Device and method for single cell capture and analysis

Abstract

The present invention provides devices and methods for single-cell capture and analysis from complex solutions. The method includes the preparation of substrates for the capture of single cells for multiplexed analysis.

Description

[0001] Device and Method for Single Cell Capture and Analysis

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 639,051, filed April 26, 2024, which is hereby incorporated by reference in its entirety.

[0004] BACKGROUND OF THE INVENTION

[0005] Traditional “single-cell” analysis has a large number of drawbacks that make its utilization unpractical. The majority of “single-cell” analyses do not actually involve the isolation and analysis of a single cell, but rather rely on bioinformatic arguments that the methodology utilized produces results that are for a single cell. Additionally, current single-cell analytical methods and instrumentation limit the analysis to only a single type (e.g., genomics, transcriptomics, proteomics, metabolomics, etc.).

[0006] Accordingly, there is a need in the art for a means of performing multiple analyses in a truly single-cell manner. The present invention addresses this long felt, but unmet need.

[0007] SUMMARY OF THE INVENTION

[0008] In some embodiments, the present disclosure provides a single-cell capture array comprising: a) a substrate; and b) a plurality of cell-binding molecule spots on the substrate, wherein the cell-binding molecule spots are between 1 pm and 100 pm in diameter and wherein the distance between each spot is between 10 pm and 500 pm. In some embodiments, the cell binding molecules are antibodies or lectins. In some embodiments, the cell-binding molecule spots are in a square grid and the distance between each spot is between 10 and 500 pm. In some embodiments, the cell-binding molecule spots are between 1 pm and 50 pm in diameter. In some embodiments, the substrate is selected from the group consisting of glass, polydimethylsiloxane (PDMS), oxygen plasma-treated PDMS, epoxysilane-coated glass,, and amine-reactive hydrogel- coated glass.

[0009] In some embodiments, all cell-binding molecules in a single cell-binding molecule spot bind a single antigen. In some embodiments, the array of cell-binding molecules spots comprises a plurality of spots against one or more antigens, wherein the cell-binding molecules in each spot bind a single antigen. In some embodiments, a cell-binding molecule spot comprises two or more cell-binding molecules that bind two or more antigens.

[0010] In some embodiments, the present disclosure provides a method of analyzing the profile of single cell comprising the steps of a) providing a single-cell capture array of any one of the present invention; b) incubating the single-cell capture array with a sample containing the cell to be analyzed; c) rinsing the single-cell capture array to remove unbound cells, leaving only a single cell on each antibody spot; d) leaving the cells unfixed or fixing the cells; e) detecting single cells through an algorithm which identifies and marks single cells; and f) scanning the single-cell capture array by mass spectrometry.

[0011] In some embodiments, the mass spectrometry is selected from the group consisting of: matrix-assisted laser desorption / ionization imaging Fourier transform ion cyclotron resonance

[0012] (MALDI-FTICR) mass spectrometry, matrix-assisted laser desorption / ionization time of flight

[0013] (MALDI-TOF) mass spectrometry, scanning microprobe MALDI (SMALDI) mass spectrometry, infrared matrix assisted laser desorption electrospray ionization (MALD-ESI) mass spectrometry, surface-assisted laser desorption / ionization (SALDI) mass spectrometry, desorption electrospray ionization (DESI) mass spectrometry, secondary ion mass spectrometry (SIMS) mass spectrometry, and easy ambient sonic spray ionization (EASI) mass spectrometry.

[0014] In some embodiments, the step of scanning the single-cell capture array is preceded by a step of spraying the single-cell capture array with a MALDI matrix material. In some embodiments, the MALDI matrix solution is selected from the group consisting of: 2,5dihydroxybenzoic acid, a-cyano-4-hydroxycinnamic acid, sinapinic acid, l,5diaminonaphthalene, and 9-aminoacridine.

[0015] In some embodiments, the step of fixing the cells is preceded by spraying the single-cell capture array with an enzymatic solution. In some embodiments, the enzymatic solution comprises one or more selected from the group consisting of: glycolytic enzymes, lipases, proteases, peptidases, nucleases, and amylases.

[0016] In some embodiments, step e) comprises scanning the single-cell capture array for one or more profiles selected from the group consisting of: a genomic profile, a proteomic profile, a glycomic profile, a lipidomic profile, a metabolomic profile, and a transcriptomic profile. In some embodiments, the present disclosure provides a kit for analyzing the profile of a single cell from a sample comprising: a single-cell capture array of any one of claims 1-7; and a MALDI matrix material. In some embodiments, the MALDI matrix material is a-cyano- 4hydroxycinnamic acid. In some embodiments, the kit further comprises an enzymatic solution. In some embodiments, the kit further comprises a non-transitory computer-readable medium with instructions stored thereon, which when executed by a processor, perform steps comprising identifying single cells.

[0017] In some embodiments, the present disclosure provides a kit for analyzing the profile of a single cell from a sample comprising: a substrate; a stamp; a blocking protein; and a MALDI matrix material. In some embodiments, the kit further comprises an antibody. In some embodiments, the blocking protein is bovine serum albumin (BSA). In some embodiments, the MALDI matrix material is a-cyano-4-hydroxycinnamic acid. In some embodiments, the kit further comprises a non-transitory computer-readable medium with instructions stored thereon, which when executed by a processor, perform steps comprising identifying single cells. In some embodiments, the kit further comprises an enzymatic solution. In some embodiments, the enzymatic solution comprises one or more selected from the group consisting of: glycolytic enzymes, lipases, proteases, peptidases, nucleases, and amylases.

[0018] DETAILED DESCRIPTION OF THE DRAWINGS

[0019] The following detailed description of exemplary embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, shown in the drawings are exemplary embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0020] Figure 1 depicts an illustrative computer architecture for a computer 100 for practicing the various embodiments of the invention.

[0021] Figure 2, comprising Figure 2A through Figure 2E, depicts the single cell capture workflow. Figure 2A depicts PDMS stamps with pillars. Figure 2B depicts PDMS stamps incubated with capture protein solution, allowing protein to bind via electrostatic interactions. Once “inked” the stamp is pressed onto a hydrogel slide where the capture protein on the PDMS to covalently bind to the slide. Once removed micro capture spots of protein are left, and the slide is blocked. Figure 2C depicts multi-well chambers attached to the slide allowing for multiple samples per slide. Figure 2D depicts cell suspension added to each well allowing for single cell capture. Figure 2E depicts single cell capture once well chambers are removed and excess cell suspension washed away and cells are fixed, leaving an array of captured single cells.

[0022] Figure 3, comprising Figure 3 A through Figure 3E, depicts representative images and schematic representations illustrating single-cell capture and spatial configuration for MALDI imaging applications. Figure 3 A depicts a human T cell approximately 8-10 pm in diameter immobilized on a capture spot measuring approximately 30-40 pm in diameter. Figure 3B depicts the arrangement of capture spots spaced at intervals ranging from 60 to 240 pm, designed to facilitate single-cell MALDT imaging without spatial interference. Figure 3C depicts a single laser burst, 60-80 pm in diameter, applied to each individual cell with no overlap from adjacent ablation regions. Figure 3D depicts concanavalin A (ConA)-mediated capture of single Jurkat cells using a polydimethylsiloxane (PDMS) stamp with pillars measuring 30 pm in diameter, 60 pm center-to-center spacing, and 40 pm in height, with an associated matrix image showing 60 pm2ablation marks centered over each single cell. Figure 3E depicts single Jurkat cells captured using a PDMS stamp with pillars measuring 40 pm in diameter, 240 pm spacing, and 40 pm in height, as well as a PDMS stamp with pillars measuring 30 pm in diameter, 60 pm spacing, and 40 pm in height, with matrix images demonstrating 80 pm2ablation marks accurately overlaid on each single cell.

[0023] Figure 4, comprising Figure 4A through Figure 4B, depicts the use of capture proteins for single-cell immobilization. Figure 4A depicts the use of antibodies or lectins as capture proteins to selectively bind target cells. Figure 4B depicts anti-CD7 capturing single Jurkat cells, and concanavalin A (ConA) capturing single Jurkat, 4T1, and peripheral blood mononuclear cells (PBMCs), all using a polydimethylsiloxane (PDMS) layout with pillars measuring 30 pm in diameter, 60 pm center-to-center spacing, and 40 pm in height.

[0024] Figure 5 depicts a representative brightfield image of cells captured in a cell capture array produced by direct antibody stamping.

[0025] Figure 6 depicts the SoloCell workflow for automated detection and classification of single cells in a patterned array. Figure 6A depicts user selection of an area on the slide. Figure 6B depicts the manual marking of 10 cells: 8 adjacent cells (colored purple) used to calculate the average distance between adjacent cells, and 2 additional cells (colored blue) that are the farthest apart while remaining in the same column or row, used to determine the grid angle. Figure 6C depicts SoloCell generating the first seeding frame based on the calculated distance and angle, then proceeding to the next position using these parameters. Because the grid may not be perfectly uniform, the next frame searches a nearby dashed region to locate the darkest area using a contour detection algorithm. Figure 6D depicts SoloCell centering the darkest region in the frame and iteratively continuing this process across the array. Figure 6E depicts this operation being repeated in four directions to determine the full grid's column and row positions. Figure 6F depicts SoloCell creating the complete frame set across the grid. Figure 6G depicts each frame being evaluated by a pretrained convolutional neural network (CNN) to classify whether it contains a solo cell. Figure 6H depicts an interactive histogram interface that enables the user to adjust the confidence threshold in real time, dynamically updating the count of solo cells to balance confidence and cell yield. Figure 61 depicts the outputs generated by SoloCell, including the original image with detected solo cells, a coordinate list of solo and non-solo cell groups, the histogram, and a log file.

[0026] Figure 7 depicts a representative convolutional neural network (CNN) architecture used to identify single cells for analysis.

[0027] Figure 8 depicts representative software used to identify single cells for analysis using HSV (Hue, Saturation, Value) thresholds for contour detection.

[0028] Figure 9 depicts the use of software that identifies, and marks single cells captured by a single cell capture array for further analysis.

[0029] Figure 10 depicts the use of a poly dimethylsiloxane (PDMS) stamp with pillar dimensions of 30 pm in diameter, 60 pm spacing, and 40 pm height to pattern a hydrogel-coated slide. Figure 15 also depicts a 24-well module placed over the slide to enable cell incubation and capture. A total of 2,223 single cells were captured within a 5 mm * 7 mm area and imaged in under 6 minutes.

[0030] Figure 11 depicts sequential lipid and N-glycan spectra acquired from a single Jurkat cell. Figure 11A depicts the single-cell lipid spectrum highlighting a peak at m / z 760.589. Figure 11B depicts the single-cell N-glycan spectrum highlighting a peak at m / z 2539.896. The same Jurkat cell was analyzed in both Figure 11 A and Figure 1 IB. Figure 12 depicts the average single-cell lipid and N-glycan signals across 3,250 single cells, comprising 40 N-glycans and 273 lipids, compared to corresponding lipid and N-glycan signals obtained from bulk cell analysis. R-squared values were calculated at 0.95 for lipids and 0.93 for N-glycans.

[0031] Figure 13, comprising Figure 13A through Figure 13C, depicts sequential brightfield images of the same cells after multiple omic measurements. Figure 13A depicts cells post fixation. Figure 13B depicts cells post lipid imaging, Carnoy’s washes and antigen retrieval. Figure 13C depicts cells post N-glycan imaging after matrix was washed away.

[0032] Figure 14, comprising Figure 14A through Figure 14C, depicts the capture of Jurkat and 4T1 cells using different capture formats. Figure 14A depicts co-capture of 4T1 and Jurkat cells using a polydimethyl siloxane (PDMS) stamp with pillars measuring 40 pm in diameter, 240 pm spacing, and 40 pm in height. Figure 14B depicts bulk capture of 4T1 cells. Figure 14C depicts bulk capture of Jurkat cells.

[0033] Figure 15, comprising Figure 15A through Figure 15D, depicts single-cell lipid validation across co-captured and bulk-captured 4T1 and Jurkat cells. Figure 15A depicts a volcano plot showing the log2 fold change of average percent relative intensity between bulk 4T1 and Jurkat cells versus the -logw P value from a two-tailed independent t-test, identifying lipids with a twofold or greater difference out of 134 analyzed lipids. Figure 15B depicts ion images of lipids at m / z 692.559 and 608.392 that are differentially expressed between the two cell types. Figure 15C depicts a UMAP visualization of 1,214 co-captured single cells alongside bulk-captured 4T1 and Jurkat cells based on percent relative intensity values of 134 lipids. Figure 15D depicts the correlation between single-cell and bulk lipid data, showing relative lipid levels expressed as 4T1 / Jurkat ratios of percent relative intensity, with a Pearson correlation coefficient indicating strong agreement between the datasets.

