Single molecule separation system for cell populations and single cells and methods and uses thereof

Abstract

The present invention relates to systems and methods for isolating single cells, capturing selected prey biomolecules within the cells, and detecting the prey biomolecules using sensitive assays. Also described herein are functionalized substrates and compositions capable of detecting selected prey biomolecules of selected cells with low detection limits.

Description

[0001] Related Applications

[0002] This patent application claims the benefit of U.S. Provisional Application No. 62 / 922,657, filed on August 22, 2019, the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] The invention described herein generally relates to the field of protein and / or cell detection. Specifically, embodiments described herein relate to systems and methods for isolating single cells, capturing selected prey biomolecules within the cells, and detecting the prey biomolecules using sensitive assays.

[0004] Incorporated by Reference

[0005] All U.S. patents, U.S. patent application publications, foreign patents, foreign and PCT published applications, articles and other documents, references and publications identified herein, as well as all documents cited as references in any one or more of the patents issued thereby, are hereby incorporated by reference in their entirety. The incorporated information is a part of this application, as if all text and other content were repeated in this application, and will be considered a part of the text and content of this application as filed. Background Art

[0006] The following includes information useful for understanding the present invention. It is not an admission that any information, publication or document explicitly or implicitly cited herein is prior art to the invention described or claimed in this application, or is essential to the invention described or claimed in this application.

[0007] A protein never acts alone: ​​it acts with other proteins that function as its regulators, auxiliary subunits, or effectors. Functional associations of proteins usually require physical interactions with other proteins, or protein-protein interactions (PPIs), although they occasionally involve only soluble factors, such as cAMP or Ca 2+ Therefore, PPIs are indispensable in nearly all aspects of diverse cellular processes, and the identification of protein-interaction partners is essential for elucidating protein function and regulation.

[0008] To globally assess PPIs, a commonly used and powerful technique is conventional co-immunoprecipitation (co-IP / pull-down) followed by western blotting. However, conventional co-IP assays cannot be used to study PPIs in rare cells such as sensory hair cells, circulating tumor cells, and embryonic stem cells: a single western blot assay typically requires about 10 4 -105 cells, and a single co-IP assay requires approximately 10 5 -10 6 Cells are rare, and rare cells cannot be easily obtained in such large quantities (Schulte R. et al., "Single Bead Affinity Detection (SINBAD) for the Analysis of Protein-Protein Interactions", PLoS ONE, 3(4): e2061 (2008). https: / / doi.org / 10.1371 / journal.pone.0002061). For example, hair cells are extremely rare; the number of cochlear hair cells is about 3,300 per mouse cochlea and about 15,000 per human cochlea, and the efficiency of isolating these cells from surrounding supporting cells is extremely low (Willott JF et al., "Modulation of Presbycusis: Current Status and Future Directions", Audiol Neurootol., 6: 231-249 (2001)). Currently, only heterologous overexpression systems are used to study PPI in hair cells, but these are affected by human factors and need to be confirmed by studies of endogenous proteins. This technical limitation of conventional co-IP assays greatly hinders the study of hair cells and other rare cells, so it is necessary to develop single-cell pull-down techniques. Other methods for studying cell-to-cell changes include single-cell RNA sequencing and single-cell Western blotting (Kang, CC et al., "Single-cell Western Blotting After Whole-Cell Imaging to Assess Cancer Chemotherapeutic Response", Anal. Chem., 86 (20): 10429-10436 (2014)).

[0009] Recently, Jain et al. developed a single-molecule pull-down assay (SiMPull) that combines the principles of conventional pull-down and single-molecule fluorescence microscopy (Jain, A. et al., "Single-molecule pull-down for studying protein interactions", Nat. Protoc., 7(3):445-452 (2012)). SiMPull enables not only the evaluation of the stoichiometry of protein complexes, but also the examination of K in the micromolar range. d The weak and short-lived PPIs are characterized by the presence of a large number of weak and short-lived PPIs (Stelzl, U. et al., "A Human Protein-Protein Interaction Network: A Resource for Annotating the Proteome", Cell, 122(6):957-968 (2005)). These weak PPIs are easily destroyed in the multiple washing steps used in conventional co-IP assays. However, SiMPull has not been widely used since its development, nor has it been commercialized on a large scale, because the method presents high technical barriers and is time-consuming.

