /or absence lum may exhibit a simplified structure appearance . The ER of colored internal bodies . In some embodiments , the imag may be amorphous, with granular or filamentous material ing of the morphological features of the contacted cells can accumulating in the cisternae . In some embodiments , frag comprise measuring an optical property of the optical con mentation and degranulation can be observed , with the trast agent. The optical property of the optical contrast agent interruption of connections between the granular endoplas can include or exclude, for example , absorbance , scattering , mic reticulum and mitochondria . A decrease of the granular fluorescence , photoluminesence , Raman emission , and pho endoplasmic reticulum from tumor cells can occur with an toluminescent lifetime. In a preferred embodiment, the opti increase of free ribosomes and polysomes . cal property detected by the optical contrast agent is the [0219 ] In cancerous cells , the Golgi apparatus can be absorbance of light . The wavelength of the absorbed light poorly developed , which indicates a lack of tumor cell can be from the ultraviolet range to the infrared range . differentiation . Cancerous cells that have completely lost Preferably , the wavelength of absorbed light is in the visible differentiation may exhibit a Golgi apparatus. range ( 300 - 800 nm ) . The optical property of the optical [0220 ] Cancer cell mitochondria can decrease in volume contrast agent can be measured under a microscope with with tumor development. Mitochondria can show a high either a light field illumination or dark field illumination . variability of shape and volume, with very large mitochon [ 0214 ] In some embodiments , the morphological features dria observed . Cancerous cell mitochondrial crystals can be identified from the cell can comprise the cell surface shape, different from those of a normal cell, with inclusions and the cell nucleus shape , the chromatin shape, the nucleolar pyknotic images present in the matrix . shape , the number of nucleolus , the grade of the cancer [0221 ] A cancer cell may exhibit secondary lysosomes , ( closeness to a normal cell ) , the arrangement of the cells , or myelinic structures and lipofuscin granules. combinations of the foregoing . In some embodiments , the (0222 ] A cancer cell membrane can exhibit an increase or morphological features identified from the cell of a subject diminution in the number of surface receptors , changing cell can be compared against a previously obtained cell of a sensitivity to the regulating mechanisms of the host; struc subject so as to determine if the cells exhibit dysplasia over tural changes of proteins or surface receptors that no longer time . react with the corresponding ligand ; and the presence of new [ 0215 ] Morphologically, a cancerous cell is characterized surface molecules , characteristic of the embryonic tissue , by a large nucleus , having an irregular size and shape , which are hidden at the surface of adult cells . Abnormal prominent nucleoli , and scarce and intensely colored or pale surface molecules are able to act as antigens and are recog cytoplasm . Changes in cell nucleus over time can be imaged nized by the mechanisms of humoral and cellular defense . of the cells surface , volume, nucleus /cytoplasm ratio , shape , Tumor cells can be covered with immune complexes , which density , structure and homogeneity . Other morphological allows the complement to destroy the cells covered by features of a cell that can be imaged are characteristics are antibodies and allows phagocytes to attack the opsonized related to nucleus segmentation , invaginations , changes in cells . In some embodiments , the immune complexes can chromatin , such as heterochromatin reduction , increase of comprise a biomarker. interchromatin and perichromatin granules , increase of [ 0223 ] In some embodiments , the distribution of receptors nuclear membrane pores , and the formation of inclusions , on the cell surface in malignant cells is altered , which etc . The nucleolus of a cancer cell can be characterized by modifies the cell agglutination behavior . hypertrophy , macro - and microsegregation , its movement [0224 ] In some embodiments , the method for detecting the towards the membrane, numerical increase and formation of biomarker -morphological profile of a cell can further com intranuclear canalicular systemsbetween the nuclear mem - prise : ( h ) diagnosing the subject' s condition based on the brane and the nucleolus. In some embodiments , malignant biomarker- morphological profile of each cell . In some US 2017 /0234874 A1 Aug . 17 , 2017 aspects the subject 's condition may include presence of a which can be added to the cells after first providing a sample hematological cancer, non - malignant hematological disor comprising cells from a subject. der, solid tumor, kidney disease , bladder disease , liver [0228 ] When functionalized nanoparticles are subject to a disease, or infectious disease . The hematological cancer can high gravitational force , they can irreversibly agglomerate . be leukemia , lymphoma, or multiple myeloma. The non In some embodiments , charge - neutral organics can be added malignant hematological disorder can be anemia or sickle to the cells during the contacting with the functionalized cell disease . The solid tumor can be breast cancer, lung nanoparticles to prevent/ reduce / inhibit functionalized nano cancer, prostate cancer, bone cancer , colorectal cancer, or particle agglomeration . The charge -neutral organics can be bladder cancer . When the solid tumor is breast cancer , the a solvent with a high dielectric constant or a charge -neutral biomarkers can be, for example , Her2 or Neu . In some surfactant. In some embodiments , the charge -neutral surfac embodiments , the kidney disease can be acute kidney injury , tants can include or exclude, for example , Tween , Brij , Span , chronic kidney disease , lupus nephritis , kidney rejection , or IGEPAL , MERPOL , Triton or Pluronic surfactant . In some preeclampsia . In some embodiments , the infectious disease aspects , the Tween surfactants can include or exclude , for can be HIV , hepatitis , sexually transmitted diseases , or example , Tween20 , Tween40 , Tween60 , or Tween85 . In sepsis . In some embodiments, the hematological cancer can some aspects , the Pluronic surfactants can include or further comprise circulating cancer cells . exclude, for example , Pluronic 408 , Pluronic P - 123 , Pluronic F -68 , Pluronic F - 127 , Pluronic L31, Pluronic L35 , [ 0225 ] In some embodiments , when the subject 's condi Pluronic F - 108 , Pluronic , L -61 , Pluronic L - 81 , Pluronic tion is a cancer, the subject' s condition can be further L -64 , Pluronic L -121 , Pluronic 10R5 , Pluronic 17R4 , identified by the lineage of the malignancy . For example , the Pluronic 31R1, or Pluronic 188 . In some aspects , the Brij lineage of the malignancy can be negative , Myeloid line , surfactants can include or exclude , for example , Brij 52 , Brij Lymphoid T cell line , or Lymphoid B cell line . 58 , Brij C10 , Brij L4 , Brij 010 , Brij S10 , Brij S20 , or Brij Contacting the Cells with Functionalized Nanoparticles S100 . In some aspects , the IDEPAL surfactants can include [ 0226 ] The term " contacting” as used herein , refers gen or exclude , for example, IGEPAL CA - 520 , IGEPAL erally to providing access of one component, reagent, ana CA -720 , IGEPAL CO - 520 , IGEPAL CO -630 , IGEPAL lyte or sample to another . For example , contacting can CO -720 , IGEPAL CO - 890 , or IGEPAL DM - 970 . In some involve mixing a solution comprising a functionalized nano aspects , the Span surfactant can be Span40 . In some aspects , particle with a sample comprising a cell. The solution the MERPOL surfactant can include or exclude , for comprising one component, reagent, analyte or sample may example , MERPOL DA , MERPOL HCS , MERPOL OJ, also comprise another component or reagent, such as dim MERPOL SE , MERPOL SE , or MERPOL A . In some ethyl sulfoxide (DMSO ) or a detergent, which facilitates aspects , the Triton surfactant can include or exclude , for mixing , interaction , uptake, or other physical or chemical example , Triton X - 100 , Triton X - 114 , or Triton X -405 . In phenomenon advantageous to the contact between compo some aspects , the surfactant can be sorbitan monooleate or nents , reagents , analytes and /or samples, in some embodi sorbitan monopalmitate . In some aspects , the solvent with a ments of the invention , contacting involves adding a solu high dielectric constant can include or exclude , for example , tion comprising a functionalized nanoparticle to a sample DMSO ( dimethylsulfoxide ), DMF ( N , N -dimethylforma comprising a cell utilizing a delivery apparatus , such as a mide ), THF (tetrahydrofuran ), ethanol , isopropanol, or any pipette -based device or syringe -based device . n - alcohol wherein n is from 3 to 8 . [ 0227] The cells can be reacted with functionalized nano Resonant Light Scattering Detection of Functionalized particles so as to create a functionalized nanoparticle -cell Nanoparticles complex . In some embodiments , the cells can be contacted with one or a plurality of functionalized nanoparticle species [ 0229 ] The term " detecting” as used herein refers to any by subjecting the cells and functionalized nanoparticles to an method of verifying the presence of a given nanoparticle or external force to increase the local concentration of the particle . The techniques used to accomplish this may functionalized nanoparticles and cells . The external force include , but are not limited to resonance light scattering or can be a gravitational, electric , or magnetic force . The plasmon resonance . gravitational force can be generated by centrifugation . The [0230 ] Resonance light scattering is a physical phenom centrifugation can be pulsed . The pulse duration can be 10 enon where a particle with a diameter less than the wave seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 length of incident light exhibits a surface plasmon wave minute , or 2 minutes . The pulse duration can be from 1 around the particle and said wave becomes coherent to the minute to 2 minutes , 2 minutes to 3 minutes , 3 minutes to 4 circumference of the particle . Particle electrons can resonate minutes , 4 minutes to 5 minutes, or any time period inbe in phase with the incident light forming an electromagnetic tween the aforementioned times . The magnetic force can be dipole that emits energy as scattered light. The wavelength effected by paramagnetic nanoparticles , wherein the core of of the reflected ( scattered ) light is a function of the compo the nanoparticle comprises a paramagnetic region and the sition , shape , and particle size . In some embodiments , the shell of the nanoparticle can include or exclude Ag, Au , Pt , composition of the particle can be a noble metal, such as Pd , Rh , Ro, Al, Cu , Ru , Cr, Cd , Zn , Si, Se or mixtures or gold or silver. In some embodiments , the size of the particle alloys thereof. The paramagnetic region can be comprised of is below the wavelength of white light (below 300 nm ) . magnetic iron oxide (Fe2O3 ) . In some embodiments , [0231 ] The scattered light from a particle exhibiting a charged species can be added to the solution comprising resonance light scattering effect can be used as the signal for cells before contacting the cells with the functionalized ultrasensitive analyte detection . (Yguerabide , J . , et al. , Ana nanoparticles so as to prevent agglomeration or enhance lytical Biochemistry, 262; 137 - 156 ( 1998 ) . The advantages functionalized nanoparticle penetration into the cell . In some of using particles with a wavelength less than the wave embodiments , the charged species can be charged polymers , length of light is that ( a ) the particles can be detected at US 2017 /0234874 A1 Aug . 17 , 2017 concentrations at low concentrations in suspension by eye observed complexed nanoparticles can be completed in and a simple illuminator, such as with dark field illumina - under, for example, 1 second , 500 milliseconds, 200 milli tion , ( b ) the particles as a light source do not photobleach , seconds, 100 milliseconds, 50 milliseconds, 25 millisec ( c ) the color of scattered light can be changed by changing onds , 10 milliseconds , 5 milliseconds, 2 milliseconds , 1 particle size or composition formulticolor multiplexing , and millisecond , or 0 . 2 milliseconds. In some embodiments , ( d ) the particles can be conjugated with biomarker - binding detection of the resonant light scattering from some of the moieties for specific analyte detection . observed complexed nanoparticles can be completed in [0232 ] In some aspects , the resonant light scattering from under , for example , from 2 seconds to 1 second , 1 second to each observed complexed nanoparticle can be detected using 500 milliseconds , 500 milliseconds to 200 milliseconds , 200 evanescent or non - evanescent light . In some aspects , the milliseconds to 100 milliseconds, 100 milliseconds to 50 non - evanescent light can be transmitted light. The resonant milliseconds , 50 milliseconds to 25 milliseconds, 25 milli light scattering of the complexed nanoparticle can be seconds to 10 milliseconds, 10 milliseconds to 5 millisec detected when imaging under a dark field illumination . In onds , 5 milliseconds to 2 milliseconds , 2 milliseconds to 1 some aspects , an illuminated slide holder can replace the millisecond , 1 millisecond to 0 . 2 milliseconds , or any time darkfield condenser in the microscope . The illuminated slide between any of the foregoing time periods . In some embodi holder can use total internal reflection ( TIRF ) to illuminate ments , the dark field illumination can comprise LEDs with the slide holder. TIRF illumination can eliminate or reduce different wavelengths. The different wavelengths can be the scatter from other light scattering elements on the applied in parallel or in series . When the different wave substrate surface . TIRF illumination will not interact with lengths are applied in series , the interrogation time can be such surface debris as transilluminated darkfield illumina varied for each different LED wavelength . In some embodi tion would . ments , detection of the resonant light scattering from some [ 0233] The TIRF illuminated slide holder can be analyzed of the observed complexed nanoparticles can be completed by TIRF microscopy . The inventors have recognized that in under, for example , 1 second , 500 milliseconds, 200 utilizing TIRF microscopy in the present disclosure reduces milliseconds, 100 milliseconds, 50 milliseconds, 25 milli background fluorescence from outside the focal plane and seconds , 10 milliseconds , 5 milliseconds, 2 milliseconds, 1 can noticeably improve the signal- to - noise ratio , and thus millisecond , or 0 .2 milliseconds when one LED wavelength the spatial resolution of the resonance light scattered from is applied , then completed in under, for example , 1 second , the nanoparticles. TIRF microscopy utilizes an induced 500 milliseconds, 200 milliseconds, 100 milliseconds, 50 evanescent wave in a limited substrate region immediately milliseconds, 25 milliseconds, 10 milliseconds, 5 millisec adjacent to the interface between two media having different onds , 2 milliseconds , 1 millisecond , or 0 . 2 milliseconds , refractive indices . In some embodiments , the utilized TIRF when a different LED wavelength is applied . In some interface can be the contact area between the substrate and embodiments , a software control system can adjust the a glass coverslip or tissue culture container. The illuminated detection time , compare the detections of two or more slide holder can comprise optical fibers to deliver light to the interrogations, normalize the relative intensities for two or edge to the slide . The illuminated slide holder can be the more different nanoparticles when interrogated two or more Darklite Vertical Illuminator (Micro Video Instruments , Inc , times , and /or normalize for binding kinetics and / or strengths Avon , Mass . ) . of functionalized nanoparticals to their targets . [ 0234 ] In some embodiments , the dark field illuminator [ 0238 ] In some embodiments , the illumination can com can provide effective low -angle lighting to targeted regions prise one or a plurality of signal exposures . There can be , for of the substrate . The dark field illuminator can be comprised example , 1 , 2 , 3 , 4 , or 5 signal exposures . Each signal of LEDs ( light emitting diodes ). The LEDs can be posi exposure can be for a different time. A software control tioned so as to provide low - angle illumination to provide a system can adjust the detection time, compare the detections high contrast image . In some embodiments , the dark field of two or more interrogations , and / or normalize the relative illuminator can be the DF -50 , DF - 150 , DF - 200 illuminators intensities for two or more different nanoparticles when from Microscan Systems, Inc . (Renton , Wash . ). interrogated two or more times . [0235 ] In some embodiments, the dark field microscopy [ 0239 ] In some embodiments , the one or a plurality of system can comprise a system with high NA ( numerical functionalized nanoparticle species can comprise nanopar aperature ) condensors, with non - evanescent illumination , ticles from 5 to 200 nm in diameter . In some embodiments , vibration reduction , and stray light reduction to improve the nanoparticles can include or exclude sizes of 5 , 7 , 10 , 12 , dark - field performance . In some embodiments , the inverted 15 , 20 , 25 , 30 , 35 , 40 , 45 , 50 , 55 , 60 , 65 , 70 , 75 , 80 , 85 , 90 , darkfield contrast system described in U . S . Pat . No . 6 , 704 , 95 , 100 , 105 , 110 , 115 , 120 , 125 , 130 , 135 , 140 , 145 , 150 , 140 , herein incorporated by reference in its entirety , can be 155 , 160 , 165, 170 , 175 , 180 , 185 , 190 , 195 , or 200 used . nanometers ( nm ) in diameter. In some embodiments , the 10236 ] In some embodiments , the illuminated slide holder nanoparticles can include or exclude sizes from 4 to 6 nm , can be illuminated by transmitted light. In some embodi 6 to 8 nm , 9 to 11 nm , 11 to 13 nm , 14 to 16 nm , 19 to 21 ments , the illuminated slide holder can be illuminated by nm , 24 to 26 nm , 29 to 31 nm , 34 to 36 nm , 39 to 41 nm , epi -illumination . In some embodiements, the source of the 44 to 46 nm , 49 to 51 nm , 54 to 56 nm , 59 to 61 nm , 64 to epi -illumination can be from a laser . 