[0034] Figure 16, comprising Figurel6A through Figure 16D, depicts single-cell N-glycan validation across co-captured and bulk-captured 4T1 and Jurkat cells. Figure 16A depicts a volcano plot showing the log2 fold change of average percent relative intensity between bulk 4T1 and Jurkat cells versus the -logw P value from a two-tailed independent t-test, identifying N- glycans with a two-fold or greater difference out of 39 analyzed N-glycans. Figure 16B depicts ion images of N-glycans at m / z 2539.899 and 1971 .697 that are differentially expressed between the two cell types. Figure 16C depicts a UMAP visualization of 1,214 co-captured single cells alongside bulk-captured 4T1 and Jurkat cells based on percent relative intensity values of 39 N- glycans. Figure 16D depicts the correlation between single-cell and bulk N-glycan data, showing relative N-glycan levels expressed as 4T1 / Jurkat ratios of percent relative intensity, with a Pearson correlation coefficient indicating strong agreement between the datasets.

[0035] Figure 17 depicts the UMAP visualization of co-captured 4T1 and Jurkat single-cell lipid data. 4T1 cells are colored in red based on N-glycan classification, highlighting the distinct lipid profiles between the two cell types.

[0036] Figure 18 depicts a peak list of lipids detected on 4T1 and Jurkat cells, showing the specific lipid species identified in both cell types.

[0037] Figure 19 depicts a peak list of N-glycans detected on 4T1 and Jurkat cells, showing the specific lipid species identified in both cell types.

[0038] Figure 20 depicts representative images of a BSA-blocked slide before (left) and after (right) heat denaturation for reverse stamping, and an enlarged image demonstrating the negative space resulting (bottom).

[0039] Figure 21 depicts a representative image of a heat-treated BSA-blocked slide demonstrating approximately 100 pm spacing between negative-space holes with a diameter of approximately 5 pm.

[0040] DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention provides methods and compositions for comprehensive analysis of single cells (“omni-omics”), including those in complex solutions such as a biological sample. The method includes the preparation of substrates for the capture of cells for multiplexed analysis. The invention further relates to the use of single-cell analysis in the diagnosis and screening of disease states and disease progression.

[0042] Definitions

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0044] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0045] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0046] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass non-limiting variations of ±40% or ±20% or ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate.

[0047] The term “abnormal” when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the “normal” (expected) respective characteristic. Characteristics that are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.

[0048] The terms “biomarker” and “marker” are used herein interchangeably. They refer to a substance that is a distinctive indicator of a biological process, biological event and / or pathologic condition.

[0049] The phrase “body sample” or “biological sample” is used herein in its broadest sense. A sample may be of any biological tissue or fluid from which biomarkers of the present invention may be assayed. Examples of such samples include but are not limited to blood, saliva, buccal smear, feces, lymph, urine, gynecological fluids, biopsies, amniotic fluid, and smears. Samples that are liquid in nature are referred to herein as “biofluids.” Body samples may be obtained from a patient by a variety of techniques including, for example, by scraping or swabbing an area or by using a needle to aspirate biofluids. Methods for collecting various body samples are well known in the art. Frequently, a sample will be a “clinical sample,” i.e., a sample derived from a patient. Such samples include, but are not limited to, biofluids which may or may not contain cells, e.g., blood (e.g., whole blood, serum, or plasma), urine, saliva, tissue or fine needle biopsy samples, and archival samples with known diagnosis, treatment, and / or outcome history. Biological or body samples may also include sections of tissues such as frozen sections taken for histological purposes. The sample also encompasses any material derived by processing a biological or body sample. Derived materials include, but are not limited to, cells (or their progeny) isolated from the sample, proteins or nucleic acid molecules extracted from the sample. Processing of a biological or body sample may involve one or more of: filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like.

[0050] As used herein, the term “carbohydrate” is intended to include any of a class of aldehyde or ketone derivatives of polyhydric alcohols. Therefore, carbohydrates include starches, celluloses, gums, and saccharides. Although, for illustration, the term “saccharide” or “glycan” is used elsewhere herein, this is not intended to be limiting. It is intended that the methods provided herein can be directed to any carbohydrate, and the use of a specific carbohydrate is not meant to be limiting to that carbohydrate only.

[0051] As used herein, the term “cell-surface glycoprotein” refers to a glycoprotein, at least a portion of which is present on the exterior surface of a cell. In some embodiments, a cell-surface glycoprotein is a protein that is positioned on the cell-surface such that at least one of the glycan structures is present on the exterior surface of the cell.

[0052] In the context of the present invention, the term “control,” when used to characterize a subject, refers, by way of non-limiting examples, to a subject that is healthy, to a patient that otherwise has not been diagnosed with a disease. The term “control sample” refers to one, or more than one, sample that has been obtained from a healthy subject or from a non-disease tissue such as normal colon.

[0053] The term “control or reference standard” describes a material comprising none, or a normal, low, or high level of one of more of the marker (or biomarker) expression products of one or more the markers (or biomarkers) of the invention, such that the control or reference standard may serve as a comparator against which a sample can be compared.

[0054] “Differentially increased levels” refers to biomarker levels which are at least 1%, 2%, 3%, 4%, 5%, 10% or more, for example, 5%, 10%, 20%, 30%, 40%, or 50%, 60%, 70%, 80%,

[0055] 90% higher or more, and / or 0.5-fold, 1.1 -fold, 1.2-fold, 1.4-fold, 1.6-fold, 1 8-fold higher or more, as compared with a control. “Differentially decreased levels” refers to biomarker levels which are at least at least 1%, 2%, 3%, 4%, 5%, 10% or more, for example, 5%, 10%, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% lower or less, and / or 0.9-fold, 0.8-fold, 0.6-fold, 0.4-fold, 0.2-fold, 0.1-fold or less, as compared with a control.

[0056] A “disease” is a state of health of a living organism animal wherein the organism cannot maintain homeostasis, and wherein if the disease is not ameliorated then the organism’s health continues to deteriorate. In contrast, a “disorder” in an living organism is a state of health in which the organism is able to maintain homeostasis, but in which the organism’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the organism’s state of health. Cells or tissues of an organism may similarly be referred to with a disease or disorder when such cells or tissues are unable to maintain homeostasis or optimal conditions compared to a cell or tissue not afflicted with the disease or disorder. While cells or tissues with a disease or disorder may be obtained directly from an organism with the disease or disorder, they may also be normal cells or tissue induced to have a disease or disorder by external means.

[0057] A disease or disorder is “alleviated” if the severity of a sign or symptom of the disease, or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.

[0058] The terms “effective amount” and “pharmaceutically effective amount” refer to a sufficient amount of an agent to provide the desired biological result. That result can be reduction and / or alleviation of a sign, symptom, or cause of a disease or disorder, or any other desired alteration of a biological system. An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0059] As used herein “endogenous” refers to any material from or produced inside the organism, cell, tissue, or system.

[0060] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0061] The “level” of one or more biomarkers means the absolute or relative amount or concentration of the biomarker in the sample. The term “level” also refers to the absolute or relative amount of glycosylation of the biomarker in the sample. As is known in the art and used herein “glycans” are sugars (e.g., oligosaccharides and polysaccharides). Glycans can be monomers or polymers of sugar residues typically joined by glycosidic bonds also referred to herein as linkages. In some embodiments, the terms “glycan,” “oligosaccharide,” and “polysaccharide” may be used to refer to the carbohydrate portion of a glycoconjugate (e.g., glycoprotein, glycolipid, or proteoglycan). A glycan may include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'fluororibose, 2'-deoxyribose, phosphomannose, 6'-sulfo N-acetylglucosamine, etc.). The term “glycan” includes homo and heteropolymers of sugar residues. The term “glycan” also encompasses a glycan component of a glycoconjugate (e.g., of a glycoprotein, glycolipid, proteoglycan, etc.). The term also encompasses free glycans, including glycans that have been cleaved or otherwise released from a glycoconjugate.

[0062] As is known in the art, and used herein, “lipids” includes a wide range of organic molecules, including fats, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, phospholipids, glycolipids, and other structures with hydrophobic tails greater than four carbons in length. The functions of lipids may include energy storage, signaling, and structural support of cell membranes.

[0063] As used herein, the term “antibody array” refers to a tool used to identify capture a wide variety of molecules, including proteins, glycans, lipids, and proteoglycans, that interact with any of a number of different antibodies linked to the array substrate. In some embodiments, antibody arrays comprise a number of immobilized antibodies, referred to herein as “antibody spots.” In some embodiments, antibody arrays comprise at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 350, at least 1000 or at least 1500 antibody spots. In some embodiments, antibody arrays may be customized to present a desired set of antibody spots. Similar arrays may comprise any affinity molecules, including, aptamers, lectins, monobodies, chemical compounds, and any other material with an affinity to a specific protein or macromolecular structure.

[0064] “Measuring” or “measurement,” or alternatively “detecting” or “detection,” means assessing the presence, absence, quantity or amount (which can be an effective amount) of either a given substance within a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise evaluating the values or categorization of a subject’s clinical parameters.

[0065] As used herein, “nucleic acid” is meant as any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil). The term “nucleic acid” typically refers to large polynucleotides.

[0066] Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5’-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5’-direction.

[0067] The direction of 5’ to 3’ addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the “coding strand;” sequences on the DNA strand that are located 5’ to a reference point on the DNA are referred to as “upstream sequences”; sequences on the DNA strand which are 3’ to a reference point on the DNA are referred to as “downstream sequences.”

[0068] The term “pre-cancerous” or “pre-neoplastic” and equivalents thereof shall be taken to mean any cellular proliferative disorder that is undergoing malignant transformation. Examples of such conditions include, in the context of colorectal cellular proliferative disorders, cellular proliferative disorders with a high degree of dysplasia and the following classes of adenomas: Level 1: penetration of malignant glands through the muscularis mucosa into the submucosa, within the polyp head; Level 2: the same submucosal invasion, but present at the junction of the head to the stalk; Level 3: invasion of the stalk; and Level 4: invasion of the stalk's base at the connection to the colonic wall. In some instances, pre-neoplastic is used to describe a normal tissue that will form tumors.

[0069] As used herein, “predisposition” refers to the property of being susceptible to a cellular proliferative disorder. A subject having a predisposition to a cellular proliferative disorder has no cellular proliferative disorder, but is a subject having an increased likelihood of having a cellular proliferative disorder.

[0070] A “polynucleotide” means a single strand or parallel and anti-parallel strands of a nucleic acid. Thus, a polynucleotide may be either a single-stranded or a double-stranded nucleic acid. In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0071] The term “oligonucleotide” typically refers to short polynucleotides, generally no greater than about 60 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T.”

[0072] As used herein, the term “providing a prognosis” refers to providing a prediction of the probable course and outcome of a cancer, including prediction of severity, duration, chances of recovery, etc. The methods can also be used to devise a suitable therapeutic plan, e.g., by indicating whether or not the condition is still at an early stage or if the condition has advanced to a stage where aggressive therapy would be ineffective.

[0073] A “reference level” of a biomarker means a level of the biomarker, for example level of a type of collagen fragment that is indicative of a particular disease state, phenotype, or lack thereof, as well as combinations of disease states, phenotypes, or lack thereof. A “positive” reference level of a biomarker means a level that is indicative of a particular disease state or phenotype. A “negative” reference level of a biomarker means a level that is indicative of a lack of a particular disease state or phenotype.

[0074] As used herein, the term “saccharide” refers to a polymer comprising one or more monosaccharide groups. Saccharides, therefore, include mono-, di-, tri- and polysaccharides (or glycans). Glycans can be branched or branched. Glycans can be found covalently linked to nonsaccharide moieties, such as lipids or proteins (as a glycoconjugate). These covalent conjugates include glycoproteins, glycopeptides, peptidoglycans, proteoglycans, glycolipids, and lipopolysaccharides. The use of any one of these terms also is not intended to be limiting as the description is provided for illustrative purposes. In addition to the glycans being found as part of a glycoconjugate, the glycans can also be in free form (i.e., separate from and not associated with another moiety). By the term “specifically binds,” as used herein, is meant a molecule, such as an antibody, which recognizes and binds to another molecule or feature, but does not substantially recognize or bind other molecules or features in a sample.