[0010] Existing single-cell pull-down methods can isolate single cells, but involve too many difficulties in expanding their use for analysis of cellular components, as evidenced by the complete lack of any commercial systems or methods that adopt their use (Wedeking, T. et al., "Single Cell GFP-Trap Reveals Stoichiometry and Dynamics of Cytosolic Protein Complexes", Nano Lett., 15(5):3610-3615 (2015); Wang, X. et al., "Toward Single-Cell Single-Molecule Pull-Down", Biophysical Journal, 115(2):283-288 (2018); Ryu, JY et al., "Profiling Protein-Protein Interactions of Single Cancer Cells with in situ Lysis and Co-Immunoprecipitation", Lab Chip., 19(11): 1922-1928 (2019)). The applicability of these methods is questionable because (1) the methods can be used to analyze molecules that diffuse slowly only in bacteria or adherent cell cultures but not in primary or suspension cultured cells (such as blood cells and circulating tumor cells); 2) the methods can only be used to analyze cell surface proteins using antibodies against the extracellular domains of cell surface proteins; and 3) the technical barriers of the methods are extremely high and therefore the methods cannot be used in general biological laboratories.

[0011] Thus, there remains a considerable unmet need for single-cell pull-downs for cellular composition analysis. Summary of the invention

[0012] The invention described and claimed herein has many attributes and embodiments, including but not limited to those stated or described or mentioned in the Summary of the Invention. However, this is not intended to be all-inclusive, and the invention described and claimed herein is not limited to, or by, the features or embodiments identified in this Preface, including that the purpose of this Preface is merely illustrative and not limiting.

[0013] The present disclosure provides the use of a substrate comprising multiple micron-sized features for analyzing isolated single cells and / or selected components thereof. In some aspects, the present disclosure provides a system comprising a glass substrate comprising multiple micron-sized features, a plurality of antibody-coated beads, a plurality of primary antibodies for bait biomolecules, a plurality of bait molecules, a plurality of target prey biomolecules, and a plurality of primary antibodies for the target prey biomolecules. A plurality of antibody-coated beads may include a plurality of secondary antibodies for a primary antibody for a target bait biomolecule. A plurality of antibody-coated beads may include a plurality of primary antibodies for a target bait biomolecule. In some aspects, a plurality of antibody-coated beads may include secondary antibodies that bind to a plurality of primary antibodies that bind to bait biomolecules.

[0014] In some aspects, the system may further comprise one or more types of cells. The cells may comprise one or more types of target prey biomolecules. A plurality of antibody coated beads may be located within a plurality of micron-sized features. A plurality of cells may be located in a plurality of micron-sized features. A plurality of cells may include cells having an average diameter of about 30 microns. The average diameter of the micron-sized features may be configured to selectively separate cells having a diameter less than the diameter of the micron-sized features.

[0015] In some aspects, the plurality of target prey biomolecules may include molecules that can be found in the cell membrane of the cell. In some aspects, the plurality of target prey biomolecules may include molecules that can be found in the cytoplasm of the cell. In some aspects, the bait biomolecules and prey biomolecules may be independently selected from the group consisting of: proteins, DNA, RNA, mRNA, tRNA, cRNA, antibodies, antibody fragments, ScFv, peptides, and small molecules. The DNA or RNA may be further modified with biotin.

[0016] In some aspects, the system can further comprise a cell lysis buffer, which can include a non-denaturing detergent.

[0017] In some aspects, a plurality of micron-sized features may include a series of holes, each of which has a diameter of about 15 microns to about 45 microns, a distance between the centers of the holes ranging from about 100 microns to about 5000 microns, and a depth of about 25 microns to about 150 microns. In some aspects, the diameter of the series of holes may be about 30 microns. The distance between the centers of the holes may be about 150 microns. The depth of the holes may be about 75 microns. In some aspects, a plurality of cells may include cells having an average diameter less than the diameter of the holes. In some aspects, a plurality of micron-sized features may be etched directly into a glass substrate. In some aspects, a plurality of micron-sized features may be formed by PDMS (polydimethylsiloxane). The substrate may include cured PDMS. The glass substrate may be a glass cover having a thickness of 0.085 mm to 0.64 mm. In some aspects, the glass substrate is configured to be measurable with TIRF (total internal reflection microscopy). The holes may be circular, annular, spherical, or other shapes similar to a circle. A series of holes may also be oval or squoval. The hole can be a flat bottom (F-hole), a round bottom (U-hole), a C-bottom (which is a combination of a flat bottom and a round bottom, such as a flat bottom hole with a curved edge at the bottom) (C-hole); a star-shaped hole (which is a hole with a modified C-shaped geometry having one or more fins disposed on the bottom and / or sides of the hole); or a V-bottom (which is a tapered hole) (V-hole).

[0018] In some aspects, the plurality of antibody-coated beads may further comprise magnetic beads, which themselves may comprise an iron oxide core and an outer layer comprising functional groups enabling binding of biomolecules (preferably capture agents, preferably antibodies). In some aspects, the system may further comprise a magnet.