66 nm , 69 to 71 nm , 74 to 76 nm , 79 to 81 nm , 84 to 86 nm , [ 0237 ] In some embodiments , the interrogation time can 89 to 91 nm , 94 to 96 nm , 99 to 101 nm , 104 to 106 nm , 109 be adjusted to detect all of the nanoparticles while minimiz - to 111 nm , 119 to 121 nm , 124 to 126 nm , 129 to 131 nm , ing the saturation of any particular nanoparticle species . 134 to 136 nm , 139 to 141 nm , 144 to 146 nm , 149 to 151 Some nanoparticles may exhibit a bloom effect when the nm , 154 to 156 nm , 159 to 161 nm , 164 to 166 nm , 169 to interrogation time is too long . In some embodiments, detec - 171 nm , 174 to 176 nm , 179 to 181 nm , 184 to 186 nm , 189 tion of the resonant light scattering from some of the to 191 nm , 194 to 196 nm , 199 to 201 nm , or in between any US 2017 /0234874 A1 Aug . 17 , 2017 of the foregoing sizes . In some embodiments , the size posix # AGCN5 - 25M , AGCN10 -25M , AGCN20 -25M , distribution of the nanoparticles can be less than 25 % AGCN30 - 25M , AGCN40 - 25M , AGCNO5 - 25M , AGCN60 coefficient of variation (CV ) , less than 20 % CV , 15 % CV , 25M , AGCN70 - 25M , AGCN75 - 25M , AGCN80 - 25M , less than 10 % CV , less than 5 % CV , or less than 4 % , 3 % , 2 % AGCN100 - 25M , or AGCN200 - 25M ) ; Au 5 , 7 , 10 , 12 , 15 , or less than 1 % CV , or any range between any two of the 20 , 30 , 40 , 50 , 60 , 70 , 80 , or 100 nm (Nanocomposix # recited percentages. In some embodiments , the diameter can AUCN5 - 25M , AUCN7 - 25M , AUCN10 - 25M , AUCN12 be measured at the maximum difference between the sides of 25M , AUCN15 - 25M , AUCN20 - 25M , AUC30 - 25M , the particle or the minimum distance between the sides of AUCN40 - 25M , AUCN50 - 25M , AUCN60 - 25M , AUCN70 the particle , when viewed from a side profile . In some 25M , AUCN80 - 25M , or AUCN100 -25M ) ; Ag 75 nm or 100 embodiments , the nanoparticles can be made from any metal nm nanocubes (Nanocomposix # SCPH75 - 1M , SCPH100 or metal composition as described herein . 1M ) ; 550 nm resonant Ag nanoplates (Nanocomposix # [ 0240 ] In some embodiments , each nanoparticle prepara SPPN550 - 25M ) ; 650 nm resonant Ag nanoplates (Nano tion has a narrow size distribution . By narrow size distri composix # SPPN650 -25M ); 750 nm resonant Ag nano bution is meant that an individual nanoparticle preparation plates (Nanocomposix # SPPN750 - 25M ) ; 850 nm resonant has a scattering spectrum whose full -width half maximum Ag nanoplates (Nanocomposix # SPPN850 - 25M ) ; 950 nm ranges from 5 to 150 nm . (See Chen et. al, Journal of resonant Ag nanoplates (Nanocomposix # SPPN950 - 25M ) ; Biomedical Optics 10 (2 ) , 024005 (March / April 2005 ) ). In 1050 nm resonant Ag nanoplates (Nanocomposix # some embodiments , an individual nanoparticle preparation SPPN1050 - 25M ) ; 1150 nm resonant Ag nanoplates (Nano has a scattering spectrum whose full -width half maximum composix custom order ) ; 660 nm resonant Au nanoshells ranges from 5 to 50 nm . In some embodiments , a spectrum (Nanocomposix # GSPN660 - 25M ), 800 nm resonant Au of light scattering is collected for each pixel in the imaged nanoshells (Nanocomposix # GSPN800 - 25M ) , 980 nm reso field . Next, the spatial distribution of each molecular target nant Au nanoshells (Nanocomposix # GSPN980 -25M ); 660 is represented by the spatial distribution of nanoparticles , nm resonant Au nanorods (Nanocomposix # GRCN660 which in turn is reported by the presence and / or absence of 10M ) , 800 nm resonant Au nanorods (Nanocomposix # its resonant light scattering peak at each pixel. The size GRCN800 - 10M ) , 980 nm resonant Au nanorods (Nanocom distribution can be combined with the compositional varia posix # GRCN980 - 10M ) ; 30 nm Au50Ag50 alloy (50 /50 ) tion of each nanoparticle preparation to achieve greater (Cytodiagnostics # GSA - 30 - 20 ), 30 nm Au80Ag20 alloy multiplexing capacity . ( 80 / 20 ) ( Cytodiagnostics # GSB - 30 - 20 ), 30 nm Au20Ag80 [ 0241] In some embodiments , the nanoparticles can be (20 / 80 ) ( Cytodiagnostics # GSC - 30 - 20 ) ; gold nanorods comprised of a noble metal. The nanoparticles can be from Cytodiagnostics (25 nm diam , 650 nm max abs. comprised metals that can include or exclude Ag, Au , Pt, Pd , GRC3K -25 -650 - 25 ) ; functionalized and non - functionalized Rh , Ro , Al, Cu , Ru , Cr, Cd , Zn , Si, Se or mixtures or alloys Nanourchins (Cytodiagnotics ) — 50 nm (GU -50 - 20 ) , 60 nm thereof. The alloy can be an alloy of gold ( Au ) and silver (GU -60 - 20 ) , 70 nm (GU - 70 -20 ) , 80 nm (GU - 80 - 20 ) , 90 nm ( Au ) . In some embodiments , the alloy can be of Copper (Cu ) (GU - 90 -20 ) , and 100 nm (GU - 100 - 20 ) . and Gold ( Au ) to modulate the intensity of the reflected light [ 0245 ] In some embodiments , the nanoparticles can ( see Su , Y . et al ., Nanoscale Research Letters, 8 :408 , 2013 ) . exhibit a peak resonance wavelength of the nanoparticle In some embodiments , the composition of the alloy can be plasmon resonance from 240 to 1150 nm . In some embodi adjusted to affect the intensity of the reflected light . In some ments, the nanoparticles can exhibit a peak resonance wave embodiments , the alloy composition can be adjusted to length of the nanoparticle plasmon resonance from 400 to modulate the wavelength of the reflected light. The nano 900 nm . particles can comprise mixtures of the listed metals in f0246 ] In some embodiments , the plurality of functional discrete shells or layers. For example , a nanoparticle may be ized nanoparticle species can be from 2 to 347 different comprised of an Au core and a Si or SiO2 (silica ) shell. In species of functionalized nanoparticle species . In some some embodiments , the core can be Fe2O3. In some embodi embodiments , the up to 347 functionalized nanoparticle ments , the nanoparticles are spherical, tubular, cylindrical, species , may comprise up to 50 different types of nanopar pyramidal, cubic , egg - shaped , t - bone - shaped , urchin - or ticles , and each plurality of functionalized nanoparticle rose - like (with spiky uneven surfaces ) or hollow shaped . In species may comprise 50 functionalized nanoparticle spe some embodiments , the nanoparticles may have a round , cies . In some embodiments , the plurality of functionalized oval , triangular, square , egg - shaped , or a t - bone - shaped nanoparticle species can be from 2 to 50 different species of cross -section . functionalized nanoparticle species . In some embodiments , [ 0242 ] In some embodiments the nanoparticles can com the plurality of functionalized nanoparticle species can be prise a chemical group to which other functional groups can from 2 to 10 different species of functionalized nanoparticle be added , such as streptavidin , biotin , amino - functionalized species . In some embodiments , the plurality of functional dextran , a biomarker binding moiety or an oligonucleotide ized nanoparticle species can be from 2 to 5 different species or other component of a releasable or displaceable nanopar of functionalized nanoparticle species . ticle system . The chemical group can be , for example , lipoic [0247 ] In some embodiments , each species of functional acid , reduced forms of lipoic acid , an amine , carboxylic ized nanoparticle species can be functionalized with a dif acid , an alkyne, an azide , or — NHS . ferent species of biomarker - binding moiety . In some [ 0243] In some embodiments , the nanoparticles can be the embodiments , each species of functionalized nanoparticle size of any nanoparticles described herein . species is functionalized with a different biomarker binding [0244 ] In some embodiments , the nanoparticles can moiety . include or exclude Pt 30 , 50 , or 70 nm (Nanocomposix # ' s [0248 ] In some embodiments , the nanoparticles can be PTCN30 -25M , PTCN50 - 25M , PTCN70 -25M ); Ag 5 , 10 , functionalized by using streptavidin -biotin binding . FIGS. 4 , 20 , 30 , 40 , 50 , 60 , 70 , 75 , 80 , 100 , or 200 nm (Nanocom 5 , and 6 depict some of the embodiments by which the US 2017 /0234874 A1 Aug . 17 , 2017 24. nanoparticles can be functinonalized . In some embodiments , a biotinylated antibody . In some embodiments , the nanopar nanoparticles coated with a carboxylic acid functional group ticle can be purchased with a functional group selected from : can be activated with EDC /NHS ( 1 - Ethyl- 3 - ( 3 - dime thylam carboxylic acid , NHS , streptavidin , amine , alkyne, or alde inopropyl) carbodiimide / N -hydroxy - succinimide ) , followed hyde . The biotinylated antibody can be reacted to the by a wash to yield an EDC - functionalized nanoparticle , via streptavidin -functionalized nanoparticle to create the the process depicted in FIG . 4 . Other amide coupling agents directly - linked antibody - functionalized nanoparticle . can be used instead of EDC , for example , DCC ( dicyclo [0250 ] The terms “ polynucleotide ” and “ nucleic acid hexylcarbodiimide ) , EDAC .HCI , ( N - ( 3 - Dimethylaminopro (molecule ) " are used interchangeably to refer to polymeric pyl) - N ' -ethylcarbodiimide .HCl ) , HOBt ( 1 -Hydroxybenzo formsof nucleotides of any length . The polynucleotides may triazole ) , HOOBt (HODhbt ) (Hydroxy - 3 , 4 -dihydro - 4 -oxo comprise deoxyribonucleotides , ribonucleotides and/ or their 1 , 2 , 3 -benzotriazine ) , HOAT ( 1 -Hydroxy - 7 - aza - 1H analogs . Nucleotides may have any three - dimensional struc benzotriazole ), DMAP (4 - (N , N -Dimethylamino )pyridine ), ture , and may perform any function , known or unknown . BOP (Benzotriazol - 1 -yloxy -tris ( dimethylamino ) -phospho The term “ polynucleotide” includes single - stranded , double nium hexafluorophosphate ) , PyBOP (Benzotriazol - 1 - yloxy stranded and triple helical molecules . " Oligonucleotide ” tripyrrolidinophosphonium hexafluorophosphate ), PyOxim refers generally to polynucleotides of between 5 and about (Ethyl cyano (hydroxyimino ) acetato - 02 ) - tri - ( 1 -pyrrolidi 100 nucleotides of single - or double - stranded nucleic acid , nyl) - phosphonium hexafluorophosphate ) , PyBrOP ( 7 - Aza typically DNA . Oligonucleotides are also known as oligom benzotriazol- 1 - yloxy -tripyrrolidinophosphonium hexafluo ers or oligos and may be isolated from genes , or synthesized rophosphate ) , DEPBT ( 3 - (Diethoxy - phosphoryloxy ) - 1 , 2 , 3 ( e . g ., chemically or enzymatically ) by methods known in the benzo [ d ]triazin - 4 (3H ) -one ) , TBTU /HBTU ( 2 - 1H art . A “ primer ” refers to an oligonucleotide , usually single Benzotriazol- 1 - yl) - N , N , N ', N ' - tetramethylaminium tetrafl stranded , that provides a 3 ' -hydroxyl end for the initiation of uoroborate /hexafluorophosphate ) , HCTU (( 2 -( 6 -Chloro - 1H enzyme- mediated nucleic acid synthesis . The following are benzotriazol- 1 - yl ) - N , N , N , N -tetramethylaminium non - limiting embodiments of polynucleotides : a gene, a hexafluorophosphate ) , HDMC ( N - [ ( 5 - Chloro - 1H -benzotri gene fragment, exons, introns , mRNA , TRNA, CRNA , azol - 1 - yl) - dimethylamino -morpholino ] -uronium hexafluo ribozymes, cDNA , recombinant polynucleotides, branched rophosphate N - oxide ), HATU ( 2 -( 7 - Aza - 1H - benzotriazol- 1 polynucleotides, plasmids, vectors , isolated DNA of any yl) - N , N , N ', N ' - tetramethylaminium hexafluorophosphate ), sequence , isolated RNA of any sequence , nucleic acid COMU ( 1- [1 - (Cyano - 2 -ethoxy -2 -oxoethylideneaminooxy ) probes and primers. A nucleic acid molecule may also dimethylamino -morpholinol - uronium hexafluorophos comprise modified nucleic acid molecules, such as methyl phate ), TOTT (( 2 -( 1 -Oxy -pyridin -2 -yl ) - 1, 1, 3 ,3 - tetramethyl ated nucleic acid molecules and nucleic acid molecule isothiouronium tetrafluoroborate ) , TFFH analogs . Analogs of purines and pyrimidines are known in ( Tetramethylfluoroformamidinium hexa - fluorophosphate ) , the art , and include , but are not limited to , aziridinycytosine , EEDQ (N - Ethoxycarbonyl- 2 -ethoxy - 1, 2 -dihydroquinoline ), 4 -acetylcytosine , 5 - fluorouracil , 5 - bromouracil, 5 - car T3P ( 2 - Propanephosphonic acid anhydride ), DMTMM ( 4 boxymethylaminomethyl- 2 - thiouracil, 5 -carboxymethyl ( 4 ,6 - Dimethoxy - 1, 3 ,5 - triazin -2 - yl) - 4 -methylmorpholinium aminomethyluracil , inosine, N6 - isopentenyladenine, salt) , or CDI ( 1 , 1 '- Carbonyldiimidazole ). In some alterna 1 -methyladenine , 1 -methylpseudouracil , 1- methylguanine , tive embodiments , the coupling can be performed in the 1 -methylinosine , 2 , 2 - dimethylguanine , 2 -methyladenine , presence of a base . The base can be organic or inorganic . The 2 -methylguanine , 3 -methylcytosine , 5 -methylcytosine , inorganic bases can include or exclude , for example , car pseudouracil , 5 -pentylnyluracil and 2 , 6 - diaminopurine. The bonate buffer , or phosphate buffer. The organic bases can be use of uracil as a substitute for thymine in a deoxyribo triethylamine , Diisopropylethylamine (DIPEA ) , or N -meth nucleic acid is also considered an analogous form ofpyrimi ylmorpholine ( NMM ) . dine. [ 0249] The wash can be a pH mild wash so as to not [0251 ] Sugar modifications ( e .g ., 2 -o -methyl , 2 - fluor and hydrolyze the NHS moieties . The mild pH wash can be with the like ) and phosphate backbone modifications ( e . g ., mor PBS buffer (phosphate buffered saline , pH around 7 . 4 ) . pholino , PNA ', thioates, dithioates , methyl phosphonates , Next, streptavidin can be reacted with the EDC - functional and the like ) can be incorporated singly, or in combination , ized nanoparticle to yield a streptavidin - functionalized into the nucleic acid molecules of the present invention . In nanoparticle . Other avidin - like molecules can be used in some embodiments, for example , a nucleic acid of the place of streptavidin , for example : avidin , neutravidin , invention may comprises a modified sugar and a modified superavidin , and streptavidin with one, two or three biotins phosphate backbone . In another embodiment, a nucleic acid already bound . In some embodiments , the biomarker bind of the invention may comprise modifications to sugar , base ing moiety can be functionalized with a biotin . In some and phosphate backbone. embodiments , the biomarker binding moiety is an antibody . [0252 ] The nucleotide sequence of the nucleic acids of the The antibody can be reacted with a cross - linker, such as present invention is of less importance than the functional Sulfo - SMCC (Pierce ) followed by a thiol- conjugated biotin roles they are required to perform . Accordingly , the to yield a biotinylated antibody . In an alternative embodi sequence of the nucleic acids, as well as the length of the ment, the antibody can be reacted with a NHS - conjugated nucleic acid component of the binding pair , may vary biotin , where the NHS - conjugated biotin can react with any considerably , provided the nucleic acid component of the free amine on the antibody (prefeable , free amines from binding pair can still perform the functional roles they are lysine residues) to yield a biotinylated antibody. In an required to perform . Importantly , the sequence and length of alternative embodiment, the antibody can be reacted with the nucleic acids of the binding pair are not limited to those DTT ( dithioerithritol) to break the di- thiol cysteine bond to exact sequences and lengths of the exemplary binding pairs yield a free sulfuryl hydryl group . The sulfuryl hydryl group disclosed herein . The nucleic acids of the binding pair thus can be reacted with a maleimide - conjugated biotin to yield can be of different lengths and or sequence . An important US 2017 /0234874 A1 Aug . 17 , 2017 25 function of the nucleic acid component of the binding pairs Am . Chem . Soc. 117 , 1863 -1872 ( 1995 ) , herein incorporated of the present invention is to provide a linker between the by reference. In some embodiments , the non - natural base biomarker - binding moiety and the functionalized nanopar can be MMO2 or SICS , as described in Leconte , A . M . et al. ticle by the ability to hybridize with a complementary strand J . Am . Chem . Soc. 130 , 2336 - 2343 (2008 ), herein incorpo of the nucleic acid to form a nucleic acid duplex . rated by reference . In some embodiments , the non -natural [0253 ] The stability of a nucleic acid duplex is dependent base can be Ds or Dioll -Px , as described in Yamashige , R . in part on the length of the region of complementarity et al. Nucl. Acids Res. 40 , 2793 - 2806 (2012 ) , herein incor between the nucleic acid strands in the duplex . A longer porated by reference . In some embodiments , the non - natural complementarity region or overlap between nucleic acids base can be Por Z , as described in Yang ; Z . , et al. , J . Am . increases the stability of the duplex that is formed . Con Chem . Soc. 133 , 15105 - 15112 ( 2011 ), herein incorporated by versely , a shorter overlap leads to a less stable duplex . The reference . In some embodiments , the non - natural base can stability of a duplex can be measured as a function of the be NaM or 5SICS , as described in Malyshev, D . A . et al. melt temperature , Tm , where a highly stable duplex results Proc . Natl Acad . Sci. USA 109 , 12005 - 12010 ( 2012 ) , herein in a high Tm and a less stable duplex results in a lower Tm . incorporated by reference . Nucleic acids of the present invention are designed to have [ 0256 ] In some embodiments , the nanoparticle can be defined stability that can be manipulated by altering length , functionalized by functionalized with a first oligonucleotide , temperature , backbone composition , base pair selection , by the process depicted in FIG . 4 . The carboxylic acid base pair structure , sugar structure , solvent and other con functionalized nanoparticle can be reacted with EDC /NHS ditions . Factors that influence the stability of the hybrid followed by a mild pH wash . Then , the EDC - functionalized include , but are not limited to , the concentration of the nanoparticle can be reacted to an amino -functionalized nucleic acid - labeled binding pairs , salt concentration , tem oligonucleotide to yield an oligonucleotide - functionalized perature , organic solvents such as ethanol, DMSO , tetram nanoparticle , as shown in FIG . 