[0075] “Standard control value” as used herein refers to a predetermined collagen peptide and / or fragment level. The standard control value is suitable for the use of a method of the present invention, in order for comparing the amounts of collagen peptide and / or fragments of interest that is present in a sample. An established sample serving as a standard control provides an average amount of a collagen peptide and / or fragment of interest that is typical for an average, healthy person of reasonably matched background, e.g., gender, age, ethnicity, and medical history. A standard control value may vary depending on the biomarker of interest and the nature of the sample.

[0076] As used herein, the term “subject” refers to a human or another mammal (e.g., primate, dog, cat, goat, horse, pig, mouse, rat, rabbit, and the like. In many embodiments of the present invention, the subject is a human being. In such embodiments, the subject is often referred to as an “individual” or a “patient.” The terms “individual” and “patient” do not denote a particular age.

[0077] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and

[0078] 6. This applies regardless of the breadth of the range.

[0079] In some aspects of the present invention, software executing the instructions provided herein may be stored on a non-transitory computer-readable medium, wherein the software performs some or all of the steps of the present invention when executed on a processor.

[0080] Aspects of the invention relate to algorithms executed in computer software. Though certain embodiments may be described as written in particular programming languages, or executed on particular operating systems or computing platforms, it is understood that the system and method of the present invention is not limited to any particular computing language, platform, or combination thereof. Software executing the algorithms described herein may be written in any programming language known in the art, compiled or interpreted, including but not limited to C, C++, C#, Objective-C, Java, JavaScript, MATLAB, Python, PHP, Perl, Ruby, or Visual Basic. It is further understood that elements of the present invention may be executed on any acceptable computing platform, including but not limited to a server, a cloud instance, a workstation, a thin client, a mobile device, an embedded microcontroller, a television, or any other suitable computing device known in the art.

[0081] Parts of this invention are described as software running on a computing device. Though software described herein may be disclosed as operating on one particular computing device (e g. a dedicated server or a workstation), it is understood in the art that software is intrinsically portable and that most software running on a dedicated server may also be run, for the purposes of the present invention, on any of a wide range of devices including desktop or mobile devices, laptops, tablets, smartphones, watches, wearable electronics or other wireless digital / cellular phones, televisions, cloud instances, embedded microcontrollers, thin client devices, or any other suitable computing device known in the art.

[0082] Similarly, parts of this invention are described as communicating over a variety of wireless or wired computer networks. For the purposes of this invention, the words “network”, “networked”, and “networking” are understood to encompass wired Ethernet, fiber optic connections, wireless connections including any of the various 802.11 standards, cellular WAN infrastructures such as 3G, 4G / LTE, or 5G networks, Bluetooth®, Bluetooth® Low Energy (BLE) or Zigbee® communication links, or any other method by which one electronic device is capable of communicating with another. In some embodiments, elements of the networked portion of the invention may be implemented over a Virtual Private Network (VPN).

[0083] Fig. 1 and the following discussion are intended to provide a brief, general description of a suitable computing environment in which the invention may be implemented. While the invention is described above in the general context of program modules that execute in conjunction with an application program that runs on an operating system on a computer, those skilled in the art will recognize that the invention may also be implemented in combination with other program modules. Generally, program modules include routines, programs, components, data structures, and other types of structures that perform particular tasks or implement particular abstract data types. Moreover, those skilled in the art will appreciate that the invention may be practiced with other computer system configurations, including hand-held devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, minicomputers, mainframe computers, and the like. The invention may also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules may be located in both local and remote memory storage devices.

[0084] Figure 1 depicts an illustrative computer architecture for a computer 100 for practicing the various embodiments of the invention. The computer architecture shown in Figure 1 illustrates a conventional personal computer, including a central processing unit 150 (“CPU”), a system memory 105, including a random-access memory 110 (“RAM”) and a read-only memory (“ROM”) 115, and a system bus 135 that couples the system memory 105 to the CPU 150. A basic input / output system containing the basic routines that help to transfer information between elements within the computer, such as during startup, is stored in the ROM 115. The computer 100 further includes a storage device 120 for storing an operating system 125, application / program 130, and data.

[0085] The storage device 120 is connected to the CPU 150 through a storage controller (not shown) connected to the bus 135. The storage device 120 and its associated computer-readable media provide non-volatile storage for the computer 100. Although the description of computer readable media contained herein refers to a storage device, such as a hard disk or CD-ROM drive, it should be appreciated by those skilled in the art that computer-readable media can be any available media that can be accessed by the computer 100.

[0086] By way of example, and not to be limiting, computer-readable media may comprise computer storage media. Computer storage media includes volatile and non-volatile, removable and non-removable media implemented in any method or technology for storage of information such as computer-readable instructions, data structures, program modules or other data. Computer storage media includes, but is not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid state memory technology, CD-ROM, DVD, or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store the desired information and which can be accessed by the computer.

[0087] According to various embodiments of the invention, the computer 100 may operate in a networked environment using logical connections to remote computers through a network 140, such as TCP / IP network such as the Internet or an intranet. The computer 100 may connect to the network 140 through a network interface unit 145 connected to the bus 135. It should be appreciated that the network interface unit 145 may also be utilized to connect to other types of networks and remote computer systems.

[0088] The computer 100 may also include an input / output controller 155 for receiving and processing input from a number of input / output devices 160, including a keyboard, a mouse, a touchscreen, a camera, a microphone, a controller, a joystick, or other type of input device. Similarly, the input / output controller 155 may provide output to a display screen, a printer, a speaker, or other type of output device. The computer 100 can connect to the input / output device 160 via a wired connection including, but not limited to, fiber optic, Ethernet, or copper wire or wireless means including, but not limited to, Wi-Fi, Bluetooth, Near-Field Communication (NFC), infrared, or other suitable wired or wireless connections.

[0089] As mentioned briefly above, a number of program modules and data files may be stored in the storage device 120 and / or RAM 110 of the computer 100, including an operating system 125 suitable for controlling the operation of a networked computer. The storage device 120 and RAM 110 may also store one or more applications / programs 130. In particular, the storage device 120 and RAM 110 may store an application / program 130 for providing a variety of functionalities to a user. For instance, the application / program 130 may comprise many types of programs such as a word processing application, a spreadsheet application, a desktop publishing application, a database application, a gaming application, internet browsing application, electronic mail application, messaging application, and the like. According to an embodiment of the present invention, the application / program 130 comprises a multiple functionality software application for providing word processing functionality, slide presentation functionality, spreadsheet functionality, database functionality and the like.

[0090] The computer 100 in some embodiments can include a variety of sensors 165 for monitoring the environment surrounding and the environment internal to the computer 100. These sensors 165 can include a Global Positioning System (GPS) sensor, a photosensitive sensor, a gyroscope, a magnetometer, thermometer, a proximity sensor, an accelerometer, a microphone, biometric sensor, barometer, humidity sensor, radiation sensor, a mass sensor, a mass spectrometer, or any other suitable sensor.

[0091] Description

[0092] The present invention is based in part on novel methods and devices that allow multiple analyses (“omni-omics”) of single cells, including those isolated from complex or homogeneous mixtures. For example, in certain aspects, the invention relates to single-cell capture array for the detection of any ionizable analyte, including, but not limited to, proteins and peptides (proteomics), carbohydrates (glycomics), lipids (lipidomics), nucleic acids (genomics), small molecules (e.g., drugs) and metabolites (metabolomics), fragments thereof, and combinations thereof. In some embodiments, the single-cell capture arrays are antibody arrays. In some embodiments, the invention relates to arrays for the simultaneous analysis of multiple types of cells.

[0093] In some embodiments, the invention provides methods of identifying biomarkers of a disease or disorder, or pathways involved in a disease or disorder.

[0094] Single-Cell Capture Array

[0095] The present invention provides, in part, single-cell capture arrays that allow for the isolation and multiple analysis (“omni-omics”) of single cells. The single-cell capture arrays utilize an efficient workflow that allows for the capture and analysis of single cells from complex or homologous solutions and analysis of the isolated cells on an individual bases by mass spectrometry, including analysis of proteins and peptides (proteomics), carbohydrates (glycomics), lipids (lipidomics), nucleic acids (genomics and transcriptomics), small molecules (metabolomics), fragments thereof, and combinations thereof.

[0096] In various embodiments, the array is formed on a substrate. The substrate can be any substrate suitable for mass spectrometric analysis. Examples of suitable substrates include, but are not limited to, glass, plastic, polymer, or ceramic slides or multiwell plates, nitrocellulose, graphene, and gold. In some embodiments, the substrate can be functionalized or coated to enhance cell adherence. For example, the substrate surface can include an indium tin oxide coating, a thiol -reactive coating, a carboxy-reactive coating, an aldehyde-reactive coating, an azide-reactive coating, a gelatin coating, a collagen coating, a poly-l-lysine coating, a polyornithine coating, an extracellular matrix coating, a protein coating (such as cadherins, immunoglobulins, selectins, mucins, integrins, streptavidin, and the like), an epoxysilane coating, surface ionization, and the like.

[0097] In various embodiments, the single-cell capture array comprises a plurality of cell binding molecules. In some embodiments, the cell-binding molecules comprise antibodies or antibody fragments (e.g., an antibody array). In some embodiments, the cell-binding molecules comprise lectins (e.g., a lectin array). In some embodiments, the cell-binding molecules comprise aptamers (e.g., an aptamer array). In certain embodiments, array comprises multiple types of cell-binding molecules, for example a combination of two or more of antibodies or antibody fragments, lectins, and aptamers.

[0098] In some embodiments, the cell-binding molecules are known to interact with a molecule on the surface of a target cell of interest. In some embodiments, the cell-binding molecules cleave a molecule on the surface of a target cell of interest. In some embodiments, the cell binding molecules are derivatives of antibodies, lectins, aptamers, or proteins that interact with a molecule on the surface of a target cell of interest. In some embodiments, the derivatives lack their native enzymatic activity. In some embodiments, the derivatives comprise the molecule binding site of the molecule on the surface of the target cell of interest.

[0099] A single-cell capture array (e.g., an antibody or lectin array) spotted on the substrate allows for capture and analysis of one to hundreds of different cells. Thus, in some embodiments, the single-cell capture arrays of the invention can comprise one to hundreds of different cell binding molecules (e.g., antibodies or lectins), each specific for one target cell of interest. In some embodiments, the single-cell capture array comprises a single cell-binding molecule (e.g., antibody or lectin) that binds a single cell type. In some embodiments, the single-cell capture array comprises a single cell-binding molecule that binds a variety of cell types.

[0100] Spotting of cell-binding molecules can be achieved through any suitable technique, including but not limited to inkjet printing, acoustic printing, fine print spotting, flow patterning on a functionalized substrate, contact printing (stamping), micro-contact printing on a functionalized substrate, incubating on coated substrates (such as a nitrocellulose coating), or microprinting using epoxy-coated glass substrate or poly-amine glass substrate with printing needles or strips with very fine feature resolution. In some embodiments, cell-binding molecules are bound to a substrate. In some embodiments, cell-binding molecules, (e.g., antibodies or lectins) are covalently bonded to a substrate. For example, the cell-binding molecules may be directly conjugated to a nucleophilic or electrophilic group of a substrate. In some embodiments, cell-binding molecules are covalently bonded to a substrate through one or more linkers. For example, a linker may be conjugated to a nucleophilic or electrophilic group of a substrate and a cell-binding molecule may be conjugated to a nucleophilic or electrophilic group of the linker. In some embodiments, cell-binding molecules are bound to a substrate by non-covalent interactions between the antibodies and the substrate. For example, a cell-binding molecule may be bound to a substrate through ionic, polar, and / or non-polar interactions. In some embodiments, cell-binding molecules are bound to a substrate by non-covalent interactions between the antibodies and one or more compounds covalently bonded to the substrate. For example, a linker may be covalently bonded to a substrate and a cell-binding molecule may interact with the linker through ionic, polar, and / or non-polar interactions. In some embodiments, cell-binding molecules are bound to a substrate by non-covalent interactions between the antibodies and one or more compounds bound to the substrate by non-covalent interactions. For example, a linker may be bound to a substrate through ionic, polar, and / or non-polar interactions and an antibody may be bound to the linker through ionic, polar, and / or non-polar interactions. In some embodiments, cell-binding molecules are bound to a substrate by a covalent bond between the cell-binding molecules and one or more compounds bound to the substrate by non-covalent interactions. For example, a linker may be bound to a substrate through ionic, polar, and / or non-polar interactions and a cell-binding molecule may be conjugated to a nucleophilic or electrophilic group of the linker.