[0019] In some aspects, the present disclosure provides a system comprising a glass substrate comprising a plurality of micron-sized features, a plurality of first capture agents binding to bait biomolecules, a plurality of bait biomolecules, a plurality of prey biomolecules, a plurality of second capture agents binding to the prey biomolecules, wherein the plurality of first capture agents binding to the bait molecules may further include magnetic beads. Bait biomolecules, prey biomolecules, or both may be attached to fluorophores. A plurality of antibody-coated beads may be located within a plurality of micron-sized features. In some aspects, the system may further include one or more types of cells, and optionally, the cells may include one or more types of prey biomolecules. In some aspects, the system may further include a magnet. In some aspects, a plurality of cells may be in a plurality of micron-sized features. The plurality of micron-sized features may include a series of holes, each of which has a diameter of about 15 microns to about 45 microns, a distance between the centers of the holes ranging from about 100 microns to about 5000 microns, and a depth of about 25 microns to about 150 microns.

[0020] The present disclosure provides a method for identifying biomolecule-biomolecule interactions, comprising the steps of: (a) contacting one or more cell types with a system as described herein, (b) providing a lysis buffer to the system, and (c) imaging the system by fluorescence microscopy. In some aspects, at least one of the system components can be fluorescently labeled. Fluorescence microscopy can be selected from TIRF or confocal microscopy.

[0021] The present disclosure provides a method for identifying biomolecule-biomolecule interactions, the method comprising the following steps: (a) contacting a substrate comprising a plurality of micron-sized features with one or more cell types, wherein a portion or all of the one or more cell types enter a portion or all of the plurality of micron-sized features; (b) providing a plurality of antibody-coated magnetic beads to the substrate, wherein the antibody-coated magnetic beads comprise antibodies that bind to bait biomolecules; (c) optionally, applying a magnetic field to the antibody-coated beads; (d) contacting the substrate with a lysis buffer; (e) forming a complex selected from the antibody-coated magnetic beads and the bait biomolecule, the bait biomolecule and the prey biomolecule, or all of the bait biomolecule and the prey biomolecule bound to the antibody-coated magnetic beads; (f) providing an antibody or antibody complex that binds to the prey biomolecule; (g) imaging the antibody or antibody complex bound to the prey biomolecule by fluorescence microscopy. In some aspects, the cell may comprise a bait biomolecule and / or a prey biomolecule. In some aspects, the antibody or antibody complex that binds to the prey biomolecule is fluorescently labeled. In some aspects, the antibody complex that binds to the prey biomolecule may comprise a primary antibody that binds to the prey biomolecule, and a secondary antibody that binds to the primary antibody that binds to the prey biomolecule, wherein the secondary antibody is fluorescently labeled. In some aspects, the prey biomolecule and the bait biomolecule may be independently selected from the group consisting of proteins, DNA, RNA, mRNA, tRNA, cRNA, antibodies, antibody fragments, ScFv, peptides, and small molecules.

[0022] In some aspects, step (g) of imaging the antibody or antibody complex bound to the prey biomolecule by fluorescence microscopy can be performed by TIRF (total internal reflection microscopy) or confocal microscopy.

[0023] In some aspects, the methods described herein can have an analyte signal-to-noise ratio greater than 10, greater than 12, greater than 15, or greater than 20.

[0024] In some aspects, the methods described herein can separate a single prey biomolecule per nanometer-sized functionalized bead.

[0025] Brief description of the attached figure

[0026] The accompanying drawings, which are incorporated herein and constitute a part of this specification, illustrate several aspects of the present disclosure and together with the description further serve to explain the principles of the aspects and enable those skilled in the relevant art to practice and use the aspects. The drawings are for illustration purposes only, show exemplary non-limiting embodiments, and are not necessarily drawn to scale.

[0027] The patent or application file contains at least one drawing executed in color. Copies of the patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0028] Figure 1 A representative schematic diagram of a nanobead-based SiMPull for a cell population is shown. Magnetic nanobeads coated with streptavidin are modified by fixing a primary antibody on their surface for capturing target proteins (bait proteins) (see detailed protocol below). The cells are lysed and magnetic nanobeads are added to the lysate and cultivated for 30 minutes, and after capturing the prey protein by the beads, the protein is detected using a specific primary antibody and a fluorescently labeled secondary antibody. The nanobeads are washed three times to remove non-specifically bound proteins, transferred to a slide and covered with a cover slip, and imaged using a TIRF microscope. In some cases, the bait protein or prey protein is fluorescently labeled and can be directly visualized.