5 . In some embodiments , ethylammonium ions ( TMA + ) , base pair mismatches and the unreacted EDC groups can be reacted to prevent cross - talk like . with other functionalized nanoparticle species when the [ 0254 ] In some embodiments , the backbone composition species are mixed by reacting the unreacted EDC with a of an oligonucleotide can be varied to produce an oligo small molecule amine . The small molecule amine can be nucleotide with a selected relative duplex strength . An ethanolamine . In another embodiment, the EDC -functional oligonucleotide backbone resulting in a more stable duplex ized nanoparticle can be reacted to streptavidin to yield a can be selected from : peptide -nucleic acids (PNA ), locked streptavidin - functionalized nanoparticle . The streptavidin nucleic acids (LNA ) , or normal deoxyribonucleic acid functionalized nanoparticle can be reacted with a biotin (DNA ) . An oligonucleotide backbone resulting in a less modified oligonucleotide to yield an oligonucleotide -func stable duplex can be selected from : unlocked nucleic acids, tionalized nanoparticle. Unreacted streptavidin can be methyl phosphonate , or thiophosphonates . PNAs have a blocked by adding free biotin before mixing one species of peptide - backbone rather than a ribose - phosphate backbone oligonucleotide - functionalized nanoparticles with other spe of normal DNA . The PNA backbone is composed of repeat cies of oligonucleotide - functionalized nanoparticles . In ing N - ( 2 - aminoethyl ) - glycine units linked by peptide bonds . some embodiments , the biomarker binding moiety can be The purine and pyrimidine bases are linked to the PNA functionalized with a second oligonucleotide , as shown in backbone by a methylene bridge ( CH2 - ) and a carbonyl FIG . 5 . In some embodiments , the biomarker binding moiety group ( C = 0 ) — ) , The PNA backbone thus lacks charged is an antibody . The antibody can be reacted with DTT phosphate groups . PNAs are not easily recognized by either (dithioerythritol ) to yield free sulfurylhydryl groups . The native nucleases or proteases, imbuing them resistance to sulfuryl hydryl groups can be reacted with a maleimide enzymatic degradation and pH stability . The LNA backbone functionalized second oligonucleotide . In some embodi comprises a ribose moiety which is modified with an extra ments , the second oligonucleotide conjugated to the func bridge connecting the 2' oxygen and 4 ' carbon locking the tionalized biomarker binding moiety (e . g. , antibody ) can ribose in the 3 ' - endo (North ) conformation . The locked comprise a portion complementary to a portion of the first ribose conformation enhances base stacking and backbone oligonucleotide and hybridization of the first oligonucle pre -organization , significantly increases duplex stability of otide to the second oligonucleotide forms a linker compris LNA /DNA duplexes . Methyl phosphonate backbones ing a double - stranded nucleic acid . In some embodiments , replace the charged anionic phosphate with a neutral methyl the first and second oligonucleotides can both comprise a phosphonate ester. The resulting decrease in backbone portion complementary to a portion of a third oligonucle charge results in a less stable duplex relative to a normal otide which can act as a bridging oligonucleotide , as shown DNA backbone , yet also confers resistance to nuclease in FIG . 6 .Modified oligonucleotides discussed herein can be activity . Thiophosphonate backbones comprise a non - bridg used with the modifier at the 3 ' or 5 ' terminus . When the first ing oxygen on the phosphate backbone to form a phospho oligonucleotide is conjugated to the nanoparticle at the 5 ' rothioate (PS ) linkage . Thiophosphonate backbones exhibit terminus , the corresponding terminus of the second oligo nuclease resistance and a less stable duplex relative to a nucleotide is selected such that the two oligonucleotides are normal DNA backbone. complementary in the proper orientation if directly hybrid [0255 ] In some embodiments , the duplex stability can be ized or indirectly hybridized by a bridging oligonucletide . adjusted by incorporating one or a plurality of non - natural [ 0257 ] In some embodiments , when the nanoparticle com base pairs . In some embodiments , the non - natural base can prises a silica (SiO2 ) shell , the nanoparticle can be func be iso - G or iso - C , as described in Richert, C . , et J . Am . tionalized with a functionalized silane . The silane can be Chem . Soc. 118 , 4518 -4531 ( 1996 ), herein incorporated by dissolved in an organic solvent. The organic solvent can be reference . In some embodiments , the non - natural base can acetonitrile , ethanol, methanol, isopropanol, dimethyl sul be diflurotoluene , as described in Schweitzer , B . A . , et al. , J . foxide (DMSO ) , N , N - dimethyl formamide , or dimethylac US 2017 /0234874 A1 Aug . 17 , 2017 etamide . The silane can be a trimethoxy , dimethoxy, types of the subject. The HIPAA - compliant computer sys monomethoxy , triethoxy , diethoxy, monoethoxy , trichlori, tem or storage system can be one which is specifically dichloro , or monochlorosilane to react with the silica shell. configured so as to comply with the United States Health In some embodiments, the silane can have an alkyl , carbox - Insurance Portability and Accountability Act (HIPAA ) ylic acid , protected carboxylic acid , amine , protected amine , requirements for computer systems. activated amine (hydroxyamine , hydrazine , hydrazide , etc . ) [0261 ] In some embodiments , a software program on a aldehyde , protected aldehyde , azido , NHS , ethoxy, maleim - HIPAA -compliant computer system can be used in the ide , thiol, or dithiol functional group . In some embodiments , method of detecting the biomarker -morphological profile of the silane can be reacted to the silica shell followed by a a cell. The step ( d ) of illuminating the nanoparticle - cell subsequent functionalization . In some embodiments , the complexes with evanescent light and detecting the resonant subsequent functionalization can be a reaction to form any light scattering from each observed complexed nanoparticle , of the foregoing functional groups. In some embodiments , to obtain a biomarker signature of each observed cell in the the functionalized silica shell can be reacted with a func method of detecting the biomarker -morphological profile of tional group present on an antibody or functionalized oli a cell can further comprise: gonucleotide . In some embodiments , the antibody functional [ 0262 ] (i ) using a software program that counts the group can be a thio , aldehyde , amine , or carboxylic acid . In number of each of the functionalized nanoparticle some embodiments , the oligonucleotide functional group species per cell and processes images in each cell in the can be an azide , alkyne, aldehyde , amine, activated amine , field of view ; carboxylic acid , aklynyl halide , or thiol. The functionalized [0263 ] ( ii ) moving the field of view digitally ; oligonucleotide can be synthesized or purchased . In some [0264 ] ( iii ) using a software program to count the embodiments , when the functionalized oligonucleotide is number of each of the functionalized nanoparticle synthesized in situ , the synthesis can involve the selected species per imaged cell in the next field of view and functionalized nucleotides available from Glen Research repeating steps ( ii ) and ( Sterling , Va . ) . In some embodiments , when the functional [0265 ] (iii ) until the entire substrate area is analyzed ; ized oligonucleotide is purchased , it can be purchased from [0266 ] ( iv ) digitally combining all images obtained to IDT (San Diego , Calif. ) , Trilink (San Diego , Calif . ), or generate a single image covering the entire selected Midland Oligos (Midland , Tex . ). substrate area ; and [ 0258 ] In some embodiments , the attachment of the oli [0267 ] (v ) generating from the data obtained for the gonucelotide or antibody to the functionalized nanoparticle entire substrate area the number of each of the func can be accomplished by the bioconjugation methods tionalized nanoparticle species per imaged cell, the described in Hermanson , G . , Bioconjugate Techniques , Aca biomarker signature, of each substrate - adhered cell . demic Press ( 1996 ) , herein incorporated by reference in its [0268 ] In some embodiments the software program stores entirety . the positional information for each imaged and /or observed 10259 ). In some embodiments , when the nanoparticle spe cell. cies are functionalized with a biomarker binding moiety , [0269 ] In some embodiments , the software on the HIPAA e . g ., an antibody or antibody fragment or other biomarker compliant computer system can count the number of each of binding moiety that binds to one of the following : CD3 , the functionalized species per cell and process images in CD22 , CD79a , Kappa , Lambda, Pax -5 , ZAP -70 , MPO , and each cell in the field of view . In some embodiments , the TdT ; the nanoparticle species can enter the cell and bind to software can identify the nanoparticle by identifying the its respective intracellular biomarker. The intracellular bio resonant light signature obtained from the nanoparticle . The marker can be in the cytosol and/ or nucleus , or on the software can identifty the circumference of the light signa nuclear membrane , or in or on another cellular compartment ture , the color of the light signature , and reduce the bloom or structure . In some embodiments, the functionalized nano of the light signature , of each nanoparticle in the field of particles are small enough to enter the cell without disrupt view . In some embodiments , the color of the light signature ing the cell membrane . The cells can be treated with a can be identified using spectral identification algorithms. In permeabilizer so as to allow the functionalized nanoparticles some embodiment, the software can identify one nanopar to enter the cell without disrupting the cell membrane . In ticle as a circular light source . some embodiments , the permeabilizer can be a surfactant. [0270 ] In some embodiments, the field of view is from about 0 .25 um ? to about 2 . 5 cm2. In some embodiments , the Obtaining a Biomarker Signature of Each Observed Cell field of view can be from about 100 um ? to about 1000 mm ? . [ 0260 ] In some embodiments , the biomarker signature can In some embodiments, the field of view is 5 microns by 5 be obtained by counting the number or proportion of each of microns. In some embodiments, the field of view is 100 mm the functionalized nanoparticle species per cell. The number by 100 mm . In some embodiments , the field of view is of cells or proportion of cells having identified normal or round . In some embodiments, the field of view is square abnormal morphological profiles in the sample can be shaped . The sides of the square - shaped field of view can be totaled , weighted , or otherwise determined . In some from 0 . 25 microns up to 2 . 5 centimeters. The field of view embodiments , the number of cells or proportion of cells can cover one cell, or a plurality of cells . In some embodi having identified normal or abnormalmorphological profiles ments , the field of view can cover the area of the entire slide . in a sample can be stored in a HIPAA -compliant computer The field of view can be digitally moved to view a different storage system and compared against a different sample field of view from a previous image . The movement can from the same subject. In some embodiments , the different occur via electronic servo -controlled motors which control samples from the same subject can be obtained at different the sample stage upon which the substrate is located . The timepoints . In some embodiments , the different samples software on the HIPAA - compliant computer system can from the same subject can be obtained from different tissue identify each field of view within the substrate . The software US 2017 /0234874 A1 Aug . 17 , 2017 can then count the number of each of the functionalized first set of functionalized nanoparticles , contacting the cells nanoparticle species per cell in the next field of view and with a next plurality of functionalized nanoparticles , and repeating steps (ii ) and ( iii ) until the entire selected substrate illuminating the nanoparticle - cell complexes with evanes area is analyzed . The substrate area selected can be the entire cent light and detecting the resonant light scattering from substract or a portion thereof. The software can combine the each observed complexed next plurality of nanoparticles. results of each field of view for the entire selected substrate [0282 ] In some embodiments, the optical contrast agent area so as to obtain a biomarker signature of the sample . can be a leuco dye or any of optical contrast agents described [ 0271 ] In some embodiments , the morphological features herein . The leuco dye can be methylene blue , methylene of each substrate - adhered cell can be associated with the green , red leuco dye , crystal violet , phenolphthalein , or biomarker signature of the contacted cells to detect the thymolphthalein . The leuco dye can be converted to a biomarker- morphological profile of each cell . The associa colorless form by the addition of one or more electrons to the tion can be made by comparing the corresponding physical dye or by any of the methods described herein . Electrons can location of the cells identified in the morphological features be added to the dye via a reduction method . The reduction analysis when imaging the optical contrast agent properties method can be effected by an electrochemical reduction , to the physical location of the nanoparticles around the same photoreduction , or reaction with a reducing agent. In some area. For example , a cell may be identified as being a embodiments , the leuco dye can be converted to a colored cancerous cell in by its morphological features in a bright form by the removal of one or more electrons from the dye field image , and the diagnosis can be confirmed by analyzing by any of the methods described herein . One or more which biomarkers are present on or within the cell by electrons can be removed from the dye by an oxidation measuring which biomarker -binding functionalized nano method . The oxidation method can be effected by an elec particle species are present at the same corresponding area trochemical oxidation , photooxidation , or reaction with an during the darkfield imaging process . In some embodiments , oxidation agent , by the methods described herein . measuring which biomarker- binding functionalized nano particle species are present at the same corresponding area Iterative Interrogations of Biomarker -Binding during the darkfield imaging process comprises associating Functionalized Nanoparticles the color of the resonant light signature of the particular size [0283 ] The cells can be analyzed with a series of func of nanoparticle with which biomarker- binding moiety was tionalized nanoparticle species . In some embodiments, the functionalized to that size of nanoparticle . In some embodi series of functionalized nanoparticle species can be iterative ments , the association is a color - to - biomarker association . interrogations of the cell with functionalized nanoparticle [0272 ] In some embodiments , a method for detecting the pluralities , where a first plurality of functionalized nanopar biomarker -morphological profile of a cell can comprise an ticles are first contacted with a cell , followed by illuminating order of steps where the cells are contacted with an optical and detecting the first plurality of functionalized nanopar contrast agent before contacting with a functionalized nano ticles on the cell, followed by removing the first plurality of particle species . When the biomarker- morphological profile functionalized nanoparticle from the cell , followed by con is detected for a sample adhered to a substrate , the substrate tacting the cell with a second plurality of functionalized may be stored for future analysis or retesting . In some nanoparticles . In some embodiments , the method for detect embodiments , the method for detecting the biomarker- mor ing the biomarker- morphological profile of a cell can further phological profile of a cell can comprise : comprise : ( d )( 2 ) removing a first plurality of functionalized [0273 ] ( a ) providing a sample comprising cells from a nanoparticles; and ( d )( 3 ) contacting the cells with a second subject ; plurality of functionalized nanoparticle species . In some [0274 ] ( b ) adhering the cells to a substrate ; embodiments , the selection of the second plurality of func [0275 ]. ( c ) contacting the substrate - adhered cells with an tionalized nanoparticles can be dependent upon the results of optical contrast agent; which first plurality of functionalized nanoparticles were [ 0276 ] ( d ) imaging morphological features of the con detected on the cell . For example , if a first plurality of tacted cells ; functionalized nanoparticles are contacted with a cell and [0277 ] ( e) converting the optical contrast agent to a found to indicate the presence of a first biomarker which colorless form ; may be indicative of a particular cell condition or disease [0278 ] (f ) contacting the cells with one or a plurality of state , the second plurality of functionalized nanoparticles to functionalized nanoparticle species, each functional be contacted with the cell can be functionalized with a ized nanoparticle species comprising a biomarker -bind second biomarker- binding moiety , which binds to a second ing moiety to form nanoparticle - cell complexes ; biomarker which is confirmatory for cell condition or dis [0279 ] ( g ) illuminating the nanoparticle -cell complexes ease state . In some embodiments , there can be additional with evanescent light and detecting the resonant light iterations of contacting the cell with a third plurality of scattering from each observed complexed nanoparticle , functionalized nanoparticles, where the third biomarker to obtain a biomarker signature of each observed cell ; binding moiety which binds to a third biomarker can also be and confirmatory of the disease or condition of the cell . In some [0280 ] (h ) associating the morphological features of the embodiments , there can be from 1 to 50 iterations of contacted cells with the biomarker signature of each successive interrogations of the cell with a next plurality of substrate - adhered cell to detect the biomarker- morpho functionalized nanoparticles, where the next plurality of logical profile of each cell . functionalized nanoparticles can be comprised of a nano [ 0281 ] In some embodiments , the step of illuminating the particle functionalized with a biomarker -binding moiety nanoparticle - cell complexes with evanescent light and which was different from the previous biomarker- binding detecting the resonant light scattering from each observed moieties on the functionalized nanoparticles . In some complexed nanoparticle can further comprise releasing a embodiments , there can be , for example, 1 to 2 , 1 to 3 , 1 to US 2017 /0234874 A1 Aug . 17 , 2017