[0101] The single-cell capture (e g., antibody or lectin) arrays can be arranged in any desired grid or pattern. In certain embodiments, individual antibody spots are spaced laterally and longitudinally in an array of rows and / or columns. In some embodiments, individual cell-binding molecule spots are regularly spaced at about 10-300 pm in separation. In some embodiments, individual cell-binding molecule spots are regularly spaced at about 10 pm, about 20 pm, about 30 pm, about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, or about 300 pm. In some embodiments, the capture arrays comprise about 10-1,000,000 individual cell-binding molecule spots. In another embodiment, the capture arrays comprise about 500-500,000 individual cell-binding molecule spots. In another embodiment, the capture arrays comprise about 100-100,000 individual cell binding molecule spots. In some embodiments, the capture arrays comprise cell binding molecule spots at a density of about 200 cell binding molecule spots per cm2to about 20,000 antibody spots per cm2. In various embodiments, arraying cell-binding molecule spots can be aided with the use of one or more grids, such as a well slide module.

[0102] In certain embodiments, each spot comprises a single specific cell-binding molecule. In another embodiment, each spot comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different cell-binding molecules (e.g., antibodies or lectins). In these embodiments, specific binding to a number of particular cell-binding molecules within the feature can be determined by use of different detectable labels on a second set of capture agents, with each label corresponding to a particular cell-binding molecule.

[0103] In some embodiments, a cell-binding molecule binds to a cell surface molecule. A cell surface molecule may be any molecule displayed on the surface of any cell. In general, any eukaryotic or prokaryotic cell can be captured and analyzed, including, but are not limited to, blood cells, immune cells, epithelial cells, endothelial cells, muscle cells, nerve cells, bone cells, fat cells, sex cells, brain cells, liver cells, and cancer cells.

[0104] In some embodiments, the cell surface molecule is a peptide, protein, lipid, polysaccharide, nucleic acid, or a combination thereof. Examples of combination molecules include, but are not limited to, lipopeptides, glycopeptides, nucleopeptides, lipoproteins, proteoglycans, nucleoproteins, glycolipids, lipo-nucleosides, and glyconucleic acids.

[0105] In some embodiments, the cell surface molecule is a protein. Examples of suitable cell surface proteins include, but are not limited to, receptors, co-receptors, transport proteins, adhesion proteins, cohesion proteins, signaling proteins, and combinations thereof.

[0106] In various embodiments, antibody arrays can include antibodies, antibody fragments, or combinations thereof. Such antibodies include polyclonal antibodies, monoclonal antibodies, Fab and single chain Fv (scFv) fragments thereof, bispecific antibodies, heteroconjugates, human and humanized antibodies. Such antibodies may be produced in a variety of ways, including hybridoma cultures, recombinant expression in bacteria or mammalian cell cultures, and recombinant expression in transgenic animals. The choice of manufacturing methodology depends on several factors including the antibody structure desired, the importance of carbohydrate moieties on the antibodies, ease of culturing and purification, and cost. Many different antibody structures may be generated using standard expression technology, including full-length antibodies, antibody fragments, such as Fab and Fv fragments, as well as chimeric antibodies comprising components from different species.

[0107] While the methods describe the use of antibody arrays, it should be understood that any suitable capture molecule having an affinity to a cell-surface molecule of interest can be used to cells as would be understood by those having skill in the art. For example, antibodies can be replaced or supplemented with one or more antigens, aptamers, affibodies, proteins, peptides, lectins, nucleic acids, carbon nanotubes, and fragments thereof. The capture molecules are also not limited to an array pattern and can be provided in any shape or form desired.

[0108] Array Preparation

[0109] In some aspects, the present invention provides methods of preparing a single-cell capture array of the present invention. In some embodiments, the method comprises a direct or indirect method of spotting cell-binding molecules (e.g., antibodies, aptamers, lectins, etc.) on a substrate.

[0110] In some embodiments, the present invention provides a direct method of producing a single-cell capture array comprising the steps of: a) providing a substrate; b) spotting cell binding molecules spots on the substrate; and c) blocking the substrate surface that is not spotted with cell-binding molecules.

[0111] In some embodiments, the substrate of step a) is any substrate suitable for mass spectrometric analysis. Examples of suitable substrates include, but are not limited to, glass, plastic, polymer, or ceramic slides or multiwell plates, nitrocellulose, graphene, and gold. In some embodiments, the substrate can be functionalized or coated to enhance cell adherence. For example, the substrate surface can include an amine-reactive coating, a hydrogel coating, an indium tin oxide coating, a thiol -reactive coating, a carboxy-reactive coating, an aldehydereactive coating, an azide-reactive coating, a gelatin coating, a collagen coating, a poly-l-lysine coating, a poly-omithine coating, an extracellular matrix coating, a protein coating (such as cadherins, immunoglobulins, selectins, mucins, integrins, streptavidin, and the like), an epoxysilane coating, surface ionization, and the like.

[0112] In some embodiments, step b) is performed by any means of cell-binding molecule spotting or printing known in the art. In some embodiments, step b) comprises one or more selected from the group consisting of inkjet printing, acoustic printing, fine print spotting, flow patterning on a functionalized substrate, contact printing (stamping), contact printing on a functionalized substrate, incubating on coated substrates (such as a nitrocellulose coating), or microprinting using epoxy-coated glass substrate or poly-amine glass substrate with printing needles or strips with very fine feature resolution. In some embodiments, step b) comprises the steps of i) providing a stamp comprising a plurality of pillars; ii) coating the stamp with one or more cell-binding molecule; and iii) contacting the cell-binding molecule-coated surface of the stamp with the substrate.

[0113] In some embodiments, the stamp of step b) comprises one or more selected from the group consisting of polyethylene glycol (PEG), polyethyleneimine (PEI), polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and polydimethylsiloxane (PDMS). In some embodiments, the stamp comprises a plurality of pillars of approximately equal size. In some embodiments, the pillars are circular, elliptical, square, rectangular, triangular, or any other suitable shape.

[0114] In some embodiments, the pillars have a width or diameter of between about 1 pm and about 100 pm. In some embodiments, the pillars have a width or diameter of between about 1 pm and about 50 pm. In some embodiments, the pillars have a width or diameter of about 1 pm, about 2 pm, about 3 pm, about 4 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, about 20 pm, about 22 pm, about 24 pm, about 26 pm, about 28 pm, about 30 pm, about 32 pm, about 34 pm, about 36 pm, about 38 pm, about 40 pm, about 42 pm, about 44 pm, about 46 pm, about 48 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, or about 100 pm.

[0115] In some embodiments, the pillars are between about 5 pm and about 100 pm tall. In some embodiments, the pillars are about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 12 pm, about 14 pm, about 16 pm, about 18 pm, about 20 pm, about 22 pm, about 24 pm, about 26 pm, about 28 pm, about 30 pm, about 32 pm, about 34 pm, about 36 pm, about 38 pm, about 40 pm, about 42 jam, about 44 pm, about 46 pm, about 48 pm, about 50 jam, about 55 pm, about 60 pm, about 65 pm, about 70 pm, about 75 pm, about 80 pm, about 85 pm, about 90 pm, about 95 jam, or about 100 jam tall.

[0116] In some embodiments, the pillars are spaced between about 10 pm and about 500 pm apart. In some embodiments, the pillars are spaced between about 50 pm and about 150 pm apart. In some embodiments, the pillars are spaced about 10 pm, about 20 pm, about 30 pm, about 40 pm, 50 pm, about 55 pm, about 60 pm, about 65 pm, about 70 pm, about 75 pm, about 80 pm, about 85 pm, about 90 pm, about 95 pm, 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 300 pm, about 400 pm, or about 500 pm apart.

[0117] In some embodiments, step b) part ii) comprises incubating the stamp in a solution of cell-binding molecules. In some embodiments, the stamp is incubated in a solution of cell binding molecules for between about 5 minutes and about 24 hours. In some embodiments, the stamp is incubated in a solution of cell-binding molecules for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 1.25 hours, about 1.5 hours, about 1.75 hours, about 2 hours, about 2.25 hours, about 2.5 hours, about 2.75 hours, about 3 hours, about 3.25 hours, about 3.5 hours, about 3.75 hours, about 4 hours, about 4.25 hours, about 4.5 hours, about 4.75 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours.

[0118] In some embodiments, the solution of cell-binding molecules is at a concentration between about 0.05 pg / mL and about 500 pg / mL. In some embodiments, the concentration is about 0.05 pg / mL, about 0.1 pg / mL, about 0.2 pg / mL, about 0.3 pg / mL, about 0.4 pg / mL, about 0.5 pg / mL, about 0.6 pg / mL, about 0.7 pg / mL, about 0.8 pg / mL, about 0.9 pg / mL, about 1 pg / mL, about 2 pg / mL, about 3 pg / mL, about 4 pg / mL, 5 pg / mL, about 10 pg / mL, about 15 pg / mL, about 20 pg / mL, about 25 pg / mL, about 30 pg / mL, about 35 pg / mL, about 40 pg / mL, about 45 pg / mL, about 50 pg / mL, about 55 pg / mL, about 60 pg / mL, about 65 pg / mL, about 70 pg / mL, about 75 pg / mL, about 80 pg / mL, about 85 pg / mL, about 90 pg / mL, about 95 pg / mL, about 100 pg / mL, about 110 pg / mL, about 120 pg / mL, about 130 pg / mL, about 140 pg / mL, about 150 gg / mL, about 160 gg / mL, about 170 gg / mL, about 180 gg / mL, about 190 gg / mL, about 200 gg / mL, about 225 gg / mL, about 250 gg / mL, about 275 gg / mL, about 300 gg / mL, about 325 gg / mL, about 350 gg / mL, about 375 gg / mL, about 400 gg / mL, about 425 gg / mL, about 450 gg / mL, about 475 gg / mL, or about 500 gg / mL.

[0119] In some embodiments, step b) part ii) further comprises a step of rinsing unbound cell binding molecule off the stamp. In some embodiments, the step of rinsing unbound cell-binding molecule off the stamp comprises one or more washes with phosphate-buffered saline (PBS). In some embodiments, the step of rinsing unbound cell-binding molecule of the stamp comprises one or more water washes. In some embodiments, the step of rinsing unbound cell-binding molecule off the stamp comprises one or more PBS washes and one or more water washes.

[0120] In some embodiments, step b) part iii) comprises incubating the substrate in contact with the stamp for between about 1 minute and about 24 hours. In some embodiments, the method comprises incubating the substrate in contact with the stamp for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 22 minutes, about 24 minutes, about 26 minutes, about 28 minutes, about 30 minutes, about 32 minutes, about 34 minutes, about 36 minutes, about 38 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 1.25 hours, about 1.5 hours, about 1.75 hours, about 2 hours, about 2.25 hours, about 2.5 hours, about 2.75 hours, about 3 hours, about 3.25 hours, about 3.5 hours, about 3.75 hours, about 4 hours, about 4.25 hours, about 4.5 hours, about 4.75 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours.

[0121] In some embodiments, step b) part iii) further comprises a step of rinsing unbound cell binding molecule off the substrate. In some embodiments, the step of rinsing unbound cell binding molecule off the substrate comprises one or more washes with phosphate-buffered saline (PBS). In some embodiments, the step of rinsing unbound cell-binding molecule of the substrate comprises one or more water washes. In some embodiments, the step of rinsing unbound cell binding molecule off the substrate comprises one or more PBS washes and one or more water washes.

[0122] In some embodiments, step c) comprises incubating the substrate in a blocking solution. In some embodiments, the blocking solution of step b) is a serum. In some embodiments, the serum is bovine serum albumin (BSA). In some embodiments, the serum is BSA in PBS. In some embodiments, the serum is 1% BSA in PBS. In some embodiments, the serum is 1% BSA (w / w) in PBS with one or more detergents. In some embodiments, step b) further comprises removing the blocking solution. In some embodiments, the step of removing of the blocking solution comprises one or more washes with PBS. In some embodiments, the removing of the blocking solution comprises one or more washes with water. In some embodiments, the removing of the blocking solution one or more PBS washes and one or more water washes.

[0123] In some embodiments, step c) further comprises drying the single-cell capture array.

[0124] In some embodiments, the present invention provides an indirect method of producing a single-cell capture array comprising the steps of: a) providing a substrate; b) printing a blocking protein on the substrate to leave “negative space” spots on the substrate; and c) incubating the substrate in cell-binding molecules to create cell-binding molecule spots in the negative space spots.