4 , 1 to 5 , 1 to 6 , 1 to 7 , 1 to 8 , 1 to 9, 1 to 10 , 1 to 11 , 1 to the duplexes . In some embodiments , a third competing 12 , 1 to 13 , 1 to 14 , 1 to 15 , 1 to 16 , 1 to 17 , 1 to 18 , 1 to oligonucleotide can be added in molar excess to disrupt the 19 , 1 to 20 , 1 to 25 , 1 to 30 , 1 to 35 , 1 to 40 , 1 to 45 , 1 to duplex of the first oligonucleotide and the second oligo 50 , 2 to 50 , 3 to 50 , 4 to 50 , 5 to 50 , 6 to 50 , 7 to 50 , 8 to nucleotide . In this approach , an oligonucleotide hybrid is 50 , 9 to 50 , 10 to 50 , 11 to 50 , 12 to 50 , 13 to 50 , 14 to 50 , dissociated by competitive binding of one member of the 15 to 50 , 20 to 50 , 30 to 50 , 40 to 50 , or any of the ranges hybrid pair to an excess of its complement. In some embodi of the foregoing, numbers of iterations of successive inter ments , the duplex can be disrupted by displacing either the rogations of the cell with a next plurality of functionalized first or second oligonucleotide to form a new duplex with nanoparticles . either the first oligonucleotide or the second oligonucleotide , [0284 ] In some embodiments , each species of functional as shown in FIG . 2 . “ Displacing, " or " releasing ” for the ized nanoparticle species can be functionalized with a dif purpose of the present invention , may be accomplished by ferent DNA oligonucleotide releasing system . such methods as strand displacement or hydrolysis of the [ 0285 ] In some embodiments , the removal of a first plu displaced strand catalyzed by a polymerase having a 3 ' to 5' rality of functionalized nanoparticles can be achieved by or 5 ' to 3 ' exonuclease activity . In some embodiments , after displacing the first plurality of functionalized nanoparticles displacing the first plurality of functionalized nanopanicle , from the biomarker binding moieties . In some embodiments , the first plurality of functionalized nanoparticle can be the linker between each nanoparticle species in the first washed away so as to eliminate the resonant light signal plurality of functionalized nanoparticles and its respective (RLS ) from the first plurality of functionalized nanopar biomarker binding moiety comprises a first oligonucleotide ticles . The same cells can then be analyzed with a second bound to a first functionalized nanoparticle species and a biomarker - binding moiety conjugated to a functionalized second oligonucleotide bound to its respective biomarker nanoparticle with the same RLS signature as the first bio binding moiety , where the second oligonucleotide comprises marker- binding moiety - nanoparticle properties. For a portion complementary to at least a portion of the first example , after displacing and washing away a 25 nm Au oligonucleotide , and hybridization of the first oligonucle nanoparticle functionalized with a first biomarker - binding otide to the second oligonucleotide forms a linker compris moiety , a next 25 nm Au nanoparticle functionalized with a ing a double -stranded nucleic acid in these oligonucleotide second biomarker- binding moiety can be contacted to the linker functionalized nanoparticle species . In some cells . In some embodiments , the cells can be stained and embodiments the first, second and third oligonucleotides imaged after displacing the functionalized nanoparticles . may be the same for each of the functionalized nanoparticle [0287 ] In some embodiments , the functionalized nanopar species and respective biomarker binding moiety in the first ticle comprising a first oligonucleotide can be connected to plurality of nanoparticles . Each functionalized nanoparticle the biomarker - binding moiety comprising a second oligo species can be displaced from its respective biomarker nucleotide via an indirect hybridization with a third oligo binding moiety by binding of a third oligonucleotide to the nucleotide , where a portion of the first oligonucleotide is first oligonucleotide with the hybrid formed by hybridization complementary to a portion of the third oligonucleotide, and of the third oligonucleotide and the first oligonucleotide a portion of the second oligonucleotide is complementary to exhibiting a melting temperature higher than the melting a different portion of the third oligonucleotide , as show in temperature of the double - stranded nucleic acid formed by FIG . 3 . The indirect hybridization duplex can be disrupted hybridization of the first and second oligonucleotide, as by displacing the third oligonucleotide from either the first shown in FIG . 1 . In other embodiments , first, second and or second oligonucleotides by adding a molar excess of a third oligonucleotides associated with each functionalized fourth oligonucleotide . In some embodiments , the fourth nanoparticle species and its respective biomarker binding oligonucleotide can be to the second oligonucleotide with to moiety may be different for each nanoparticle species and its form a stronger duplex (relatively higher Tm ) than the respective biomarker binding moiety . For example , in the duplex between the second oligonucleotide and the third first plurality of functionalized nanoparticles , the second oligonucleotide, as shown in FIG . 3 . In some embodiments , functionalized nanoparticle species may comprise a fourth a fifth oligonucleotide can be added in molar excess , option oligonucleotide , its respective biomarker binding moiety ally with the fourth oligonucleotide also present, where the may comprise a fifth oligonucleotide, and the displacing fifth oligonucleotide can be complementary to the first or oligonucleotide may be a sixth oligonucleotide . third oligonucleotides and form a stronger duplex between [0286 ] In some embodiments , the first plurality of func the fifth and first or third oligonucleotides thanthe first or tionalized nanoparticles comprises a first oligonucleotide third oligonucleotides with the second oligonucleotide . In and the biomarker binding moiety comprises a second some embodiments , the displaced functionalized nanopar oligonucleotide which is hybridized to the first oligonucle ticle can be removed . The removal can be effected by otide to form a duplex , as shown in FIG . 1 , After scanning washing the cells with an aqueous solution . to detect the resonant light scattering of the first plurality of 10288 ] In some embodiments , one or more iterations of functionalized nanoparticles, the first plurality of function interrogating biomarkers can be achieved by successive alized nanoparticles can be displaced from the biomarker contacts with at least a second , third , up to ten or more binding moieties by dissociating the duplex . In some plurality of functionalized nanoparticle species. In some embodiments of the invention , dissociation may be accom embodiments , the one or more iterations of interrogating plished by heating the complexes above the melting tem biomarkers can be , for example , one , two, three , four, five , perature of the nucleic acid duplex . In some embodiments , six , seven , eight, nine , ten , fifteen , twenty , thirty, forty , or the mixture comprising the complex can be warmed or the fifty or more times . In embodiments where oligonucleotide ionic strength reduced sufficiently to cause the hybridized linker functionalized nanoparticles are used , each plurality duplex to dissociate . In some embodiments , a chemical or of functionalized nanoparticle species and respective bio biological agent may be added to the complex to dissociate marker binding moiety may comprise the same first , second US 2017 /0234874 A1 Aug . 17 , 2017 third oligonucleotide for each oligonucleotide - linker func nucleotide , modified polynucleotide , polyribonucleotide, tionalized nanoparticle species in a given plurality of oligo modified polyribonucleotide, peptide , or glycan . The poly nucleotide -linker functionalized functionalized nanoparticle nucleotide can comprise a DNA restriction enzyme species. Alternatively , each oligonucleotide linker function sequence . The modified polynucleotide can comprise a alized nanoparticle species and its respective biomarker di- thiol, diol, abasic , or uracil moiety within the polynucle binding moiety in each plurality of oligonucleotide -linker otide sequence . functionalized nanoparticle species may comprise a unique set of first, second and third oligonucleotides such that each [0296 ] In some embodiments , the linker can comprise a biomarker binding moiety is associated with a unique set of peptide that further comprises a protease sequence. The first , second and third oligonucleotides . In this embodiment, protease sequence can be a trypsin or chymotrypsin protease from one , to ten or more successive rounds of displacement recognition sequence . In some embodiments , the linker can and contact with a new plurality of oligonucleotide - linker comprise a glycan that further comprises an alpha - fucosi functionalized nanoparticle species can take place . Practi dase recognition site . The alpha - fucosidase recognition site tioners in the art will recognize that the ultimate limit to the can be an alpha - 1 , 2 fucoside bond . In some aspects, the number of iterations will be limited by the available cell area linker can be cleaved with a peptidase , DNAase, and / or to contact a successive biomarker -binding moiety . The limit RNAse . will be large , as a cell can range , for example , from 1 to 15 micrometers in diameter while a biomarker -binding moiety Substrate Features ( e . g . , antibody ) is about 150 nanometers ( 0 . 15 micrometers ) [0297 ] In some embodiments , the substrate can be com in diameter . prised of glass silica , clear polymer ( plastic ) , gold , or [ 0289 ] In some embodiments , after displacing the second alumina . In some embodiments, the substrate can be ITO or previous plurality of functionalized nanoparticles from ( indium tin - oxide ) . In some embodiments , the substrate can the biomarker binding moieties , the following steps are be FTO ( fluoride tin - oxide ) . The substrate can be function performed : alized . The substrate functionalization can be patterned . The [0290 ] (i ) the biomarker- binding moieties of the func substrate functionalization can be a silane - linked cell bio tionalized nanoparticles which bound to the cell are marker, polymer -linked cell biomarker, silane - linked amine , associated with the biomarker binding moiety function silane- linked carboxylic acid , silane - linked biotin , polyfluo alized -nanoparticle is classified , and rinated alkyl- linked amine , polyfluorinated alkyl- linked bio [0291 ] (ii ) the cells are contacted with a next plurality of tin , polymer- linked amine , polymer -linked carboxylic acid , nanoparticles functionalized with different biomarker polyethylene glycol ( PEG ) , gold , polysaccharides ( e . g . , binding moieties , and each nanoparticle species of the amine - functionalized dextran ), teflon , fluorinated silane , sil next plurality of nanoparticles are functionalized with ver , alumina , or glass silica. In some embodiments , the different biomarker binding moieties that bind to a polysaccharides can be selected from : amino - functionalized biomarker which is suspected of being associated with dextran , amino - functionalized pullulan , amino - functional samples in which the first biomarker is present. ized dextrin , and combinations thereof. In some embodi [ 0292 ] In some embodiments , the biomarkers targeted by ments , the substrate can comprise features to identify which the biomarker binding moieties in the second plurality of region of the substrate is being imaged . The features can functionalized nanoparticle species each bind to a biomarker vary per region of the substrate so as to enable which region suspected of being associated with samples or conditions, is being imaged . The features can comprise physical differ diseases , or disorders that are also associated with the first ences in the substrate at specific parts of the substrate . In biomarker. In this aspect, the methods of this disclosure are some embodiments , the features can be: mirrors, lines , dots , useful in detecting whether the associated biomarkers are particular shapes , barcodes , 2 - D barcodes , or patterns or present on the same or different cells , or populations of cells . combinations thereof. 10293 ]. In some embodiments , the methods are useful in determining an association between the biomarkers that Compositions/ Combinations/ Kits were bound during the first iteration and the biomarkers bound during the next or subsequent iterations. In some [0298 ] A composition is described for the detection of a embodiments , the assocation can be made of the biomarkers cellular biomarker signature . In some embodiments , the bound during any iteration , and biomarkers bound during composition can comprise a plurality of functionalized any other iteration . In some embodiments , the association nanoparticles where the nanoparticles are functionalized can be made of all , or a portion , of the biomarkers bound to with a biomarker -binding moiety . In some embodiments , the the cells in any iteration with biomarkers bound in a different functionalized nanoparticles can further comprise : a nano iteration . particle functionalized with a first oligonucleotide ; a bio [0294 ] The association can be based on a systemic or marker -binding moiety functionalized with a second oligo tissue -based assay . The association can be a presumed nucleotide , and the first oligonucleotide is complementary to biological correlation . In some embodiments , the method a portion of the second oligonucleotide , and the first and can determine whether two or more biomarkers which have second oligonucleotide form a hybridized duplex . In an been assumed to associate with the same cell are truly alternative embodiment, the functionalized nanoparticles associated with the same cell or associated with different can further comprise a nanoparticle functionalized with a cells . first oligonucleotide ; a biomarker -binding moiety function [0295 ] In some embodiments , the removing a first plural alized with a second oligonucleotide ; and a third oligonucle ity of functionalized nanoparticles can be achieved by otide , where the first oligonucleotide is complementary to a cleaving a linker between the nanoparticle and the bio portion of the third oligonucleotide , the second oligonucle marker- binding moiety . The linker can comprise a poly - otide is complementary to a separate portion of the third US 2017 /0234874 A1 Aug . 17 , 2017 oligonucleotide , and the first and second oligonucleotides released from their respective biomarker - binding moieties . form a hybridized duplex to the third oligonucleotide . A next plurality of functionalized nanoparticles comprising [0299 ] In some embodiments , a combination or kit for the a fourth functionalized nanoparticle species comprising the detection of a cellular biomarker signature can comprise a first nanoparticle and a fourth biomarker - binding moiety , a plurality of biomarker- binding moiety functionalized nano fifth functionalized nanoparticle species comprising the sec particle species . In some embodiments , the combination of ond nanoparticle and a fifth biomarker -binding moiety , and functionalized nanoparticle species can further include : a a sixth functionalized nanoparticle species comprising the nanoparticle species bound to a first oligonucleotide and a third nanoparticle and a sixth biomarker - binding moiety can biomarker - binding moiety bound to a second oligonucle be contacted to the cell . In some embodiments, the first otide , where the first oligonucleotide is complementary to a plurality of particles may comprise 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , or portion of the second oligonucleotide , and the first and any integer up to 50 functionalized nanoparticle species . second oligonucleotide form a hybridized duplex . In some Alternatively, in some embodiments , the fourth functional embodiments , the combination of functionalized nanopar ized nanoparticle species comprising a fourth nanoparticle ticle species can further comprise : a nanoparticle function and a fourth biomarker -binding moiety , a fifth functionalized alized with a first oligonucleotide; a biomarker - binding nanoparticle species comprising a fifth nanoparticle and a moiety functionalized with a second oligonucleotide ; and a fifth biomarker -binding moiety , and a sixth functionalized third oligonucleotide , where the first oligonucleotide is nanoparticle species comprising a sixth nanoparticle and a complementary to a portion of the third oligonucleotide , the sixth biomarker - binding moiety . second oligonucleotide is complementary to a separate por [ 0302 ] The combination or kit can comprise , pluralities of tion of the third oligonucleotide, and the first and second functionalized nanoparticle species, each plurality compris oligonucleotides form a hybridized duplex to the third ing , for example , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , oligonucleotide . In some embodiments , the plurality of 15 , 16 , 17 , 18 , 19 , 20 , 21, 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , functionalized nanoparticle species can comprise a mixture . 31, 32, 33 , 34 , 35 , 36 , 37 , 38, 39, 40 , 41, 42 , 43 , 44 , 45 , 46 , In some embodiments , the plurality of functionalized nano 47 , 48 , 49, 50 , or more pluralities of functionalized nano particle species can be segregated before use . In some particle species. A kit may comprise , for example , 1, 2 , 3 , 4 , embodiments , the plurality of functionalized nanoparticle 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21, species can be segregated , for example , into separate vessels 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , and contacted separately with cells , or combined before 38, 39, 40 , 41, 42, 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , or more contacting a mixture with cells . pluralities of functionalized nanoparticle species . The num [ 0300 ] In some embodiments , a combination or kit for the ber of pluralities of functionalized nanoparticles species in a detection of a cellular morphological biomarker signature kit will depend on the number of biomarkers to be detected can comprise a plurality of functionalized nanoparticle spe and the number of biomarkers detected in each multiplex cies and an optical contrast agent. In some embodiments , the assay , and the number of replicates , controls , or duplicates plurality of functionalized nanoparticle species can com in the assay design . prise a mixture . In some embodiments, the plurality of [0303 ] In some embodiments , the combination can com functionalized nanoparticle species can be segregated before prise 2 to 5 functionalized nanoparticle species . In some use . embodiments , the next plurality of nanoparticles , function [0301 ] A combination or kit is one featured embodiment alized with a different biomarker -binding moiety than used for the detection of a cellular morphological biomarker in a previous iteration . In some embodiments , a subsequent signature using iterations of pluralities of functionalized plurality of functionalized nanoparticle species may com nanoparticles with biomarker- binding moieties, where each prise a different plurality of nanoparticles , functionalized plurality of functionalized nanoparticle species with bio with the same or a different biomarker -binding moiety used marker - binding moieties can be releasable by the methods in a previous plurality of functionalized nanoparticle spe described herein . Each plurality of functionalized nanopar cies . This embodiments can be used , for example , to confirm ticle species with biomarker -binding moieties can be segre the presence of a biomarker using a different functionalized gated from the other pluralities of functionalized nanopar nanoparticle species that may comprise the same or a ticle species. In successive iterations, the plurality of different nanoparticle and / or the same or a different binding functionalized nanoparticle species can comprise the same moiety for the same biomarker in a different plurality of plurality of nanoparticles , but functionalized with different functionalized nanoparticle species used in different steps . biomarker- binding moieties than those in the functional In some embodiments , the pluralities of functionalized nanoparticle species comprising a previous plurality of nanoparticles can be segregated before use . In some embodi functionalized nanoparticle species . As a non - limiting ments each functionalized nanoparticle comprising each example , a first plurality of functionalized nanoparticles can plurality of functionalized nanoparticle species may be comprise a first functionalized nanoparticle species com combined in a mixture . Alternatively , each functionalized prising a first nanoparticle with a first biomarker -binding nanoparticle comprising each plurality of functionalized moiety , a second plurality of functionalized nanoparticle nanoparticle species may be segregated until use or added to species comprising a second nanoparticle with a second cells one at a time or in submixtures . biomarker - binding moiety , and a third functionalized nano [0304 ] As described herein , “ segregated ” means physi particle species comprising a third nanoparticle with a third cally separate . Segregated components can be in separate biomarker - binding moiety . After contacting the first , second , containers or vessels , or in separate sections of a container , and third species of functionalized nanoparticles with their or separated by a seperatable medium , which can be respective biomarker - binding moieties to a cell and imaging removed to yield a mixture . the cell - functionalized nanoparticle complexes , the first [0305 ] A composition is described for the detection of a second , and third functionalized nanoparticles can be cellular biomarker morphological profile , the composition US 2017 /0234874 A1 Aug . 17 , 2017 31 comprising a plurality of functionalized nanoparticles and an nanoparticles. This method can be used to identify cells , optical contrast agent. The optical contrast agent can be the cellular morphologies , and biomarker signatures from the optical contrast agents described herein . image of the functionalized nanoparticles. [ 0306 ] A “ kit” for detecting the presence of an analyte in a sample by the methods of the invention may, by way of Automated Robot System example , comprise at least one container means having disposed therein a functionalized nanoparticle specific for [ 0314 ] In some embodiments , the sample handling and the selected analyte . The kit may further comprise other detection steps , including, without limitation , reagent con container means comprising one or more of the following : tact and mixing steps , and application of external force , buffers , solutions or other reagents and materials necessary complex formation , and detection can be performed by an for performing biological morphological profiling of a cell ; automated robot system . In some embodiments , applying and buffers , solutions or other reagents and materials nec the external force to the cells contacted with the function essary for detecting the optical properties of an optical alized biomarker binding moieties can be handled by an automated robot system . In some embodiments, for contrast agent contacted with a cell . Preferably , the kit example , the steps of providing a sample comprising cells further comprises instructions for use . The kit, if intended from a subject, contacting the cells with one or a plurality of for diagnostic use , may also includes notification of a FDA functionalized nanoparticle species, and adhering the func approved use and instructions therefor , tionalized nanoparticle - cell complexes to a substrate , can be (0307 ] A kit is described for the detection of a cellular performed by the automated liquid handling robot system . In biomarker signature . In some embodiments , the kit can some embodiments , the step of contacting the adhered cells comprise a plurality of functionalized nanoparticles, an with an optical contrast agent can be performed by the optical contrast agent, and a mountant. The mountant can automated liquid handling robot system . The automated have a refractive index (RI ) substantially the same, or within liquid handling robot system can comprise a controller , a 0 .1 RI to that of the fixed cells . In some embodiments , the servo mechanism , fluid lines , and optionally solenoids . The mountant can have a RI of 1 .52 . automated liquid handling robot system can be programmed Homogenous Assay to deliver the reagents described herein at selected times , for selected durations, to deliver selected volumes of reagents . [0308 ] In some embodiments , the biomarker signature of In some embodiments, the controller can be programmed a cell can be detected in a homogeneous assay , the assay using Labview software . In some embodiments , the auto comprising the steps : mated liquid handling robot system can include or exclude, [ 0309 ] (a ) providing a sample comprising cells from a for example, one or a plurality of automatic pipettes , one or subject; a plurality of automatic pipettes syringe pumps, a Hamilton [ 0310 ] (b ) contacting the cells with one or a plurality of Microlab NIMBUS 96 channel liquid handling robot , a functionalized nanoparticle species, each functional Hamilton Microlab STAR liquid handling robot, a Hamilton ized nanoparticle species comprising a biomarker- bind VANTAGE liquid handling system , a Tecan Freedom EVO ing moiety , and forming nanoparticle - cell complexes liquid handling system , a Tecan Fluent liquid handling through binding of the nanoparticle species comprising system , a Beckman Biomek liquid handling system , a Beck a biomarker- binding moiety to its respective bio man BioRAPTR FRD liquid handling system , a Perkin marker ; Elmer JANUS liquid handling system , a Hudson Robotics [0311 ] (c ) adhering the functionalized nanoparticle - cell SOLO liquid handling system , a Hudson Rbootcs Micro 10x complexes to a substrate ; liquid handling system , a QiaCube liquid robot system , an [0312 ] ( d ) illuminating the nanoparticle -cell complexes Aurora VERSA liquid handling system , or an Epppendorf with evanescent light and detecting the resonant light epMotion liquid handling system . scattering from each observed complexed nanoparticle , [0315 ] In some embodiments , detecting cell -functional to obtain a biomarker signature of each observed cell, ized nanoparticle complexes and detecting morphological where unbound functionalized nanoparticles are not images can also be handled by an HIPPA compliant auto removed from the field of view . In some embodiments , not mated system with cell recognition software . In some removing the unbound functionalized nanoparticles from the embodiments , images of the cell - functionalized nanoparticle field of view can be to not wash the field of view . Often , complexes and morphological images of the cells are unbound species are washed from a target to reduce back obtained and stored in an electronic medium , for example in ground noise . Eliminating the wash step had the advantage a HIPPA compliant system . In some embodiments the of a faster overall operation time . The functionalized nano images are accessed by , or provided to a doctor or patholo particles are specific to the biomarker on the cell , and can gist for review in the doctor' s or pathologist ' s office . substantially contact the cell such that little to none signal is [ 0316 ] In some embodiments , the methods of this inven observed for the unbound functionalized nanoparticles . tion are useful in obtaining images of cell -functionalized [ 0313] In some embodiments , the functionalized nanopar nanoparticle complexes under ambient conditions which do ticles contacted to the cells can be used to identify the not require use of a darkroom , in contrast to fluorescent cellular features and morphology when the functionalized labeling systems. In some embodiments , the samples may be nanoparticle is functionalized with a biomarker- binding moiety that binds to the biomarkers in the interrogated viewed on a microscope in a doctor' s or pathologist' s office . morphological feature . FIG . 8b shows an expanded image of Examples the functionalized nanoparticle - contacted cells . The cells were not washed to remove the functionalized nanoparticles [0317 ] The instant disclosure and examples herein docu which did not contact the cells . The cellular shape is clearly ments the features of the methods of this invention , includ identifiable from the relative location of the functionalized ing the detection of cell -functionalized nanoparticle com US 2017 /0234874 A1 Aug . 17 , 2017 32 plexes to detect a biomarker signature of a cell, and the solution was vortexed and sonicated every 15 minutes for integrated morphological biomarker signature of a cell. one hour. One percent BSA in water was added before a 30 Operating procedures for the presently disclosed method of minute incubation period . Particles were washed three times detecting the biomarker -morphological profile of a cell with 1 % BSA / 1 % PBS . After washing , solutions were using resonance - light scattering are set forth in the following sonicated and vortexed to resuspend particles in final vol examples. ume of 100 ul (microliters ) 1 % BSA / 1 % PBS. [0318 ] The following Examples have been included to [0326 ] To label the cells , particles were diluted 1 : 10 in 1 % provide guidance to one of ordinary skill in the art for BSA / 1 % PBS . One ul (microliter ) of Au and Ag particles practicing representative embodiments of the presently dis were added 500 (microliters ) , approximately 10 ,000 cells , of closed subject matter. In light of the present disclosure and CCRF -CEM cells (ATCC # CRM - CCL - 119 ) . The CEM cell the general level of one of skill in the art , those of skill can line is a leukemia cell line . The solution was centrifuged for appreciate that the following Examples are intended to be one minute at 500xg three times , followed by vortexing the exemplary only of the embodiments and features described cells for resuspension . throughout this application , and appreciated by those skilled 0327 ] Labeled cells were spread by applying 2 ul (micro in the art , and that numerous changes , modifications, and liters ) to a glass slide in an area of 1 cm ' and dried for five alterations can be employed without departing from the minutes. To fix cells , the slide was soaked in Coplin Jar with scope of the presently disclosed subject matter. 100 % MeOH for five minutes . The slide was transferred to a tube containing diluted 1 : 20 Giemsa stain (Ricca Chemical Materials and Methods # 3250 - 16 ) in water for one minute . The slide was washed with water to remove excess stain and allowed to air dry . General. 0328 ] FIG . 7 shows stained cells were imaged for mor [ 0319 ] All materials were purchased from the vendors phology detection in Bright- Field using a 20x objective , indicated , with the part numbers indicated . Centrifugation Olympus BX60M microscope and DP71 color camera . steps were performed as units of 1 gravitational force (“ xg ” ) . [0329 ] FIG . 8 shows the same field imaged for phenotype [0320 ] Sample Preparation detection using a 20x objective on Olympus BX60M micro [0321 ] Settling solution at 6 % Dextran /0 .32M Potassium scope in Dark - field utilizing DarkLite Illuminator light oxalate was prepared by mixing 0 .6 mL 10 % Dextran 500 source . Some of the functionalized nanoparticles can be ( Sigma # 31392 ) in PBS, 0 . 16 mL 2M Potassium oxalate observed which are not bound to the cells . In this exemplary ( Sigma# P0963 ) in water, and 0 . 24 mL PBS . Whole blood homogeneous assay , the functionalized nanoparticles spe sample in 5 mM EDTA was added to settling solution at a cies which are introduced to the cells but do not bind to the ratio of 4 : 1 . After a ten minute incubation , the supernatant cells have not been removed from the field of view . was removed and centrifuged at 500xg for five minutes . Supernatant was removed down to 10 ul (microliters ) to give Example 2 leukocyte rich fraction (LRF ) . Cell - functionalized nanopar ticle complexes can be formed through settling or throught Cell Staining /Destaining application of external force , as disclosed herein . Other [0330 ] Labeled cells were spread by applying 2 ul of the compositions for contacting cells with functionalized nano cell suspension to a glass slide in an area of 1 cm² and dried particles , as set forth in this disclosure can be used . for five minutes . To fix cells , the slide was soaked in a Coplin Jar with 100 % MeOH for five minutes . The slide was Example 1 transferred to a tube containing diluted 1: 20 Giemsa stain (Ricca Chemical # 3250 - 16 ) in water for one minute . The Cell Labeling for Simultaneous Detection of Morphology slide was washed with water to remove excess stain and and Phenotyping allowed to air dry . Other stains, as disclosed herein may be [ 0322] As a non - limiting example of detecting the bio used to stain cells. marker- morphology profile of a cell , cells were labeled with [ 0331 ] FIG . 9 shows an initial Brightfield image of a -CD4 (BD # 555344) and a -CD8 (BD # 555631) nanopar Giemsa stained cells imaged for morphology detection in ticles. Other combinations of antibodies described herein Bright- Field using 20x objective , Olympus BX60M micro and nanoparticles described herein can be used to detect the scope and DP71 color camera . cell biomarker- morphology profile . 0332 ] The cells were then destained as follows: Destain [ 0323] Coating Particles with Antibody solution , pH 11 . 3 , was prepared by adding 50 ul (microliters ) [0324 ] Particles were first concentrated by centrifuging of 100 mM sodium phosphate to one mL of 60 % MeOH / 1 .0 ml of 150 nm Au particles (Cytodiagnostics # G150 - 20 ) 40 % Glycerol . 500 ul of destain was added to slide , incu to 100 ul at 800xg for five minutes , and separately, one ml bated for 30 seconds, and washed with water . Before imag of 100 nm Ag particles (NanoComposix # ECP1095 ) to 100 ing , 411 DPX mountant ( Sigma # 06522 ) was applied to cell ul at 1200xg for five minutes. Particles were resuspended by area followed by 18x18 mm cover glass . Amountant is any sonication and followed by an addition of 500 ul (microli substance in which a specimen is suspended between a slide ters ) of 5 mM Sodium Bicarbonate . Particles were concen and a cover glass for microscopic examination . In some trated again to 100 ul (microliters ) by centrifugation and embodiments , the mountant can be comprised from a solu resuspended by sonication . tion with about the similar refractive index of the cells . In [0325 ] To the Au and Ag concentrated particles, 20 ul of some embodiments , the refractive index of the fixed cells is a -CD4 and a -CD8 were added , respectively ( separately ) , at about 1 . 52 . In some embodiments , the mountant can be approximately 0 . 1 mg/ml Ab for 10 OD particles . An OD of immersion oil. FIG . 10 shows destained cells imaged in particles is the reported concentration (per mL) at the Bright- Field using 20x objective , Olympus BX60M micro maximum absorbance wavelength for the particles . Each scope and DP71 color camera . The same field was imaged US 2017 /0234874 A1 Aug . 17 , 2017 33 for residual Giemsa stain using 20x objective on Olympus Example 4 BX60M microscope in Dark - field utilizing DarkLite Illumi nator light source (FIG . 