[0125] In some embodiments, the substrate of step a) is any substrate suitable for mass spectrometric analysis. Examples of suitable substrates include, but are not limited to, glass, plastic, polymer, or ceramic slides or multiwell plates, nitrocellulose, graphene, and gold. In some embodiments, the substrate can be functionalized or coated to enhance cell adherence. For example, the substrate surface can include an amine reactive coating, a hydrogel coating, an indium tin oxide coating, a thiol-reactive coating, a carboxy-reactive coating, an aldehydereactive coating, an azide-reactive coating, a gelatin coating, a collagen coating, a poly-l-lysine coating, a poly-ornithine coating, an extracellular matrix coating, a protein coating (such as cadherins, immunoglobulins, selectins, mucins, integrins, streptavidin, and the like), an epoxysilane coating, surface ionization, and the like.

[0126] In some embodiments, step b) comprises: i) obtaining a stamp comprising a plurality of holes; ii) coating the stamp with a blocking protein; and iii) contacting the stamp with the substrate. In some embodiments, the stamp of step b) comprises one or more selected from the group consisting of polyethylene glycol (PEG), polyethyleneimine (PEI), polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and polydimethylsiloxane (PDMS). In some embodiments, the stamp comprises a plurality of holes of approximately equal size. In some embodiments, the holes are circular, elliptical, square, rectangular, triangular, or any other suitable shape.

[0127] In some embodiments, the holes have a width or diameter of between about 1 pm and about 100 pm. In some embodiments, the holes have a width or diameter of between about 1 pm and about 50 pm. In some embodiments, the holes have a width or diameter of about 1 pm, about 2 pm, about 3 pm, about 4 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 1 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, about 20 pm, about 22 pm, about 24 pm, about 26 pm, about 28 pm, about 30 pm, about 32 pm, about 34 pm, about 36 pm, about 38 pm, about 40 pm, about 42 pm, about 44 pm, about 46 pm, about 48 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, or about 100 pm.

[0128] In some embodiments, the holes are between about 1 pm and about 100 pm deep. In some embodiments, the holes are about 1 pm, about 2 pm, about 3 pm, about 4 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 12 pm, about 14 pm, about 16 pm, about 18 pm, about 20 pm, about 22 pm, about 24 pm, about 26 pm, about 28 pm, about 30 pm, about 32 pm, about 34 pm, about 36 pm, about 38 pm, about 40 pm, about 42 pm, about 44 pm, about 46 pm, about 48 pm, about 50 pm, about 55 pm, about 60 pm, about 65 pm, about 70 pm, about 75 pm, about 80 pm, about 85 pm, about 90 pm, about 95 pm, or about 100 pm deep.

[0129] In some embodiments, the holes are spaced between about 10 pm and about 500 pm apart. In some embodiments, the holes are spaced between about 50 pm and about 150 pm apart. In some embodiments, the holes are spaced about 10 pm, about 20 pm, about 30 pm, about 40 pm, about 50 pm, about 55 pm, about 60 pm, about 65 pm, about 70 pm, about 75 pm, about 80 pm, about 85 pm, about 90 pm, about 95 pm, 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 300 pm, about 400 pm, or about 500 pm apart. In some embodiments, step b) part ii) comprises incubating the substrate in a blocking solution. In some embodiments, the blocking solution is a serum. In some embodiments, the serum is BSA. In some embodiments, the serum is BSA in PBS. In some embodiments, the serum is 1% (w / w) BSA in PBS. In some embodiments, the serum is 1% BSA in PBS with one or more detergents.

[0130] In some embodiments, the stamp is incubated in a solution of blocking protein for between about 5 minutes and about 5 hours. In some embodiments, the stamp is incubated in a solution of cell-binding molecules for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 1.25 hours, about 1.5 hours, about 1 .75 hours, about 2 hours, about 2.25 hours, about 2.5 hours, about 2.75 hours, about 3 hours, about 3.25 hours, about 3.5 hours, about 3.75 hours, about 4 hours, about 4.25 hours, about 4.5 hours, about 4.75 hours, or about 5 hours.

[0131] In some embodiments, the solution of blocking protein is at a concentration between about 5 pg / mL and about 500 pg / mL. In some embodiments, the concentration is about 5 pg / mL, about 10 pg / mL, about 15 pg / mL, about 20 pg / mL, about 25 pg / mL, about 30 pg / mL, about 35 pg / mL, about 40 pg / mL, about 45 pg / mL, about 50 pg / mL, about 55 pg / mL, about 60 pg / mL, about 65 pg / mL, about 70 pg / mL, about 75 pg / mL, about 80 pg / mL, about 85 pg / mL, about 90 pg / mL, about 95 pg / mL, about 100 pg / mL, about 110 pg / mL, about 120 pg / mL, about 130 pg / mL, about 140 pg / mL, about 150 pg / mL, about 160 pg / mL, about 170 pg / mL, about 180 pg / mL, about 190 pg / mL, about 200 pg / mL, about 225 pg / mL, about 250 pg / mL, about 275 pg / mL, about 300 pg / mL, about 325 pg / mL, about 350 pg / mL, about 375 pg / mL, about 400 pg / mL, about 425 pg / mL, about 450 pg / mL, about 475 pg / mL, or about 500 pg / mL.

[0132] In some embodiments, step b) part ii) further comprises a step of rinsing unbound blocking protein off the stamp. In some embodiments, the step of rinsing unbound blocking protein off the stamp comprises one or more washes with phosphate-buffered saline (PBS). In some embodiments, the step of rinsing unbound blocking protein of the stamp comprises one or more water washes. In some embodiments, the step of rinsing unbound blocking protein off the stamp comprises one or more PBS washes and one or more water washes.

[0133] In some embodiments, step b) part iii) comprises incubating the substrate in contact with the stamp for between about 1 minute and about 2 hours. In some embodiments, the method comprises incubating the substrate in contact with the stamp for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 22 minutes, about 24 minutes, about 26 minutes, about 28 minutes, about 30 minutes, about 32 minutes, about 34 minutes, about 36 minutes, about 38 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 1.25 hours, about 1.5 hours, about 1.75 hours, or about 2 hours.

[0134] In some embodiments, step b) part iii) further comprises a step of rinsing unbound blocking protein off the substrate. In some embodiments, the step of rinsing unbound blocking protein off the substrate comprises one or more washes with phosphate-buffered saline (PBS). In some embodiments, the step of rinsing unbound blocking protein of the substrate comprises one or more water washes. In some embodiments, the step of rinsing unbound blocking protein off the substrate comprises one or more PBS washes and one or more water washes.

[0135] In some embodiments, step b) part iii) further comprises a step of drying the substrate. In some embodiments, step b) part iii) further comprises a step of heating the substrate to denature the blocking protein.

[0136] In some embodiments, step c) comprises incubating the substrate in a solution of cell binding molecules. In some embodiments, the substrate is incubated in a solution of cell-binding molecules for between about 5 minutes and about 5 hours. In some embodiments, the substrate is incubated in a solution of cell-binding molecules for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 1.25 hours, about 1.5 hours, about 1.75 hours, about 2 hours, about 2.25 hours, about 2.5 hours, about 2.75 hours, about 3 hours, about 3.25 hours, about 3.5 hours, about 3.75 hours, about 4 hours, about 4.25 hours, about 4.5 hours, about 4.75 hours, or about 5 hours.

[0137] In some embodiments, the solution of cell-binding molecules is at a concentration between about 5 pg / mL and about 500 pg / mL. In some embodiments, the concentration is about 5 pg / mL, about 10 pg / mL, about 15 pg / mL, about 20 pg / mL, about 25 pg / mL, about 30 pg / mL, about 35 pg / mL, about 40 pg / mL, about 45 pg / mL, about 50 pg / mL, about 55 pg / mL, about 60 pg / mL, about 65 pg / mL, about 70 pg / mL, about 75 pg / mL, about 80 pg / mL, about 85 pg / mL, about 90 ug / mL, about 95 pg / mL, about 100 pg / mL, about 110 pg / mL, about 120 gg / mL, about 130 gg / mL, about 140 gg / mL, about 150 gg / mL, about 160 gg / mL, about 170 gg / mL, about 180 gg / mL, about 190 gg / mL, about 200 gg / mL, about 225 gg / mL, about 250 gg / mL, about 275 gg / mL, about 300 gg / mL, about 325 gg / mL, about 350 gg / mL, about 375 gg / mL, about 400 gg / mL, about 425 gg / mL, about 450 gg / mL, about 475 gg / mL, or about 500 gg / mL.

[0138] In some embodiments, step c) further comprises a step of rinsing unbound cell-binding molecule off the substrate. In some embodiments, the step of rinsing unbound cell-binding molecule off the substrate comprises one or more washes with phosphate-buffered saline (PBS). In some embodiments, the step of rinsing unbound cell-binding molecule of the substrate comprises one or more water washes. In some embodiments, the step of rinsing unbound cell binding molecule off the substrate comprises one or more PBS washes and one or more water washes.

[0139] Method of Analysis

[0140] Traditional single-cell analysis typically relies on a bioinformatics-based argument that the results of the analysis are representative of a single cell, rather than actually performing the analysis on a single isolated cell. Further, these methods are limited to a single type of analysis on a cell (e g., proteomics, genomics, metabolomics, etc.). Utilizing a single-cell capture array, the present invention provides methods of performing comprehensive analysis (omni-omics) on single isolated cells.

[0141] In various aspects, the present invention provides a method of isolating and performing omni-omics analysis of single cells isolated from a sample. In some embodiments, the method comprises the steps of: a) providing a single-cell capture array according to the present invention; b) incubating the single-cell capture array with a sample containing the target cell to be analyzed; c) rinsing the single-cell capture array to remove unbound cells, leaving only a single cell on each cell-binding molecule spot; d) fixing the cells or leaving the cells unfixed; e) detecting single cells through an algorithm that identifies and marks single cells; and f) scanning the single-cell capture array by mass spectrometry.

[0142] In some embodiments, the sample of step b) is any liquid comprising a target cell of interest. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is a biofluid. Examples of biofluids include, but are not limited to, serum, plasma, whole blood, bronchial lavage, urine, cerebrospinal fluid (CSF), synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal fluid, otic fluid, gastric fluid, ascitic fluid, nasal mucus, and breast milk. In some embodiments, the sample comprises at least one additive. In some embodiments, the additive is an aqueous additive. In some embodiments, the additive is a non-aqueous additive. Examples of suitable additives include, but are not limited to, a diluent, a solvent, a base, an acid, a buffer, a salt, or any combination thereof. In some embodiments, the sample is a fluid derived from a solid tissue.

[0143] In some embodiments, step b) comprises incubating the single-cell capture array with the sample for between about 1 minute and about 24 hours. In some embodiments, the single-cell capture array is incubated with the sample for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 32 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 1 hour, about 1.25 hours, about 1.5 hours, about 1.75 hours, about 2 hours, about 2.25 hours, about 2.5 hours, about 2.75 hours, about 3 hours, about 3.25 hours, about 3.5 hours, about 3.75 hours, about 4 hours, about 4.25 hours, about 4.5 hours, about 4.75 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours.

[0144] In some embodiments, step c) comprises rinsing away unbound cells off the array. In some embodiments, the step of rinsing unbound cells off the array comprises one or more washes with phosphate-buffered saline (PBS). In some embodiments, the step of rinsing unbound cells of the array comprises one or more water washes. In some embodiments, the step of rinsing unbound cells off the array comprises one or more PBS washes and one or more water washes.

[0145] In some embodiments, step d) comprises fixing the cells to the single-cell capture array. In some embodiments, the cells are heat-fixed to the array. In some embodiments, the cells are chemically fixed to the array. In some embodiments, the cells are chemically fixed to the array by formaldehyde or formalin. In some embodiments, the formalin is neutral buffered formalin (NFB). In some embodiments, the NFB is aqueous 10% NFB (v / v). In some embodiments, the cells are chemically fixed to the array for between about 1 minute and about 1 hour. In some embodiments, the cells are chemically fixed to the array for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 32 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about3 7 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, or about 1 hour.

[0146] In some embodiments, step e) comprises identifying captured single cells by an algorithm that detects and assigns coordinates for subsequent downstream analysis of only single cells. In some embodiments, identifying captured single cells comprises utilizing a software package that identifies single cells and marks them for further analysis.