11) . Color Multiplexing [ 0336 ] This example demonstrates the ability to detect Example 3 functionalized nanoparticles comprising four differentnano particles. [0337 ] To 0 . 2 mL of 1 OD particles , addition of 10 ul of 3 Color Multiplexing 20 mM CTPEG mixtures (Nanocs # PG2 -CATH - 10k ) was made to yield approximately 1 mM PEG . After 30 minute [0333 ] This example demonstrates the ability to detect incubation at room temperature, 0 . 1 % w / v Pluronic® F127 functionalized nanoparticles comprising three different (BASF # 51181981 ) was added and allowed to stand for an nanoparticles . To 0 . 2 mL of 1 OD particles, addition of 10 additional 30 minutes . Particles were spun at 3000xg for 10 ul of 20 mM CTPEG mixtures (Nanocs # PG2 -CATH - 10k ) minutes and resuspended in 200 ul mM MES , pH 6 , 0 . 1 % was made to yield approximately 1 mM PEG . CTPEG F127 . Ten mg of concentrated EDC ( Thermo # 77149 ) was mixtues are thiol carboxylic acid functionalized PEG , dissolved in onemL 5 mM MES , PH 6 , 0 . 1 F127 to yield 52 Molecular Weight of 10000 . After 30 minute incubation at mM . An addition of 11 . 5 ul of 52 mM EDC was made to room temperature , 0 . 1 % w / v Pluronic® F127 yield 2 mM EDC . Sulfo - NHS ( Thermo# 24520 ) at 2 mg was (BASF # 51181981 ) was added and allowed to stand for an dissolved in 40 ul of 5 mM MES , pH 6 , 0 . 1 % F127 to yield additional 30 minutes. Pluronic® block copolymers are 230 mM Sulfo -NHS . Another addition of 6 . 5 ul of 230 mm synthetic copolymers of ethylene oxide and propylene oxide Sulfo -NHS was added to yield 5 mM NHS . After allowing represented by the following chemical structure : it to react for five minutes , mixtures were spun at 3000xg for HO (C2H40101 (C3H60 ) 56 ( C2H40 ) 101H . Particles were ten minutes, resuspended in 0 . 2 mL of 5 mM HEPES, PH centrifuged at 3000xg for 10 minutes and resuspended in 7 . 4 , 0 . 1 % F127 and anti- CD45 antibody (BD Biosci 200 ul mM MES , pH 6 , 0 . 1 % F127 . Next, 10 mg of ences # 555480 ; 0 . 5 mg/ ml (milligram /milliliter )) to final concentrated EDC ( Thermo # 77149 ) in distilled water was concentration of 100 ug /ml (microgram /milliliter ) . Mixtures dissolved in one mL 5 mM MES , pH 6 , 0 . 1 F127 to yield 52 were reacted overnight at room temperature . One percent mM . An addition of 11 . 5 ul (microliters ) of 52 mM EDC BSA was added and incubated for one hour. Next, particles solution was made to yield 2 mM EDC . Sulfo -NHS were washed with 5 mM HEPES , PH 7 .4 , 0 . 1 % F127, 0 .1 % ( Thermo # 24520 ) at 2 mg was dissolved in 40 ul (microli BSA . Monodispersity was checked by imaging with 40x ters ) of 5 mM MES , pH 6 , 0 . 1 % F127 to yield 230 mM objective and DarkLite Illuminator light source . Sulfo -NHS . Another addition of 6 . 5 ul (microliters ) of 230 [0338 ] A multiplex cocktail containing yellow Anti -CD45 mM Sulfo -NHS was added to yield 5 mM NHS. After 70 nm Au particles (Nanocomposix # AUCN70 -25M ), allowing it to react for five minutes , mixtures were spun at orange Anti - CD45 70 nm Au Nanourchins (Cytodiagnos 3000xg for ten minutes , resuspended in 0 .2 mL of 5 mM tics # GU70 - 20 ) , green Anti - CD45 50 nm Au particles HEPES , PH 7 .4 , 0 . 1 % F127 and anti -CD45 antibody (BD (Nanocomposix # AUCN50 - 25M ), and blue Anti -CD45 50 Biosciences # 555480 ; 0 . 5 mg/ ml ) to final concentration of nm Ag particles (Nanocomposix # AGCN50 - 25M ) was pre 100 ug /ml (microgram /milliliter ) was added . Mixtures were pared by mixing the particles at 1 : 2 : 2 : 4 parts, respectively, reacted overnight at room temperature . One percent BSA into 5 mM HEPES , PH 7 . 4 , 0 . 1 % F127 , 0 . 1 % BSA . To 10 was added and incubated for one hour . Then , particles were ul of CCRF - CEM ( ATCC # CRM - CCL - 119 ) cell suspension washed with 5 mM HEPES , PH 7 . 4 , 0 . 1 % F127 , 0 . 1 % BSA . of approximately 1 , 000 cells , 1 ul (microliter ) of multiplex Monodispersity was checked by imaging with 40x objective cocktail particle suspension was added . Centrifugation at and DarkLite Illuminator light source . 3000xg for one minute was performed . 03391 Labeled cells were then added to 200 ul (microli [0334 ] Anti - CD45 40 nm Ag particles (Nanocomposix # ter ) of a 1 :2000 dilution of whole blood in 5 % BSA /PBS , AGCN40 - 25M ) were diluted 1 : 10 into 5 mM HEPES , PH and washed three times with 200 ul (microliter ) of 5 % 7 . 4 , 0 . 1 % F127 , 0 . 1 % BSA . Anti- CD45 50 nm Au particles BSA /PBS at 100xg for 4 min . The entire sample was then (Nanocomposix # AUCN50 - 25M ) and anti -CD45 80 nm Au applied to an assembled CytoFuge device containing a particles (Nanocomposix # AUCN80 -25M ) were each SuperFrost Plus slide (Fisher # 12 -550 - 15 ) , and spun at 1500 diluted 1 : 20 into the same solution . To 10 ul (microliters ) of rpm for 3 minutes to adhere cells to the slide using a concentrated 10x CCRF - CEM ( ATCC # CRM - CCL - 119 ) Cytofuge 12 (Beckman - Coulter # X00 -006082 - 001) . The cell suspension of approximately 10 , 000 cells , 1 ul (micro slide was then fixed for five minutes in 100 % MeOH soak liter) of appropriate particle suspension was added . Cen in a Coplin jar . Type HF Immersion oil (Cargille # 16245 ) trifugation at 3000xg for one minute three times was per was applied at 7 ul ( microliter ) followed by a coverslip and formed . To a glass slide , 1 ul ( microliter ) of the cell -particle imaged in Dark - field with DarkLite Illuminator light source suspension per slide was applied to an approximate 1 cm ? and 40x objective ( 100 ms exposure ) ( image shown in FIG . diameter area . The slide was then fixed for one minute in 13 ). 100 % MeOH soak in a Coplin jar. DPX mountant was [0340 ] The coverslip and oil were removed by washing applied at 5 ul (microliters ) followed by a coverslip and with propanol, and the slide was air dried . Next, 400 ul imaged in Dark - field with DarkLite Illuminator light source (microliter ) of Giemsa stain ( Ricca # 3250 - 16 ) was applied and 40x objective (200 ms exposure ), as shown in FIG . 12 . to the cell area and incubated 4 minutes . Stain was removed [0335 ] Any of the functionalized nanoparticles and or with water wash . Next , 10 ul ( microliter ) of DPX Mountant other features disclosed in this application can be used to (Sigma # 06522 ) was applied with coverslip and the same multiplex detection of the biomarker signature and /or bio cell area imaged in Brightfield using 40x objective ( 0 . 1 ms marker -morphological profile . exposure ) ( image shown in FIG . 14 ) . US 2017 /0234874 A1 Aug . 17 , 2017 34

[ 0341 ] Any of the functionalized nanoparticles and or a microscope slide (SuperFrost Plus ), allowed to air dry, then other features disclosed in this application can be used to fixed in 100 % MeOH for 1 minute and air dried . The slide multiplex detection of the biomarker signature and / or bio was blocked with 10 % BSA in PBS for 10 minutes at room marker- morphological profile . In accordance with the meth temperature . The blocking solution was removed , and 70 nm ods of this invention , it will be appreciated that 5 , 6 , 7, 8 , 9 Au nanoparticles functionalized with streptavidin were 10 , 11, 12 , 13 , 14 , 15 , or up to 50 different nanoparticles applied to the cell area . One slide was placed into a slide comprising functionalized nanoparticle species may be used holder and centrifuged 2 minutes at 500xg in a swinging in the multiplexed methods of this invention . bucket centrifuge ( centrifuge labeling ) . A second slide was incubated for 5 minutes at room temperature (passive label Example 5 ing ) . Both slides were rinsed extensively with PBS , then water , and air dried . DPX mounting medium and coverglass Mountant Refractive Index Matching was applied , and the cells were imaged using a dark - field [ 0342 ] FIG . 15 shows an image of the CEM cells con microscope and color camera (Olympus ) . FIG . 16 shows the tacted with the functionalized nanoparticles as described in passive labelling slide ( no centrifuge ) , and FIG . 17 shows Example 4 , with no mountant added . 20x Objective, 100 ms the active labelling slide (with centrifugation ) . Significantly exposure, Darklite . The lack of a matching refractive index more streptavidin particles are bound to cells in the centri medium yields excessive white light scattering , thereby fuge labeling slide compared to the passive labeling slide , preventing imaging of the RLS signal of the functionalized demonstrating that subjecting cells contacted with function nanoparticles . alized nanoparaticles to an external force that increases the local concentration of the functionalized nanoparticles Example 6 results in enhanced labeling of the cells . HIV Assessment Using Cellular Biomarker Signature Example 8 [ 0343 ] In some embodiments , the biomarkers identified on Enhanced Labeling by Centrifugation of Blood Smear the cells can be used to identify the cell type and countof the Example cell type by adding all of the identified cell types exhibiting [0346 ] To demonstrate the effects of application of an a particular biomarker. A cell from a subject ’ s blood sample external force on labeling of cells in a blood sample , a buffy is contacted with a multiplex cocktail containing yellow coat from fresh EDTA treated blood was obtained , and a anti - CD3 70 nm Au particles (Nanocomposix # AUCN70 blood smear was prepared on a microscope slide using the 25M ), orange anti -CD -4 70 nm Au Nanourchins (Cytodi wedge technique . After air drying , the blood smear was fixed agnostics # GU70 - 20 ), and green anti - CD8 50 nm Au in methanol for 10 minutes and allowed to dry . A silicone particles (Nanocomposix # AUCN50 -25M ). The cocktail is gasket was applied to create a reaction well to hold assay comprised of equal parts 1 : 1 : 1 of each of the functionalized reagents . The slide was blocked with 10 % BSA in PBS for nanoparticle pluralities . The particles are prepared in a 10 minutes at room temperature . The blocking solution was buffer of 5 mM HEPES , PH 7 . 4 , 0 . 1 % F127 , 0 . 1 % BSA . To removed , and 50 uL of biotinylated anti -CD45 antibody 10 ul of isolated cells from subject in a suspension adjusted (Becton Dickinson ) was applied and incubated two hours at to approximately 1000 cells , 1 microliter of the cocktail room temperature . The antibody solution was removed , the particle suspension is added . The solution is centrifuged at slide washed extensively with PBS - Tween -BSA buffer. 70 3000xg for one minute to accelerate the cell - functionalized nm Au nanoparticles functionalized with streptavidin were nanoparticle contacting . The cells are adhered to a slide and applied and the slide was placed into a slide holder and contacted with HF immersion oil as an RImatched mountant centrifuged 2 minutes at 250xg in a swinging bucket cen as described in Example 4 . trifuge (centrifuge labeling ) . The slide was rinsed exten [0344 ] The total cell count of cells exhibiting all of CD3, sively with PBS - Tween - BSA buffer, then water, and air CD4 , and CD8 are compared to the amount of cells which dried . DPX mounting medium and coverglass was applied , do not exhibit CD4. The amount of cells which are CD4 and the cells were imaged using a dark - field microscope and positive is compared against a threshold amount per volume color camera ( Olympus) , as shown in FIG . 18 . Lymphocytes of blood . If the amount of cells are below a threshold and granulocytes, both known to express CD45 surface number, the subject is diagnosed with having HIV (Human antigens, were labeled with Au functionalized nanoparticles Immunodeficiency Virus) . while red blood cells were not. Example 7 Example 9 Enhanced Labeling by Centrifugation Multiplex Labeling of Blood Smear Example [0345 ] To determine the effects of subjecting cells con [0347 ] An exemplary method for multiplex labeling of tacted with functionalized nanoparticles to an external force , cells in a sample was performed as described below . A Buffy in order to increase the local concentration of the function coat from fresh EDTA blood was obtained from which a alized nanoparticles and cells , CCRF - CEM cells (ATCC ) at blood smear was prepared on a microscope slide using the 1x106 cells per mL were suspended in RPMImedia ( ATCC ) wedge technique . After air drying, the blood smear was fixed supplemented with Fetal Cal Serum were reacted with in Neutral Buffered Formalin for 30 minutes, rinsed with biotinylated anti - CD45 antibody (BD Biosciences ) at room water, and allowed to dry . A silicone gasket was applied to temperature for 30 minutes. The cells were then washed via create a reaction well to hold assay reagents. The slide was centrifugation at 500xg for 5 minutes with PBS + 0 . 1 % BSA blocked with 10 % BSA in PBS for 10 minutes at room five times . One microliter of cell suspension was applied to temperature . The blocking solution was removed and 0 .1 US 2017 /0234874 A1 Aug . 17 , 2017 35

OD 70 nm Au nanoparticles functionalized with anti -CD3 Example 11 antibody and 0 . 1 OD 50 nm Ag particles functionalized with anti - CD4 antibody were applied . The slide was placed into Electrically Enhanced Labeling a slide holder and centrifuged 1 minute at 500xg three times [0349 ] A BSA - biotin solution at a concentration of 5 in a swinging bucket centrifuge (centrifuge labeling ). The micrograms/ mL in a solution of free BSA at a concentration slide was rinsed extensively with PBS- Tween buffer , then of 5 milligrams/mL in 10 mM MES buffer was prepared . water, and air dried . Index matching mounting medium and The BSA -biotin / BSA solution was coated onto conductive , coverglass was applied , and the cells were imaged using a Indium - Tin - Oxide (ITO ) glass slides (Nanocs ) by immers dark - field microscope and color camera (Olympus ) . The ing the slide into the solution for 30 minutes, then allowing index matching medium was removed , and the slide was the slide to air dry . In some embodiments , the conductive stained with Giemsa stain for 3 minutes , rinsed with water , slide can be ITO . In some embodiments , the conductive slide and air dried . Mounting medium was applied , and the slide can be Fluorine doped tin -oxide ( e . g . , TEC GlassTM mate was imaged using a bright- field microscope and color cam rials from Pilkington (NJ , USA ) ) . The free BSA and the era (Olympus ). White blood cells were first identified by BSA on the BSA - biotin adhered to the ITO surface , and the their morphology in the stained , Brightfield image , as seen free BSA prevented biotin saturation at the surface . A 500 in FIGS . 19E - 19J. Several Neutrophils , with their multi micron thick silicone gasket (Grace Bio -Labs , Inc . ) was lobed nucleus, were visible in the field of view , as seen in placed around the BSA -biotin area , and the slides were FIG . 19E . Lymphocytes , which lack the lobed nucleus in the blocked with 1 % Casein in PBS for 10 minutes . After Brightfield image , were further differentiated in their bind removal of the blocking solution , 50 microliters of 0 . 1 OD , ing of anti - CD3 Au functionalized nanoparticles and anti 70 nm Au nanoparticles functionalized with streptavidin was CD4 Ag functionalized nanoparticles that are visible in the applied . One slide was incubated passively for 2 minutes while a second slide was covered with an additional con Darkfield image, as seen in FIGS. 19G - 19J. Several T cells , ductive slide so that the conductive surfaces of the slides known to be CD3 + , bound the anti- CD3 Au functionalized faced one another . Alligator clips from a power supply (BK nanoparticles , as seen in FIG . 19H and FIG . 19 ). As Precision ) were attached to the two conductive slides facing observed in FIG . 19H , one T helper cell, known to be both one another, and a voltage of 400 mV was applied for 2 CD3 + and CD4 + , bound both the anti -CD3 Au functional minutes . At the end of two minutes , both the passively ized nanoparticles and the anti - CD4 Ag functionalized nano incubated and electrically enhanced slides were washed with particles . One T cell , known to be CD3 + yet lack CD4, TBS / 0 .05 % Tween - 20 , water, and air dried . Imaging oil and bound to the anti - CD3 Au functionalized nanoparticles but coverglass were applied , and the BSA -biotin coated regions did not appreciably bind the anti - CD4 Ag functionalized on each slide were imaged using a dark - field microscope and nanoparticles . As observed in FIG . 19F, Neutrophils , which color camera (Olympus ) . While the passively incubated are known to lack CD3 and CD4 surface antigens, remained slide as shown in FIG . 21A has very few particles attached unlabeled with respect to functionalized particles . to its surface , the electrically enhanced slide is covered heavily with particles as shown in FIG . 21B . Example 10 Example 12 Assay of Whole Blood Cell Suspension Multiplex Labeling of FFPE Tissue [0348 ] A Buffy coat from fresh EDTA blood was obtained , 10350 Fixed Formalin , Paraffin Embedded tissue arrays and cells were exchanged into PBS via centrifugation for 2 ( Biomax -US ) were depariffinized according to the following min at 500xg . 70 nm Au nanoparticles functionalized with procedure: the slides were heated to 60° C . for 30 minutes, anti -CD3 antibody was added and incubated 30 min with soaked in xylene 10 minutes , soaked in a fresh xylene wash occasional mixing . The labeled cells were washed with for another 10 minutes , soaked in 100 % ethanol for 5 twice with human plasma at low speed centrifugation (70xg ) minutes , soaked in 95 % ethanol/ water for 5 minutes , soaked to remove unbound functionalized nanoparticles. Three in 70 % ethanol/ water for 5 minutes , then soaked in water for microliters of the cell suspension was smeared on a Super 5 minutes. A 500 micron thick silicone gasket (Grace frost slide and air dried , then fixed for 5 min in 100 % Bio - Labs ) was placed around the tissue array , and 50 nm methanol. Index matching mounting medium and coverglass ( green ) and 70 nm ( Yellow ) Au functionalized nanoparticles , was applied , and the cells were imaged using a dark - field passively coated with BSA , were applied to the tissue array microscope and color camera (Olympus ). The index match via centrifugation at 1000xg for 3 minutes. After washing ing medium was removed , and the slide was stained with with TBS / 0 . 05 % Tween - 20 and water, the slide was air Giemsa stain for 3 minutes , rinsed with water , and air dried . dried . An index matching mountant and coverglass was Mounting medium was applied , and the slide was imaged applied , and the slide was imaged using a dark - field micro using a bright- field microscope and color camera ( Olym scope and color camera (Olympus ). Both colors of func pus ). In the field of view , 13 out of 14 lymphocytes showed tionalized nanoparticles , the green 50 nm Au and yellow 70 bound Au functionalized nanoparticles. Neutrophils , which nm Au , were clearly visible with distinguishable colors in are known to lack CD3 surface antigens , remained unla the tissue sample , as shown in FIG . 22 . beled . FIG . 20A - D shows (clockwise from top left: A - D ) Au [0351 ] The inventions described and claimed herein have anti -CD3 functionalized nanoparticles (yellow / lighter col many attributes and embodiments including , but not limited ors ) bind to 13 out of 14 lymphocytes in the field . No to , those set forth or described or referenced in this Detailed functionalized nanoparticles were observed to bind to neu Description . It is not intended to be all - inclusive and the trophils . inventions described and claimed herein are not limited to or US 2017 /0234874 A1 Aug . 17 , 2017 36 by the features or embodiments identified in this Detailed [0355 ] The invention has been described broadly and Description , which is included for purposes of illustration generically herein . Each of the narrower species and sub only and not restriction . generic groupings falling within the generic disclosure also form part ofthe invention . This includes the generic descrip [ 0352 ] All patents , publications , scientific articles, web tion of the invention with a proviso or negative limitation sites , and other documents and materials referenced or removing any subject matter from the genus , regardless of mentioned herein are indicative of the levels of skill of those whether or not the excised material is specifically recited skilled in the art to which the invention pertains , and each herein . such referenced document and material is hereby incorpo [0356 ] Other embodiments are within the following rated by reference to the same extent as if it had been claims. In addition , where features or aspects of the inven incorporated by reference in its entirety individually or set tion are described in terms of Markush groups, those skilled forth herein in its entirety . Applicants reserve the right to in the art will recognize that the invention is also thereby physically incorporate into this specification any and all described in terms of any individual member or subgroup of materials and information from any such patents , publica members of the Markush group . tions, scientific articles, web sites, electronically available 1 . A method for detecting the biomarker -morphological information , and other referenced materials or documents . profile of a cell , the method comprising : [0353 ] The specific methods and compositions described ( a ) providing a sample comprising cells from a subject ; herein are representative of preferred embodiments and are ( b ) contacting the cells with one or a plurality of func exemplary and not intended as limitations on the scope of tionalized nanoparticle species , each functionalized the invention . Other objects , aspects , and embodiments will nanoparticle species comprising a biomarker- binding occur to those skilled in the art upon consideration of this moiety , and forming nanoparticle - cell complexes specification , and are encompassed within the spirit of the through binding of the functionalized nanoparticle spe invention as defined by the scope of the claims. It will be cies comprising a biomarker -binding moiety to its readily apparent to one skilled in the art that varying respective biomarker; substitutions and modifications may be made to the inven ( c ) adhering the functionalized nanoparticle - cell com tion disclosed herein without departing from the scope and plexes to a substrate ; spirit of the invention . The invention illustratively described ( d ) illuminating the functionalized nanoparticle - cell com herein suitably may be practiced in the absence of any plexes with epi -illumination or evanescent light and element or elements , or limitation or limitations, which is detecting the resonant light scattering from each not specifically disclosed herein as essential . Thus , for observed complexed functionalized nanoparticle , to example , in each instance herein , in embodiments or obtain a biomarker signature of each imaged cell ; examples of the present invention , any of the terms " com ( e ) contacting the substrate - adhered cells with an optical prising ” , “ consisting essentially of” , and “ consisting of" contrast agent; may be replaced with either of the other two terms in the ( f ) imaging morphological features of the contacted cells ; specification . Also , the terms “ comprising ” , “ including ” , and containing ” , etc . are to be read expansively and without ( g ) associating the morphological features of the con limitation . The methods and processes illustratively tacted cells with the biomarker signature of each sub described herein suitably may be practiced in differing strate - adhered cell to detect the biomarker- morphologi orders of steps, and that they are not necessarily restricted to cal profile of each cell . the orders of steps indicated herein or in the claims. It is also 2 . The method of claim 1 , wherein the biomarker is that as used herein and in the appended claims, the singular present on the cell surface . forms “ a ," " an , ” and “ the” include plural reference unless 3 . The method of claim 1 , wherein the biomarker is the context clearly dictates otherwise . Under no circum present within the cell . stances may the patent be interpreted to be limited to the 4 . The method of claim 1, wherein the biomarker is specific examples or embodiments or methods specifically selected from the following : CD1, CD2, CD3, CD4 , CD5 , disclosed herein . Under no circumstances may the patent be CD6 , CD7 , CDS, CD9 , CD10 , CD11a , CD11b , CD11c , interpreted to be limited by any statement made by any CD13 , CD14 , CD15 , CD16 , CD19 , CD20 , CD21 , CD22 , Examiner or any other official or employee of the Patent and CD23 , CD25 , CD30 , CD33 , CD34 , CD38 , CD41 , C43 , Trademark Office unless such statement is specifically and CD45 , CD56 , CD57 , CD58, CD61, CD64 , C71, CD79a , without qualification or reservation expressly adopted in a CD99, CD103 , CD117 , CD123 , CD138 , CD138 , CD163 , responsive writing by Applicants . CD235a , HLA -DR , Kappa , Lambda , Pax - 5 , BCL - 2 , Ki- 67 , [0354 ] The terms and expressions that have been ZAP -70 , MPO , TDT, and FMC- 7 . employed are used as terms of description and not of 5 . The method of claim 1, wherein the optical contrast limitation , and there is no intent in the use of such terms and agent is a leuco dye . expressions to exclude any equivalent of the features shown 6 . The method of claim 5 , wherein the leuco dye is red and described or portions thereof, but it is recognized that leuco dye , methylene blue , crystal violet, phenolphthalein , various modifications are possible within the scope of the thymolphthalein , or methylene green . invention as claimed . Thus, it will be understood that 7 . The method of claim 1 , wherein the optical contrast although the present invention has been specifically dis agent is a cell stain selected from : Giemsa stain , Wright closed by preferred embodiments and optional features , stain , Wright- Giemsa stain , May -Grünwald stain , Mallory modification and variation of the concepts herein disclosed trichrome, Periodic acid - Schiff reaction stain , Weigert ' s may be resorted to by those skilled in the art , and that such elastic stain , Heidenhain ' s AZAN trichrome stain , Orcein modifications and variations are considered to be within the stain , Masson ' s trichrome, Alcian blue stain , May -Grün scope of this invention as defined by the appended claims. wald -Giemsa , van Gieson stain , Hansel stain , Reticulin US 2017 /0234874 A1 Aug . 17 , 2017 37

Stain , Gram stain , Bielschowsky stain , Ferritin stain , Fon 24 . The method of claim 22 , wherein the nanoparticles tana -Masson stain , Hales colloidal iron stain , Pentachrome comprising mixtures of the listed metals further comprises stain , Azan stain , Luxol fast blue stain , Golgi' s method discrete shells or layers . ( reduced silver) , reduced gold , Chrome alum /haemotoxylin 25 . The method of claim 22 , wherein the nanoparticles are stain , Isamin blue stain , Argentaffin stains, Warthin - Starry spherical, tubular , cylindrical, pyramidal , cubic , egg - shaped , silver stain , Nissl stain , Sudan Black and osmium stain , t - bone -shaped , urchin - or rose - like (with spiky uneven sur osmium tetroxide stain , hematoxylin stain , Uranyl acetate faces ) or hollow shaped . stain , lead citrate stain , Carmine stain , safranin stain , and 26 . The method of claim 22 , wherein the nanoparticles Ziehl- Neelsen stain . comprising Si have a Si or SiO , shell . 8 . The method of claim 1 , wherein the optical contrast 27 . The method of claim 22 , wherein the nanoparticles agent is a dye r colorant selected from : eosin Y , eosin B , comprising Si have a Au core. azure B , pyronin G , malachite green , toluidine blue , copper 28 . The method of claim 1 , wherein the biomarker phthalocyanin , alcian blue, auramine - rhodamine , acid binding moiety is selected from the following : an antibody fuschin , aniline blue , orange G , acid fuschin , neutral red , or fragment thereof, nanobody, DNA aptamer, DNA oligo Sudan Black B , acridine orange , Oil Red 0 , Congo Red , Fast nucleotide , RNA aptamer, PNA aptamer, peptide aptamer, green FCF, Perls Prussian blue reaction , nuclear fast red , LNA aptamer , carbohydrate , and a lectin . alkaline erythrocin B , and naphthalene black . 29 . The method of claim 1 , wherein the plurality of 9 . The method of claim 1 , wherein the cells contacted with functionalized nanoparticle species is from 2 to 50 different one or a plurality of functionalized nanoparticle species are species of functionalized nanoparticle species . subjected to an external force to increase the local concen 30 . The method of claim 29 , wherein each species of tration of the functionalized nanoparticles and cells . functionalized nanoparticle species is functionalized with a 10 . The method of claim 9 , wherein the external force is different species of biomarker -binding moiety . a gravitational, electric , or magnetic force . 31. The method of claim 30 , wherein each species of 11 . The method of claim 10 , wherein the gravitational functionalized nanoparticle species is functionalized with a force is generated by centrifugation . different antibody. 12 . The method of claim 10 , wherein the magnetic force 32 . The method of claim 31 , wherein the antibody is a is effected by paramagnetic nanoparticles, wherein the core monoclonal or polyclonal antibody , or fragment thereof, or of the nanoparticle comprises a paramagnetic region and the ScFv, or single- domain antibody (nanobody ). shell of the nanoparticle comprises Ag , Au , Pt, Pd , Rh , Ro , 33 . The method of claim 32 , wherein the monoclonal Al, Cu , Ru , Cr, Cd , Zn , Si, Se , SiO , , or mixtures or alloys antibody is an antibody to a cell - surface expressing protein , thereof. protein fragment, protein glycosylation pattern , or protein 13 . The method of claim 9 , wherein charged polymers are carbohydrate . added to the cells after step ( a ). 34 . The method of claim 33 , wherein the monoclonal 14 . The method of claim 1, wherein imaging morphologi antibody is selected from an antibody that binds to : CD1, cal features of the contacted cells comprises measuring an CD2 , CD3 , CD4 , CD5 , CD6 , CD7 , CD8 , CD9, CD10 , optical property of the optical contrast agent . CD11a , CD11b , CD11c , CD13 , CD14 , CD15 , CD16 , CD19 , 15 . The method of claim 14 , wherein an optical property CD20 , CD21, CD22 , CD23 , CD24 , CD25 , CD30 , CD33 , of the optical contrast agent is selected from the following : CD34 , CD38 , CD41, CD43 , CD44 , CD45, CD56 , CD57 , absorbance , scattering , fluorescence , photoluminesence , CD58 , CD61, CD64 , C71, CD79a , CD99 , CD103 , CD117 , Raman emission , and photoluminescent lifetime . CD123 , CD138 , CD138 , CD163 , CD235a , Her - 2 , HLA - DR , 16 . The method of claim 15 , further comprising measur Kappa , Lambda , Pax - 5 , BCL - 2 , Ki- 67 , ZAP -70 , MPO , TDT , ing an optical property of the optical contrast agent under a and FMC -7 . light field illumination with a microscope . 35 . The method of claim 30 , when the functionalized 17 . The method of claim 1 , wherein the illuminating the nanoparticle species is functionalized with an antibody functionalized nanoparticle - cell complexes with evanescent selected from the following an antibody that binds to one of light and detecting the resonant light scattering from each the following : CD3, CD22 , CD79a , Kappa , Lambda , Pax - 5 , observed functionalized complexed nanoparticle is done ZAP -70 , MPO , and TdT ; the functionalized nanoparticle under a dark field illumination with a microscope . species enters the cell and binds to their respective intrac 18 . The method of claim 17, further comprising using an ellular biomarkers. illuminated slide holder to replace the darkfield condenser in 36 . The method of claim 35 , wherein the intracellular the microscope . biomarkers are in the cytosol and / or nucleus , or on the 19 . The method of claim 18 , further the illuminated slide nuclear membrane. holder uses total internal reflection to illuminate the slide 37 . The method of claim 35 , wherein the functionalized holder. nanoparticles are small enough to enter the cell without 20 . Themethod of claim 18 , wherein the illuminated slide disrupting the cell membrane . holder comprises optical fibers to deliver light to the edge to 38 . The method of claim 35 , wherein the functionalized the slide . nanoparticles are smaller than 16 nm . 21. The method of claim 1 , wherein the nanoparticles are 39 . The method of claim 30 , wherein each species of from 10 to 200 nm in diameter . functionalized nanoparticle species is functionalized with a 22 . The method of claim 1 , wherein the nanoparticles are different DNA oligonucleotide . comprised of Ag , Au , Pt, Pd , Rh , Ro, Al, Cu , Ru, Cr, Cd , Zn , 40 . The method of claim 1 , wherein the morphological Si, Se or mixtures or alloys thereof. features comprise the cell surface shape , the cell nucleus 23 . The method of claim 22 , wherein the alloy is an alloy shape , the chromatin shape, the nucleolar shape , the number of Au and Ag. of nucleolus, or combinations of the foregoing . US 2017 /0234874 A1 Aug . 17 , 2017 38

41 . The method of claim 40 , further comprising : 59 . The method of claim 58 , wherein the field of view is ( h ) diagnosing the subject ' s condition based on the bio - from 5 microns by 5 microns to 100 mm by 100 mm . marker -morphological profile of each cell . 60 . The method of claim 58 , wherein imaging morpho 42 . The method of claim 41 , wherein the subject 's con - logical features of the contacted cells in step ( f ) further dition is having a hematological cancer, non -malignant comprises: hematological disorder, solid tumor, kidney disease , bladder ( i) using a software program that processes images of disease , liver disease , or infectious disease . morphological features of each cell in the field of view ; 43 . The method of claim 42 , wherein the hematological ( ii ) moving the field of view digitally ; cancer is selected from : leukemia , lymphoma , and multiple ( iii) using a software program to process images of myeloma. morphological features of each cell in the next field of 44 . The method of claim 42 , wherein the non -malignant view and repeating steps ( ii ) and ( iii ) until the entire hematological disorder is selected from : anemia and sickle substrate area is analyzed ; cell disease . ( iv ) digitally combining all images obtained to generate a 45 . The method of claim 42 , wherein the solid tumor is single image covering the entire selected substrate area ; selected from : breast cancer, lung cancer, prostate cancer , and colorectal cancer , and bladder cancer . (v ) generating from the data obtained for the entire 46 . The method of claim 45 , wherein the solid tumor is substrate area the morphological features of each sub breast cancer and the biomarker is Her2 . strate -adhered cell to detect the biomarker -morphologi 47 . The method of claim 42 , wherein the kidney disease cal profile of each cell . is selected from : acute kidney injury , chronic kidney disease , 61. The method of claim 1 , wherein the method further lupus nephritis , kidney rejection , and preeclampsia . comprises the steps of ( d ) ( 2 ) removing a first plurality of 48 . The method of claim 42 , wherein the infectious functionalized nanoparticles ; and ( d ) ( 3 ) contacting the cells disease is selected from : HIV , hepatitis , sexually transmitted with a second plurality of functionalized nanoparticle spe diseases, and sepsis . cies . 