[0147] In some embodiments, the present invention relates to a software and method of identifying single cells. In some examples referred to as a software package (e.g., a software stored on a computer-readable medium) for identifying single cells, the software may be stored on a non-transitory computer-readable medium with pre-programmed instructions stored thereon, which when executed by a processor, perform steps comprising i) prompting the user to select an area of an image of a slide; ii) prompting the user to mark eight adjacent single cells and two additional single cells that are in the same column or row but as far from each other as allowable; iii) calculating the average distance between cells from the distance between the eight adjacent cells; iv) calculating the angle of the grid structure from the two distal cells; v) creating a frame around one of the selected cells; vi) moving to the next location on the grid using the angle and average distance previously calculated, wherein the darkest are is selected as the next location by a contour detection algorithm; vii) centering the darkest area in the frame; viii) repeating steps vi) and vii) in a single direction until reaching the edge of the selected area; ix) repeating steps vi) through viii) in four directions to determine the column and row positions of the entire grid; x) creating frames at each point in the grid according to the determined column and row positions; xi) evaluating each frame by a pre-trained CNN model to identify locations as containing single cells or not; xii) producing an interactive histogram for user selection of confidence boundaries; xiii) outputting result files including the original image with single cells marked, a coordinate list of single cells and grouped cells, the histogram of step xii), and a log file.

[0148] In some embodiments, step f) comprises analyzing the substrate by mass spectrometry. Examples of mass spectrometric methods include matrix-assisted laser desorption / ionization mass spectrometry (MALDLMS), and matrix-assisted laser desorption / ionization tandem mass spectrometry (MALDI-MS / MS), fast atom bombardment mass spectrometry (FAB-MS), liquid chromatography mass spectrometry (LC-MS), liquid chromatography tandem mass spectrometry (LC-MS / MS).

[0149] Mass spectrometry imaging is a powerful tool that has been used to correlate various peptides, proteins, lipids, and metabolites with their underlying histopathology in tissue sections. One significant advantage is that matrix-assisted laser desorption / ionization (MALDI) imaging combined with tandem mass spectrometry reveals detailed structural information about the molecules and fragments in a sample. A wide range of molecular weights can be detected by mass spectrometry imaging. Also, the high mass resolution allows distinguishing two peaks with close molecular weights, which subsequently improves the detection specificity. In addition, tens or even hundreds of single molecules and fragments can be detected at femtomole levels in one single image, allowing detection of low concentrations of molecules. Therefore, MALDI imaging facilitates high-throughput analysis of single cells. MALDI imaging can also be used for performing quantitative assays. Another significant advantage of MALDI imaging is that it has the capability of detecting an unknown compound without any prior knowledge of the analytes.

[0150] Therefore, this technique is particularly suitable for biomarker discovery research.

[0151] MALDI is a soft ionization mass spectrometric technique that is suitable for use in the analysis of biomolecules, such as proteins, peptides, nucleic acids, sugars, and the like, which tend to be fragile and fragment when ionized by conventional ionization methods. This makes the coupling of MALDI and the single-cell capture arrays particularly amenable to omni-omics, i.e., simultaneous or sequential proteomic, glycomic, lipidomic, metabolomic, genomic, and transcriptomic analysis.

[0152] Generally, MALDI comprises a two-step process. In the first step, desorption is triggered by an ultraviolet (UV) laser beam. The matrix material absorbs the UV laser radiation, which leads to the ablation of an upper layer of the matrix material, thereby producing a hot plume. The hot plume contains many species: neutral and ionized matrix molecules, protonated and deprotonated matrix molecules, matrix clusters, and nanodroplets. In the second step, the analyte molecules are ionized, e.g., protonated or deprotonated, in the hot plume.

[0153] The matrix material comprises a crystallized molecule capable of absorbing the UV laser radiation. Common matrix materials include, but are not limited to, a-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid / 2-hydroxy-5-methoxybenzoic acid, 2,4,6-trihydroxyacetophenone, 6-aza-2-thiothymine, 3-hydroxypicolinic acid, 3 -aminoquinoline, anthranilic acid, 5-chloro-2-mercaptobenzothiazole, 2, 5 -dihydroxy acetophen one, ferulic acid, and 2-(4-hydroxyphenylazo) benzoic acid. A solution of the matrix material is made in highly purified water and an organic solvent, such as acetonitrile or ethanol. In some embodiments, a small amount of trifluoroacetic acid (TFA) also can be added to the solution.

[0154] The matrix solution can then be mixed with the analyte, e.g., a protein sample. This solution is then deposited onto a MALDI plate, wherein the solvents vaporize leaving only the recrystallized matrix comprising the analyte molecules embedded in the MALDI crystals.

[0155] Kits

[0156] In some aspects, the present invention provides a kit for performing a method of the present invention. In some embodiments, the kit comprises a) a single-cell capture array of the present invention; and b) a MALDI matrix material. Examples of suitable MALDI matrix materials include, but are not limited to, a-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid / 2-hydroxy-5-methoxybenzoic acid, 2,4,6- trihydroxyacetophenone, 6-aza-2-thiothymine, 3 -hydroxypicolinic acid, 3- aminoquinoline, anthranilic acid, 5-chloro-2-mercaptobenzothiazole, 2,5- dihydroxyacetophenone, ferulic acid, and 2-(4-hydroxyphenylazo) benzoic acid.

[0157] In some embodiments, the kit further comprises an enzymatic solution. In some embodiments the enzymatic solution comprises one or more selected from the group consisting of: lipases, proteases, peptidases, nucleases, and amylases.

[0158] In some embodiments, the kit further comprises instructional materials.

[0159] In some embodiments, the kit further comprises a non-transitory computer-readable medium with instructions stored thereon, which when executed by a processor, perform steps comprising identifying single cells.

[0160] In some embodiments, a kit of the present invention comprises: a) a substrate; b) a stamp; c) a blocking solution; and d) a MALDI matrix material. In some embodiments, the stamp comprises pillars for direct cell-binding molecule stamping of the substrate as described above. In some embodiments, the stamp comprises holes for the indirect stamping of the substrate as described above. In some embodiments, the blocking solution is BSA. In some embodiments, the BSA solution is a 10% (w / w) aqueous BSA solution.

[0161] In some embodiments, the kit further comprises one or more cell-binding molecules. In some embodiments, the kit further comprises instructional materials. In some embodiments, the kit further comprises a non-transitory computer-readable medium with instructions stored thereon, which when executed by a processor, perform steps comprising identifying single cells.

[0162] EMBODIMENTS

[0163] Embodiment l is a single-cell capture array comprising: a) a substrate; and b) a plurality of cell-binding molecule spots on the substrate, wherein the cell-binding molecule spots are between 1 pm and 100 pm in diameter and wherein the distance between each spot is between 10 pm and 500 pm. Embodiment 2 is the single-cell capture array of embodiment 1, wherein the cell-binding molecules in the cell-binding molecule spots are selected from the group consisting of antibodies, lectins, and aptamers.

[0164] Embodiment 3 is the single-cell capture array of embodiment 1 or 2, wherein the cellbinding molecule spots are in a square grid and the distance between each spot is between 10 and 300 pm.

[0165] Embodiment 4 is the single-cell capture array of any one of embodiments 1-3, wherein the cell-binding molecule spots are between 1 pm and 50 pm in diameter.

[0166] Embodiment 5 is the single-cell capture array of any one of embodiments 1-4, wherein the substrate is selected from the group consisting of glass, polydimethylsiloxane (PDMS), epoxysilane-coated glass, oxygen plasma-treated PDMS, and amine-reactive hydrogel -coated glass.

[0167] Embodiment 6 is the single-cell capture array of any one of embodiments 1-5, wherein all cell-binding molecules in a single cell-binding molecule spot bind a single antigen.

[0168] Embodiment 7 is the single-cell capture array of any one of embodiments 1-6, wherein the array of cell-binding molecule spots comprises a plurality of spots against one or more antigens, wherein the cell-binding molecules in each spot bind a single antigen.

[0169] Embodiment 8 is the single-cell capture array of any one of embodiments 1-7, wherein a cell-binding molecule spot comprises two or more cell-binding molecules that bind two or more antigens.

[0170] Embodiment 9 is a method of analyzing the profile of single cell comprising the steps of: a) providing a single-cell capture array of any one of embodiments 1-8; b) incubating the single-cell capture array with a sample containing the cell to be analyzed; c) rinsing the single-cell capture array to remove unbound cells, leaving only a single cell on each antibody spot; d) fixing the cells; e) detecting single cells through an algorithm which identifies and marks single cells; and f) scanning the single-cell capture array by mass spectrometry. Embodiment 10 is the method of embodiment 9, wherein the mass spectrometry is selected from the group consisting of matrix-assisted laser desorption / ionization imaging Fourier transform ion cyclotron resonance (MALDLFTICR) mass spectrometry, matrix-assisted laser desorption / ionization time of flight (MALDI-TOF) mass spectrometry, scanning microprobe MALDI (SMALDI) mass spectrometry, infrared matrix assisted laser desorption electrospray ionization (MALD-ESI) mass spectrometry, surface-assisted laser desorption / ionization (SALDI) mass spectrometry, desorption electrospray ionization (DESI) mass spectrometry, secondary ion mass spectrometry (SIMS) mass spectrometry, and easy ambient sonic spray ionization (EASI) mass spectrometry.

[0171] Embodiment 11 is the single-cell capture array of embodiment 9 or 10, wherein the step of scanning the single-cell capture array is preceded by a step of spraying the single-cell capture array with a MALDI matrix material.

[0172] Embodiment 12 is the method of embodiment 11, wherein the MALDI matrix solution is selected from the group consisting of 2,5-dihydroxybenzoic acid, a-cyano-4-hydroxycinnamic acid, sinapinic acid, 1,5-diaminonaphthalene, and 9-aminoacridine.

[0173] Embodiment 13 is the single-cell capture array of any one of embodiments 9-12, wherein the step of fixing the cells is preceded by spraying the single-cell capture array with an enzymatic solution.

[0174] Embodiment 14 is the method of embodiment 13, wherein the enzymatic solution comprises one or more selected from the group consisting of glycolytic enzymes, lipases, proteases, peptidases, nucleases, and amylases.

[0175] Embodiment 15 is the single-cell capture array of any one of embodiments 9-14, wherein step e) comprises scanning the single-cell capture array for one or more profiles selected from the group consisting of a genomic profile, a proteomic profile, a glycomic profile, a lipidomic profile, a metabolomic profile, and a transcriptomic profile.

[0176] Embodiment 16 is a kit for analyzing the profile of a single cell from a sample comprising: a single-cell capture array of any one of embodiments 1-8; and a MALDI matrix material.

[0177] Embodiment 17 is the kit of embodiment 16, wherein the MALDI matrix material is a- cyano-4hydroxy cinnamic acid. Embodiment 18 is the single-cell capture array of embodiment 16 or 17, wherein the kit further comprises an enzymatic solution.

[0178] Embodiment 19 is the single-cell capture array of any one of embodiments 16-18, wherein the kit further comprises a non-transitory computer-readable medium with instructions stored thereon, which when executed by a processor, perform steps comprising identifying single cells.

[0179] Embodiment 20 is a kit for analyzing the profde of a single cell from a sample comprising: a substrate; a stamp; a blocking protein; and a MALDI matrix material.

[0180] Embodiment 21 is the kit of embodiment 20, wherein the kit further comprises a cellbinding molecule.

[0181] Embodiment 22 is the kit of embodiment 21, wherein the cell binding molecule is selected from the group consisting of antibodies, lectins, and aptamers.

[0182] Embodiment 23 is the single-cell capture array of any one of embodiments 20-22, wherein the blocking protein is bovine serum albumin (BSA).

[0183] Embodiment 24 is the single-cell capture array of any one of embodiments 20-23, wherein the MALDI matrix material is a-cyano-4hydroxy cinnamic acid.

[0184] Embodiment 25 is the single-cell capture array of any one of embodiments 20-24, further comprising a non-transitory computer-readable medium with instructions stored thereon, which when executed by a processor, perform steps comprising identifying single cells.

[0185] Embodiment 26 is the single-cell capture array of any one of embodiments 20-25, wherein the kit further comprises an enzymatic solution.

[0186] Embodiment 27 is the kit of embodiment 26, wherein the enzymatic solution comprises one or more selected from the group consisting of: glycolytic enzymes, lipases, proteases, peptidases, nucleases, and amylases.

[0187] EXAMPLES

[0188] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples, therefore, specifically point out exemplary embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0189] Example 1: Affinity Array Capture for Single Cell Multi-Omic MALDI-MSI

[0190] Single-cell analysis offers an unprecedented lens into the complexity of biological systems, enabling researchers to observe cellular behaviors and differences that would otherwise be lost in the noise of bulk analysis. By isolating and examining individual cells, it becomes possible to uncover cellular heterogeneity, identify rare populations, and decode nuanced biological processes at a resolution inaccessible to traditional methods.