49 . The method of claim 42 , wherein the hematological 62 . The method of claim 61, wherein the method further cancer further comprises circulating cancer cells . comprises after contacting the cells with a second plurality 50 . The method of claim 42 , wherein the subject' s con of functionalized nanoparticle species , ( d ) ( 4 ) removing the dition is further identified by the lineage of the malignancy. second plurality of functionalized nanoparticles ; ( d ) (5 ) con 51 . The method of claim 50 , wherein the lineage of the tacting the cells with a third plurality of functionalized malignancy is negative ,Myeloid line, Lymphoid T cell line, nanoparticle species; and , optionally (d )( 6 ) removing the or Lymphoid B cell line . previous plurality of functionalized nanoparticle species ; 52 . The method of claim 1 , wherein the cells are white and ( d ) ( 7 ) contacting the cells with a next plurality of blood cells . functionalized nanoparticles , and optionally , repeating steps 53 . Themethod of claim 1 ,wherein at least 50 % of the red ( d ) (6 ) and ( d ) ( 7 ) an number of times from none to ten . blood cells are removed before contacting the cells with 63 . The method of claim 61 or 62 , wherein the removing plurality of functionalized nanoparticle species . a first plurality of functionalized nanoparticles is achieved 54 . The method of claim 1 , wherein obtaining the bio by cleaving a cleavable linker between each species of marker signature further comprises counting the number or functionalized nanoparticle and each species of a function proportion of each of the functionalized nanoparticle species alized nanoparticle -associated biomarker -binding moiety . per cell. 64. The method of claim 61 , wherein the removing a first 55 . The method of claim 54 , wherein the number or plurality of functionalized nanoparticles is achieved by proportion of cells having identified normal or abnormal displacing the first plurality of functionalized nanoparticles morphological profiles in the sample are totaled . from the biomarker binding moieties . 56 . The method of claim 1 , wherein the adhering the 65 . The method of claim 62 , wherein the removing the functionalized nanoparticle cell complexes to a substrate second or previous plurality of functionalized nanoparticles further comprises adding a mountant. is achieved by displacing the second or previous plurality of 57 . The method of claim 56 , wherein the volume of the functionalized nanoparticles from the biomarker binding mountant is 2 microliters . moieties . 58 . The method of claim 55 , where detecting the resonant 66 . The method of claim 63 , wherein the linker between light scattering from each observed complexed functional each nanoparticle species in the first plurality of function ized nanoparticle in step ( d ) further comprises: alized nanoparticles and its respective biomarker binding ( i ) using a software program that counts the number of moiety comprises a first oligonucleotide bound to a first each of the functionalized nanoparticle species per cell nanoparticle species and a second oligonucleotide bound to and processes images in each cell in the field of view ; its respective biomarker binding moiety , wherein the second (ii ) moving the field of view digitally ; oligonucleotide comprises a portion complementary to a ( iii ) using a software program to count the number of each portion of the first oligonucleotide and hybridization of the of the functionalized nanoparticle species per cell in the first oligonucleotide to the second oligonucleotide forms a next field of view and repeating steps ( ii ) and ( iii ) until linker comprising a double -stranded nucleic acid . the entire substrate area is analyzed ; 67 . The method of claim 66 , wherein the linker between ( iv ) digitally combining all images obtained to generate a each functionalized nanoparticle species in the second or single image covering the entire substrate area ; and next plurality of functionalized nanoparticles and its respec ( v ) generating from the data obtained for the entire tive biomarker binding moiety comprises a second or next substrate area the biomarker signature of each sub oligonucleotide bound to a second or next functionalized strate - adhered cell . nanoparticle species, respectively , and a third or next oligo US 2017 /0234874 A1 Aug . 17 , 2017 39

nucleotide bound to its respective biomarker binding moiety , polyethylene glycol (PEG ) , amino - functionalized dextran , wherein the third or next oligonucleotide comprises a por gold , silver , alumina , or glass silica . tion complementary to a portion of the second or next 84 . The method of claim 1 , wherein the sample is from oligonucleotide and hybridization of the second or next blood , bone marrow , fine needle aspirate , or tissue . oligonucleotide to the third or next oligonucleotide forms a 85 . The method of claim 84 , wherein the tissue sample is linker comprising a double - stranded nucleic acid . FFPE ( formalin - fixed , paraffin -embedded ) tissue samples. 68 . The method of claim 64 , wherein each functionalized 86 . The method of claim 84 , wherein when the sample is nanoparticle species is displaced from its respective bio tissue, the optical contrast agent is H & E (hematoxylin and marker binding moiety by binding of a third oligonucleotide eosin ) stain . to the first oligonucleotide wherein the hybrid formed by 87 . A method for detecting the biomarker- morphological hybridization of the third oligonucleotide and the first oli gonucleotide exhibits a melting temperature higher than the profile of a cell , the method comprising : melting temperature of the double -stranded nucleic acid ( a ) providing a sample comprising cells from a subject ; formed by hybridization of the first and second oligonucle ( b ) contacting the cells with one or a plurality of func otide . tionalized nanoparticle species, each functionalized 69 . The method of claim 68 , where the steps ( b ) - ( d ) are nanoparticle species comprising a biomarker -binding repeated up to one, two , three , four , five , six , seven , eight, moiety , and forming nanoparticle -cell complexes nine, ten , fifteen , twenty , thirty , forty, or fifty or more times . through binding of the nanoparticle species comprising 70 . The method of claim 65 , wherein after displacing the a biomarker - binding moiety to its respective bio second or previous plurality of functionalized nanoparticles marker ; from the biomarker binding moieties , the following steps are (c ) adhering the functionalized nanoparticle -cell com performed : plexes to a substrate; ( i) the biomarker - binding moieties of the functionalized ( d ) illuminating the functionalized nanoparticle - cell com nanoparticles which bound to the cell are associated plexes with non - evanescent light and detecting the with the biomarker binding moiety functionalized resonant light scattering from each observed com nanoparticle is classified , and plexed nanoparticle, to obtain a biomarker signature of ( ii ) the cells are contacted with a next plurality of nano each observed cell ; particles functionalized with different biomarker bind ( e ) contacting the substrate - adhered cells with an optical ing moieties , and each functionalized nanoparticle spe contrast agent ; cies of the next plurality of nanoparticles are ( f) imaging morphological features of the contacted cells ; functionalized with different biomarker binding moi and eties that binds to a biomarker which is suspected of ( g ) associating the morphological features of the con being associated with samples in which the first bio tacted cells with the biomarker signature of each sub marker is present. strate - adhered cell to detect the biomarker -morphologi 71 . The method of claim 1, wherein the cells may be the cal profile of each cell . same type or different types from each other . 88 . The method of claim 87 , wherein the non -evanescent 72 . The method of claim 71 , wherein the cells are different light is transmitted light. types from each other . 89 . A method for detecting the biomarker -morphological 73 . The method of claim 72 , wherein the cells can be from profile of a cell , the method comprising : different conditions. ( a ) providing a sample comprising cells from a subject; 74 . The method of claim 73 , wherein the different con ( b ) adhering the cells to a substrate ; ditions are selected from : having a hematological cancer , ( c ) contacting the substrate - adhered cells with an optical non -malignant hematological disorder, solid tumor, bladder contrast agent ; disease , liver disease , kidney disease , or infectious disease . ( d ) imaging morphological features of the contacted cells ; 75 . The method of claim 74 , wherein the different con ( e ) converting the optical contrast agent to a colorless ditions are having a different type of solid tumor. form ; 76 . The method of claim 1 , wherein the biomarker ( f) contacting the cells with one or a plurality of func binding moiety is anti- CD45 , and the biomarker signature tionalized nanoparticle species , each functionalized obtained is the white blood cell count. nanoparticle species comprising a biomarker -binding 77 . The method of claim 1, wherein the cells are live . moiety to form nanoparticle - cell complexes ; 78 . The method of claim 1 , wherein the cells are fixed . ( g ) illuminating the nanoparticle -cell complexes with 79 . The method of claim 78 , wherein the cells are fixed evanescent light and detecting the resonant light scat with formaldehyde . tering from each observed complexed nanoparticle , to 80 . The method of claim 1 , wherein the substrate is obtain a biomarker signature of each observed cell; and selected from : glass silica , clear polymer , gold , or alumina . ( h ) associating the morphological features of the con 81 . The method of claim 1 , wherein the substrate is tacted cells with the biomarker signature of each sub functionalized . strate - adhered cell to detect the biomarker- morphologi 82 . The method of claim 81 , wherein the substrate func cal profile of each cell . tionalization is patterned . 90. The method of claim 89 , wherein the optical contrast 83. The method of claim 82, wherein the functionalization agent is a leuco dye . is a silane - linked cell biomarker , polymer - linked cell bio 91 . The method of claim 90 , wherein the leuco dye is marker, silane - linked amine , silane - linked carboxylic acid , methylene blue, methylene green , red leuco dye , crystal polymer- linked amine, polymer -linked carboxylic acid , violet, phenolphthalein , or thymolphthalein . US 2017 /0234874 A1 Aug . 17 , 2017 40

92 . The method of claim 90 , wherein the leuco dye is moiety , wherein an external force is used to accelerate converted to a colorless form by the addition of one ormore the formation of nanoparticle - cell complexes through electrons to the dye . binding of the nanoparticle species comprising a bio 93 . The method of claim 91, wherein the leuco dye is marker- binding moiety to its respective biomarker ; converted to a colored form by the removal of one or more ( c ) adhering the functionalized nanoparticle -cell com electrons from the dye . plexes to a substrate; 94 . A kit for the detection of a biomarker signature , the ( d ) detecting the functionalized nanoparticle cell com combination comprising a plurality of biomarker- binding plexes by illuminating the nanoparticle - cell complexes moiety functionalized nanoparticle species . with evanescent light and detecting the resonant light 95 . The kit of claim 94 , wherein the functionalized scattering from each observed functionalized nanopar nanoparticle species further comprise: ticle cell complex , to obtain a biomarker signature of ( a ) a nanoparticle species bound to a first oligonucleotide ; each observed cell ; and and ( e ) associating the biomarker signature of each substrate ( b ) a biomarker -binding moiety bound to a second oligo adhered cell to a known disease , condition , or state of nucleotide , a cell exhibiting substantially the same biomarker sig wherein the first oligonucleotide is complementary to a nature to identify the disease , condition , or state of the portion of the second oligonucleotide , and the first and cell from a subject. second oligonucleotide form a hybridized duplex . 108 . A homogeneous assay for detecting functionalized 96 . The kit of claim 94 , wherein the functionalized nanoparticle species further comprise : nanoparticle cell complexes, the assay comprising : ( a ) a nanoparticle functionalized with a first oligonucle (a ) providing a sample comprising cells from a subject ; otide; ( b ) contacting the cells with one or a plurality of func ( b ) a biomarker - binding moiety functionalized with a tionalized nanoparticle species, each functionalized second oligonucleotide ; and nanoparticle species comprising a biomarker - binding a third oligonucleotide , moiety , and forming functionalized nanoparticle -cell wherein the first oligonucleotide is complementary to a complexes through binding of the nanoparticle species portion of the third oligonucleotide , the second oligonucle comprising a biomarker -binding moiety to its respec otide is complementary to a separate portion of the third tive biomarker ; oligonucleotide , and the first and second oligonucleotides ( c ) adhering the functionalized nanoparticle - cell com form a hybridized duplex to the third oligonucleotide. plexes to a substrate; 97. A kit for the detection of a biomarker morphological ( d ) illuminating the functionalized nanoparticle -cell com profile , the combination comprising a plurality of function plexes with evanescent light and detecting the resonant alized nanoparticle species and an optical contrast agent. light scattering from each observed complexed nano 98 . The kit of any one of claim 94 or 97 , wherein the particle , to obtain a biomarker signature of each plurality of functionalized nanoparticle species comprise a observed cell, mixture . wherein unbound functionalized nanoparticles are not 99 . The kit of any one of claim 94 or 97 , wherein the removed from the field of view . plurality of functionalized nanoparticle species are segre 109. A method for detecting functionalized nanoparticle gated before use . cell complexes , the method comprising : 100 . A kit comprising a plurality of functionalized nano ( a ) providing a sample comprising cells from a subject ; particle species , and a mountant . ( b ) contacting cells which have been fixed with one or a 101 . The kit of claim 100 , wherein the mountant has a plurality of functionalized nanoparticle species, each refractive index ofwithin 0 . 1 of the refractive index of fixed functionalized nanoparticle species comprising a bio cells . marker- binding moiety , and forming nanoparticle -cell 102 . The method of claim 56 , wherein the mountant has complexes through binding of the nanoparticle species a refractive index of within 0 . 1 of the refractive index of comprising a biomarker - binding moiety to its respec fixed cells . tive biomarker ; 103 . The kit of any one of claims 100 - 102 , further ( c ) adhering the nanoparticle - cell complexes to a sub comprising an optical contrast agent . strate , wherein the adhered functionalized nanopar 104 . A method for increasing the loading amount of a ticle - cell complexes are placed in contact with a moun functionalized nanoparticle onto a cell by using an external tant, wherein the refractive index of the mountant is force to increase the local concentration of the nanoparticles within about 0 .1 of the refractice index of the fixed and cells. cells ; 105 . The method of claim 104 , wherein the external force ( d ) detecting the functionalized nanoparticle cell com is a centrifugal, electrical or magnetic force . plexes by illuminating the nanoparticle - cell complexes 106 . The method of claim 1 , wherein the plurality of with evanescent light and detecting the resonant light nanoparticles exhibit a peak resonance wavelength of the scattering from each observed functionalized nanopar nanoparticle plasmon resonance between 400 to 900 nm . ticle cell complex, to obtain a biomarker signature of 107 . A method for detecting functionalized nanoparticle each observed cell; and cell complexes, the method comprising : ( e) associating the biomarker signature of each substrate (a ) providing a sample comprising cells from a subject; adhered cell to a known disease , condition , or state of ( b ) contacting the cells with one or a plurality of func a cell exhibiting substantially the same biomarker sig tionalized nanoparticle species, each functionalized nature to identify the disease , condition , or state of the nanoparticle species comprising a biomarker- binding cell from a subject . US 2017 /0234874 A1 Aug . 17 , 2017 41

110 . The method of claim 109, wherein the mountant has lished between the reference cell biomarker signature an refractive index of from 1 .51 to 1 . 54 . and a disease , disorder, condition or state of the refer 111 . A method for detecting functionalized nanoparticle ence subject. cell complexes to obtain a biomarker signature, the method 112 . The method of any one of claim 1 , 87 , 89 , 104 , 108 , 109 , or 111 , wherein the steps of providing a sample comprising: comprising cells from a subject, contacting the cells with (a ) providing a sample comprising cells from a subject; one or a plurality of functionalized nanoparticle species, and (b ) contacting the cells with one or a plurality of func adhering the functionalized nanoparticle - cell complexes to a tionalized nanoparticle species , each functionalized substrate , are performed by an automated liquid handling nanoparticle species comprising a biomarker -binding system . moiety , and forming nanoparticle -cell complexes 113 . The method of any one of claim 107, 108, 109 , or through binding of the nanoparticle species comprising 111 , further comprising obtaining and storing the positional a biomarker -binding moiety to its respective bio information for each observed cell is stored . marker ; 114 . The method of any one of claim 1 , 87 , or 89 , further ( c ) adhering the functionalized nanoparticle -cell com comprising obtaining and storing the positional information plexes to a substrate ; for each observed and imaged cell is stored . ( d ) illuminating the functionalized nanoparticle -cell com 115 . The method of claim 1 , wherein the nanoparticle plexes with evanescent light and detecting the resonant preparations are of a narrow size distribution , such that an light scattering from each observed complexed nano individual nanoparticle preparation has a scattering spec particle , to obtain a biomarker signature of each trum whose full -width half maximum ranges from 5 to 150 observed cell ; and nm . ( e ) associating the biomarker signature of an imaged cell 116 . The method of claim 1 , wherein the functionalized from the subject with a biomarker signature of a nanoparticle is present on the cell surface . reference cell exhibiting substantially the same bio 117 . The method of claim 1 , wherein the functionalized marker signature as the imaged cell biomarker signa nanoparticle is present within the cell . ture , wherein a diagnostic concordance has been estab * * * * *