[0191] Techniques such as single-cell RNA sequencing (scRNA-seq), single-cell proteomics, single-cell DNA sequencing (scDNA-seq), high-resolution imaging, and flow cytometry have all been employed to dissect the individuality of single cells. These approaches have expanded the understanding of cellular dynamics, yet the sophistication and cost of these methods often pose barriers to accessibility. Instrumentation for purification and analysis is typically specialized, demanding both technical expertise and advanced bioinformatics for multi-omic integration — especially when different types of molecular data must be co-analyzed from the same cell. Despite these challenges, the impact of single-cell technologies across research, diagnostics, and therapeutic development continues to grow.

[0192] Matrix-assisted laser desorption / ionization mass spectrometry imaging (MALDI-MSI) presents a unique opportunity to expand single-cell analysis. Its capacity to ionize a wide range of analytes — including proteins, lipids, and glycans — makes it an adaptable tool for molecular imaging across diverse biological formats. MALDI-MSI has historically been used to analyze tissues, serum, captured cells, and even individual biomolecules. More recently, efforts have shifted toward enhancing the spatial resolution required to image individual cells within complex tissue environments. However, these high-resolution applications often come at the cost of throughput, with long acquisition times, large data volumes, and substantial instrument wear limiting scalability. Described in this workflow is a new approach that utilizes an affinity array-based MALDI-MSI platform to detect and analyze single cells. This method leverages antibody or lectin microarrays created using polydimethylsiloxane (PDMS) stamps, where each micro-spot captures one to four cells. Once cells are captured, a trained convolutional neural network scans the slide, identifies single-cell locations, and logs their coordinates.

[0193] Unlike traditional raster-based MALDI imaging — which requires the laser to scan entire regions — this coordinate-targeted approach confines each ablation to a specific cell. The result is a dramatic increase in throughput and spectral clarity, as each pixel directly corresponds to a single cell. Notably, this workflow allows for multiple rounds of molecular analysis on the same captured cell, opening the door to multi-omic interrogation at scale.

[0194] To construct the capture arrays, PDMS stamps are coated with antibody or lectin solutions, which bind electrostatically to the stamp surface. These stamps are then pressed onto hydrogel-coated slides, where the protein layer covalently transfers to the substrate in defined micro-patterns. Slides are fitted with well chambers to accommodate multiple samples, and cell suspensions are applied and incubated to promote capture (Figure 2). By spacing capture spots evenly, the laser can target each cell individually without overlap, significantly improving acquisition speed and reducing noise (Figure 3). Following a gentle wash and fixation step, captured cells remain anchored in precise positions, ready for imaging.

[0195] Both antibodies and lectins offer distinct advantages for cell immobilization. Antibodies enable selective capture based on surface epitopes, while lectins — such as concanavalin A (ConA) — serve as universal capture agents, binding a variety of cell types across species and phenotypes. Experiments have shown that ConA effectively captures Jurkat cells, human PBMCs, and murine 4T1 cells, demonstrating compatibility with both adherent and suspension cells from diverse origins (Figure 4).

[0196] Following capture and fixation, cells are arranged in a defined grid across the slide (Figure 5). Determining the position of each single cell, excluding multi-cell and empty spots, is a critical step for efficient data acquisition. To automate this process, a Python-based machine learning tool named SoloCell was developed. SoloCell offers two complementary approaches for cell identification. The first is a grid-aware approach (Figure 6) where the user selects an area on the slide. The user then marks ten cells: eight adjacent cells (colored purple) and two additional cells (colored blue) that are the farthest away from each other while being in the same column or row. The eight adjacent cells are used to calculate the average distance between adjacent cells, while the other two cells are used to calculate the angle of the grid structure. SoloCell then creates the first frame (i.e., seeding frame) around one of the selections. It then moves to the next location by using the angle and average distance calculated in the previous step. Since the grid may not have a perfect geometry, the next frame searches the nearby region (dashed region) to find the darkest area by utilizing a contour detection algorithm. SoloCell then centers the darkest area in the frame and continues to the next location until reaching the end of the area. This task is performed in four directions to determine the column and row positions of the entire grid. SoloCell then creates the rest of the frames using the column and row positions. Each frame is scored by a convolutional neural network trained to distinguish between single cells, multiple cells, and empty spots (Figure 7). Users are then shown a histogram of confidence scores and can set a threshold to select single cells for imaging

[0197] The second approach (Figure 8) provides enhanced flexibility by enabling user-adjustable parameters for cell detection without requiring reference point selection. In this method, users can fine-tune HSV (Hue, Saturation, Value) thresholds for contour detection, specify minimum and maximum cell sizes, set minimum circularity requirements for each detected cell location, and define the burst area size. Users can also enhance image quality for improved detection by adjusting contrast, sharpness, brightness, and applying blurring as needed, making the cell selection process more adaptable to varying sample qualities and imaging conditions. The burst area size is particularly significant as it represents the region that will be targeted by the mass spectrometer. By precisely defining this parameter, SoloCell can eliminate conflicting burst areas to prevent multiple shots on a single region and avoid capturing multiple cells in one shot, thereby enhancing the specificity of single-cell analysis.

[0198] Upon final selection of single cells SoloCell’s algorithm calculates optimal ablation paths across thousands of cells by solving a variant of the traveling salesman problem, significantly reducing the laser ablation path and overall instrument run time (Figure 9). Once finalized, SoloCell outputs coordinate files compatible with mass spectrometry imaging software, such as Fleximaging, for targeted ablation.

[0199] Fleximaging setup involves defining probe spot diameters and using teach points to calibrate the system. A single 7 mm x 5 mm capture region — representing one well from a 24- well format — can yield over 2,000 single-cell coordinates (Figure 10). These cells can be ablated in under six minutes, enabling ultra-high-throughput analysis with a true 1 : 1 pixel-to-cell ratio.

[0200] Although single cells were captured and identified to “teach” the instrument to examine them, the question remained if anything associated with those cells could be detected. Figure 20 demonstrate that two major “omic” methods can be performed on these captured cells: lipidomics and glycomics, (Figure 11 top and bottom, respectively). Both of these methods were performed on the same antibody captured cell. As Figure 11 shows, just from a single cell, lipid signals were successfully obtained from cells captured by the antibody array. Indeed, over 200 lipid species were detected. Subsequently, because the cells are fixed and remained attached to the antibody, they can be washed and re-analyzed for the presence of N-linked glycan using enzyme-based glycan imaging. Figure 12 compares averaged single-cell signal to 'bulk signal,' where thousands of captured cells are imaged in one spot. The average single-cell signal is comparable to bulk analysis, exhibiting R2values greater than 0.93 for both lipids and N-glycans, highlighting the sensitivity of the method. To ensure cells remain intact through multiple rounds of fixation, laser ablation, antigen retrieval and various washes, Figure 13 shows images of cells taken after each round of imaging to confirm that they remained intact and adhered to the slide.

[0201] Importantly, co-capture experiments involving human Jurkat and murine 4T1 cells were conducted to assess the accuracy of classification using molecular profiles. Cells were mixed and captured on ConA arrays in both bulk and single cell formats (Figure 14), then analyzed for 134 lipid species and 39 N-glycans previously identified in bulk samples (Figure 18 and Figure 19). Differential expression patterns emerged clearly between the two cell types, with several lipids and glycans showing strong enrichment in one population over the other.

[0202] Dimension reduction using UMAP on single-cell data revealed two distinct molecular clusters for both N-glycans and lipids that corresponded with the Jurkat and 4T1 bulk profiles. While the single cells were not orthogonally phenotyped, overlaying the bulk-derived profiles onto the UMAP plot aligned each cell type with its respective cluster. This agreement was further validated by comparing fold-changes in expression levels, showing high correlation between bulk and single-cell data (Figure 15 and Figure 16).

[0203] To assess cross-modality consistency, cells identified as 4T1 based on their N-glycan signature were labeled and mapped onto the lipid UMAP. Over 91% of these cells are localized within the expected 4T1 lipid cluster, highlighting strong agreement between lipid- and gly canbased classification strategies (Figure 17).

[0204] Other methods of single cell capture using reverse antibody array fabrication were tested. This involves the stamping of a blocking protein, such as BSA on the whole slide, excluding capture spots that can be incubated and filled in with capture protein in subsequent steps. Denatured BSA was found to provide superior uniformity compared to native BSA. When stained with Coomassie, 5 pm holes were visible (Figure 20 and Figure 21).

[0205] The methods and materials used are described herein.

[0206] Materials

[0207] PDMS stamps in 30 pm Diameter * 60 pm Spacing x 40 pm Tall and 40 pm Diameter x 240 pm Spacing x 40 pm Tall configurations were obtained by Research Micro Stamps (Clemson, SC). Amine reactive slides (Nexterion Slide H Hydrogel Coated Glass Slides) were obtained from Applied Microarrays (Tempe, AZ). 4 Path Buffer and Well modules were obtained from Grace Bio-Labs (Bend, OR), a-cyano-4-hydroxycinnamic acid was obtained from Sigma Aldrich (St. Louis, MO). Peptide-N-glycosidase F Prime™ (PNGase F) and Sialidase Prime™ were obtained from N-Zyme Scientifics (Doylestown, PA). Human anti-CD7 (Clone 124-1D1) was obtained from Thermo Fisher Scientific (Waltham, MA). Concanavalin A (ConA) was obtained from Vector Laboratories (Newark, CA). Jurkat cells (Clone E6-1) and 4T1 cells were obtained from ATCC (Manassas, VA).

[0208] Bulk Capture Spot Preparation

[0209] ConA or antibodies were spotted onto Nexterion slide H hydrogel-coated glass slide to facilitate “bulk” capture where thousands of cells are captured per spot. Either 500 ng of ConA or 200 ng of antibody in 1 xPath buffer was spotted on pre-equilibrated Nexterion slide H hydrogel-coated glass slides in 1.5 pl spots. Spotted slides were placed in a preheated humidity chamber for 1 hour to facilitate covalent binding of the protein to the hydrogel. Post incubation spots were dried in a desiccator. A 24 well slide adaptor was positioned over the slide with each capture spot positioned in the middle of each well. The slide was blocked by adding 100 pL of 100 mM ammonium bicarbonate to each well chamber and incubated for 30 minutes in a humidity chamber. Post blocking, the slide was washed 2* with PBS and 1 x with HPLC water and dried in a desiccator.

[0210] Single Cell Array Preparation by direct stamping

[0211] Two different configurations of PDMS stamps were used to capture cells. One configuration consisted of pillars that are 30 pm Diameter x 60 pm Spacing x 40 pm Tall, the other was 40 pm Diameter x 240 pm Spacing x 40 pm Tall. The PDMS stamp was initially washed prior to inking with a capture protein to remove debris and any bound protein from previous uses. The following wash steps were used, 5 minutes 100% ethanol sonication, 5 minutes 100% isopropanol sonication and 5 minutes HPLC water sonication. The stamp was dried under a stream of nitrogen gas. Antibody diluted in PBS at a concentration of 50 pg / ml or Lectin diluted in PBS to a concentration of 100 pg / ml was pipetted on the surface of the PDMS stamp. PDMS is extremely hydrophobic, which will initially repel the protein solution, causing it to bead. A cover slip cut to the proportions of the PDMS stamp was placed over top of the capture solution to ensure even coating with the PDMS. The PDMS stamp was placed in a preheated humidity chamber for 30 minutes, allowing the antibody or lectin to fully bind to the PDMS. Post incubation the coverslip was removed, using tweezers to pick up the PDMS stamp at the nonfunctional edges, the stamp was dipped in HPLC water for 10 seconds to remove any unbound protein. The PDMS stamp was dried under a stream of nitrogen gas.

[0212] The inked PDMS stamp was carefully placed on the surface of a room temperature equilibrated Nexterion slide H hydrogel-coated glass slide. Once placed, the stamp location was marked with a marker on the opposite side of the slide. A 10-gram scale calibration weight was placed on top of the PDMS stamp to ensure contact with the hydrogel surface. The slide and PDMS stamp were placed in a preheated humidity chamber for 1 hour. Post incubation the 10- gram weight and PDMS stamp were removed and a ProPLate Multi-Array Slide Chamber, 24- well (Grace Biolabs, Bend, OR) was attached, using the marked positions to ensure the designated chamber was attached over the stamped region. The slide was blocked with 100 pl of 100 mM ammonium bicarbonate in each well chamber. During blocking the slide was placed in a humidity chamber for 30 minutes. Post blocking the blocking solution was removed and the slide was washed with 300 pL PBS 2x and 300 pL HPLC water lx. Slides with attached well chambers were dried in a desiccator prior to cell capture.

[0213] Single Cell Array Preparation by reverse stamping In order to achieve true single-cell capture, a second method of producing a single-cell capture array was developed, taking advantage of the “negative space” left over from stamping a surface. A substrate, such as an epoxysilane-coated glass slide, is printed with negative space by contacting the substrate with a stamp that has been coated with a blocking protein, such as bovine serum albumin (BSA). Heat denature BSA was found to provide a much more uniform stamping. By contacting the surface with the coated stamp, the substrate is covered with blocking protein except for the negative space, which corresponds to holes in the stamp (Figure 20 and Figure 21).

[0214] The substrate is then treated with a solution of antibodies for a desired cell-surface antigen, with antibodies only binding to the substrate surface in the negative space, enabling precise spotting of antibodies of the desired size, shape, and pattern.

[0215] Cell Culture

[0216] Jurkat cells (Clone E6-1, ATCC, Manassas, VA) and 4T1 cells (ATCC) were cultured in RPMI 1640 medium supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin and streptomycin. Cell culture conditions were 37 °C at a 5% CO2 humidified atmosphere.

[0217] Cell Capture

[0218] Cells were washed 3x in 1% BSA solution and resuspended at 1 x 106cells / mL for cell capture using capture spots 30 pm in diameter and 5 x 105cells / mL if capture spots were 40 pm in diameter. Reduced seeding density was required for larger spot diameters to mitigate multiple cells binding to the same capture spot. 100 pL of the cell suspension was added to one 24 well chamber containing the PDMS stamped capture array resulting in either 100,000 or 50,000 cells seeded per well chamber. Seeded slides were placed in a 10 cm diameter cell culture dish in cell culture incubator set to 37 °C at a 5% CO2 humidified atmosphere. Here the slides underwent static incubation for 30 minutes if 30 pm diameter capture spots were used and 20 minutes for 40 pm diameter capture spots. Reducing the incubation time for larger capture spots reduced multiple cells from binding to the same capture spot. For cell types other than Jurkat cells, it is recommended to perform a dilution series to determine the optimal cell concentration for effective single-cell capture, as variations in cell size and behavior can influence capture efficiency. Post incubation cells were removed from the incubator and excess cell suspension was dumped. The slide chambers were carefully removed, and the slide was slowly dipped into a 50 mL conical tube containing l x PBS for 1 minute to remove any unbound cells. Slides should not be washed with PBS by pipetting directly into the well chambers or by pouring PBS directly onto the slide, otherwise captured cells will be dislodged. Slides were then fixed for 20 minutes by carefully dipping the slide into a 50 mL conical tube containing 10% neutral buffered formalin. Post fixation, slides were washed 2x in HPLC water for 1 minute. Teach points were etched on the edges of each capture well. The slide was dipped once more in HPLC water for 10 seconds to remove any glass debris created from etching. The slides were then dried in a desiccator. Post drying, slides were imaged on a Nanozoomer 2.0RS high resolution scanner (Hamamatsu, Hamamatsu city, Japan) at 40x magnification across each individual 5 mm x 7 mm capture well.

[0219] Lipid Imaging

[0220] Slides were dipped in cold 50 mM ammonium acetate solution 2x to eliminate lipid adducts and then dried in a desiccator. Slides were sprayed with CHCA (70% acetonitrile / 0.1% trifluoracetic acid, 5 mg / mL) using a M5 TM-Sprayer (HTX Technologies) with 10 passes at 50 pL / min, 1000 mm / s, 75 C, 3 mm spacing with a dry time of 30 seconds. Slides were then dipped in cold ammonium phosphate monobasic (5 mM) solution two times and vertically dried in a desiccator. Imaging experiments were conducted using a TimsTOF Flex mass spectrometer (Bruker, Bremen, Germany) using a SmartBeam 3D 10 kHz tripled Nd:YAG laser. A resulting field size of 60 pm coupled with 600 laser shots was used for captured cells spaced apart 60 pm. A wider resulting field size of 80 pm coupled with 800 laser shots was used for capture spots spaced apart 240 pm. Imaging was conducted in positive ion mode over a mass range of 250- 1650 Da. Data was analyzed in SCiLS Lab 2024a and normalized to total ion count. METASPACE was used for putative lipid identification using the CoreMetabolome-v3 database. Glycan Imaging

[0221] Post lipid imaging slides were dipped in 100% ethanol (30 secondsx1) to remove the CHCA matrix. Captured cells were then delipidated using wash steps of Camoy’s solution (10% glacial acetic acid, 30% chloroform and 60% 200 proof ethanol) (10 minutes x 2), 100% ethanol washes (2 minutesx2), and HPLC water washes (3 minutesx2). Post delipidation slides were then taken through antigen retrieval in which slides were placed in a slide mailer containing citraconic buffer pH 3 and antigen retrieved for 20 minutes in a vegetable steamer. Following antigen retrieval slides cooled by letting stand for 5 minutes. After 5 minutes half of the solution was dumped from the slide mailer and replaced with HPLC water and let stand for another 5 minutes. This was repeated for a total of 3 x, followed by a HPLC water wash. Slides were dried in a desiccator. N-glycans and their terminal sialic acids were released from captured cells by dual spraying PNGase F PRIME™ and Sialidase PRIME™. 100 pg of each PNGase F and sialidase were mixed in 1 mL of HPLC water. Spraying parameters consisted of 10 passes at 25 pL / min, 1200 mm / s, 45 °C, 3 mm spacing between passes, with 10 psi nitrogen gas. Slides were then incubated at a relative humidity of 85% for 2 hours at 37 °C using a custom-built humidity chamber consisting of a HumidiCup Ml electronic Cigar humidifier placed in a sealed Tupperware container set to 85% relative humidity. Higher relative humidity will result in delocalization of N-glycans. a-cyano-4-hydroxyxinnamic acid (50% acetonitrile / 0.1% trifluoracetic acid, 5 mg / mL) was sprayed with 34 passes at 25 pL / min, 2000 mm / s, 79 °C, 3 mm spacing. Imaging experiments were conducted using a TimsTOF Flex mass spectrometer (Bruker, Bremen, Germany) using a SmartBeam 3D 10 kHz tripled Nd:YAG laser. A resulting field size of 60 pm coupled with 600 laser shots (10,000 Hz) was used for captured cells spaced apart 60 pm. A wider resulting field size of 80 pm coupled with 800 laser shots (10,000 Hz) was used for capture spots spaced apart 240 pm. Imaging was conducted in positive ion mode over a mass range of 700-4000 Da. Data was analyzed in SCiLS Lab 2024a and normalized to total ion count. Our in-house N-glycan database was used to putatively identify N-glycans.

[0222] SoloCell CNN

[0223] SoloCell’s CNN follows a sequential architecture beginning with a 2D convolutional layer (32 filters, 3^3 kernel, ReLU activation) and batch normalization, followed by successive convolutional layers with 64, 128, and 256 filters. Each layer is paired with batch normalization and interspersed with max pooling layers. The network employs global average pooling and two fully connected layers (256 and 128 neurons) with ReLU activation and 50% dropout, concluding with a sigmoid activation layer for binary classification (Figure 7). The model was trained with 20,000 manually labeled frames, with 90% of the data used as training, 5% as validation, and 5% as test sets, achieving 98.5% accuracy on the test set, with a precision of 98.3%, recall of 98.7%, and Fl score of 98.5%. During training, extensive data augmentation was applied, including rotations (90°, 180°, 270°), horizontal flipping, brightness adjustments, and contrast reduction, with all images normalized to [0, 1 ], The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

[0224] While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

What is claimed is:

1. A single-cell capture array comprising: a) a substrate; and b) a plurality of cell-binding molecule spots on the substrate, wherein the cell-binding molecule spots are between 1 pm and 100 pm in diameter and wherein the distance between each spot is between 10 pm and 500 pm.

2. The single-cell capture array of claim 1, wherein the cell-binding molecules in the cellbinding molecule spots are selected from the group consisting of antibodies, lectins, and aptamers.

3. The single-cell capture array of claim 1, wherein the cell-binding molecule spots are in a square grid and the distance between each spot is between 10 and 300 pm.

4. The single-cell capture array of claim 1, wherein the cell-binding molecule spots are between 1 pm and 50 pm in diameter.

5. The single-cell capture array of claim 1, wherein the substrate is selected from the group consisting of glass, polydimethylsiloxane (PDMS), epoxysilane-coated glass, oxygen plasma-treated PDMS, and amine-reactive hydrogel-coated glass.

6. The single-cell capture array of claim 1, wherein all cell-binding molecules in a single cell-binding molecule spot bind a single antigen.

7. The single-cell capture array of claim 1, wherein the array of cell-binding molecule spots comprises a plurality of spots against one or more antigens, wherein the cell-binding molecules in each spot bind a single antigen.

8. The single-cell capture array of claim 1 , wherein a cell-binding molecule spot comprises two or more cell-binding molecules that bind two or more antigens.

9. A method of analyzing the profile of single cell comprising the steps of: a) providing a single-cell capture array of any one of claims 1-8; b) incubating the single-cell capture array with a sample containing the cell to be analyzed; c) rinsing the single-cell capture array to remove unbound cells, leaving only a single cell on each antibody spot; d) fixing the cells; e) detecting single cells through an algorithm which identifies and marks single cells; and f) scanning the single-cell capture array by mass spectrometry.

10. The method of claim 9, wherein the mass spectrometry is selected from the group consisting of: matrix-assisted laser desorption / ionization imaging Fourier transform ion cyclotron resonance (MALDLFTICR) mass spectrometry, matrix-assisted laser desorption / ionization time of flight (MALDI-TOF) mass spectrometry, scanning microprobe MALDI (SMALDI) mass spectrometry, infrared matrix assisted laser desorption electrospray ionization (MALD-ESI) mass spectrometry, surface-assisted laser desorption / ionization (SALDI) mass spectrometry, desorption electrospray ionization (DESI) mass spectrometry, secondary ion mass spectrometry (SIMS) mass spectrometry, and easy ambient sonic spray ionization (EASI) mass spectrometry.

11. The method of claim 10, wherein the step of scanning the single-cell capture array is preceded by a step of spraying the single-cell capture array with a MALDI matrix material.

12. The method of claim 11, wherein the MALDI matrix solution is selected from the group consisting of: 2,5-dihydroxybenzoic acid, a-cyano-4-hydroxycinnamic acid, sinapinic acid, 1,5 -di ami nonaphthalene, and 9-aminoacridine.

13. The method of claim 9, wherein the step of fixing the cells is preceded by spraying the single-cell capture array with an enzymatic solution.

14. The method of claim 13, wherein the enzymatic solution comprises one or more selected from the group consisting of: glycolytic enzymes, lipases, proteases, peptidases, nucleases, and amylases.

15. The method of claim 9, wherein step e) comprises scanning the single-cell capture array for one or more profdes selected from the group consisting of: a genomic profile, a proteomic profile, a glycomic profile, a lipidomic profile, a metabolomic profile, and a transcriptomic profile.

16. A kit for analyzing the profile of a single cell from a sample comprising: a single-cell capture array of any one of claims 1-8; and a MALDI matrix material.

17. The kit of claim 16, wherein the MALDI matrix material is a-cyano- 4hydroxycinnamic acid.

18. The kit of claim 16, wherein the kit further comprises an enzymatic solution.

19. The kit of claim 16, wherein the kit further comprises a non-transitoiy computer- readable medium with instructions stored thereon, which when executed by a processor, perform steps comprising identifying single cells.

20. A kit for analyzing the profile of a single cell from a sample comprising: a substrate; a stamp; a blocking protein; and a MALDI matrix material.

21. The kit of claim 19, wherein the kit further comprises a cell-binding molecule.

22. The kit of claim 20, wherein the cell binding molecule is selected from the group consisting of antibodies, lectins, and aptamers.

23. The kit of claim 20, wherein the blocking protein is bovine serum albumin (BSA).

24. The kit of claim 20, wherein the MALDI matrix material is a-cyano- 4hydroxycinnamic acid.

25. The kit of claim 20, further comprising a non-transitory computer-readable medium with instructions stored thereon, which when executed by a processor, perform steps comprising identifying single cells.

26. The kit of claim 20, wherein the kit further comprises an enzymatic solution.

27. The kit of claim 26, wherein the enzymatic solution comprises one or more selected from the group consisting of: glycolytic enzymes, lipases, proteases, peptidases, nucleases, and amylases.

Images