[0029] Figure 2 a-2c show GFP pull-down using nanobead-based SiMPull. Figure 2 a (labeled "a") shows the pull-down of GFP in preformed lysates of GFP-expressing HEK293T cells by streptavidin-coated magnetic nanobeads, which were surface-modified with a biotinylated second antibody plus anti-GFP, anti-FLAG, or anti-HA antibodies. In addition, cell lysates of untransfected HEK293T cells were used as negative controls. The very high signal-to-noise (S / N) ratio indicates that GFP was specifically pulled down by the anti-GFP antibody. Scale bar, 10 μm. Figure 2 b (labeled as “b”) shows the average intensity of fluorescent molecules per imaging area. Error bars represent standard deviation (sd) Figure 2 c (marked with "c") shows Figure 2 Distribution of photobleaching steps of GFP molecules in a. Most fluorescent spots are single molecules.

[0030] Figure 3 a-3c show the pull-down method applied to the PKA complex by using a nanobead-based approach. Figure 3 a (labeled "a") shows a schematic diagram of the PKA complex and its activation by cAMP. R, regulatory subunit; C, catalytic subunit. Figure 3 b (labeled as "b") shows the upper image: pull-down of PKA-C-eGFP (green) also pulls down PKA-R-mCherry (red). Figure 3 b and Figure 3Lower image in c (labeled "c"): Corresponding surface plot of upper image. Figure 3 c shows the upper image: pull-down of PKA-C-eGFP (green) without pull-down of mCherry (red) after addition of cAMP. All analyses were performed using confocal microscopy. Scale bar: 3 μm.

[0031] Figure 4 Representative schematics showing single-cell microwell arrays. A schematic top view (left) and cross-section (right) of a microwell chip are depicted. The microwells are 30 microns wide and 70 microns deep.

[0032] Figure 5 a and Figure 5 b shows the fabrication of the microporous substrate. Figure 5 a (labeled "a") shows in the left panel, microposts made on a silicon wafer by standard soft lithography. Features are arranged in a square configuration containing 9 10x10 microposts (30 micrometers in diameter and 70 micrometers in height). Right panel; magnification of the framed area. Figure 5 b (labeled "b") is shown in the left panel; the corresponding feature in the PDMS gel of panel a. Right panel; magnification of the framed area. Scale bars: 1.5 mm in the left panel; 400 μm in the right panel.

[0033] Figure 6 a and Figure 6 b shows the number of cells per microwell. 300 μl of suspended cells (about 10 6 cells / ml) were applied to the surface of a microwell chip (see Methods for more details). Figure 6 a (labeled as "a") shows cells trapped in the microwells in the field of view (indicated by white arrows). Scale bar, 100 μm. Figure 6 b (labeled as "b") shows the total data of the number of cells in each well of the chip (n=3). About 40% of the total microwells are occupied by cells; about 85% of the microwells occupied by cells are single cells, and it is speculated that this ratio will increase as more diluted cells are applied.

[0034] Figure 7 a and Figure 7 b shows the application of magnetic beads to the microwells. Figure 7 a (labeled as "a") shows a top view of a microwell with only HEK293T cells. Figure 7 b (labeled as "b") shows a top view of the microwells with HEK293T cells after applying magnetic nanobeads to the chip.

[0035] Figure 8 a and Figure 8b shows a representative schematic diagram of the single-cell pull-down assay. Figure 8 a (labeled as "a") shows that after the single cell is captured through the microwell, the surface-modified magnetic nanobeads (diameter 100±30 nm) are applied to the microwell chip. The nanobeads are streptavidin-coated magnetic nanobeads modified with the biotinylated second antibody and the first antibody of the bait protein. Figure 8 b (labeled as "b") shows that, for in situ cell lysis, lysis buffer is added to the top of the chip. After cell lysis and protein capture, magnetic nanobeads are pulled to the bottom of the microwells by a magnet and imaged by TIRF or confocal microscopy.

[0036] Fig. 9 a-9f show GFP pull-down on single cells in microwells. Fig. 9 a (labeled as "a") shows GFP pulled down by GFP-antibody-coated magnetic nanobeads after cell lysis. Most of the fluorescent spots are small, but some are larger. Fig. 9 b (labeled "b") shows a magnification of the framed area in panel d. Small spots are indicated by arrows, and large spots are indicated by arrows. Fig. 9 c (labeled as "c") shows that in a negative control experiment, GFP was not pulled down by magnetic nanobeads without GFP-antibody coating after cell lysis. Fig. 9 d (marked with "d") shows Fig. 9 Magnification of the boxed area in c. Fig. 9 e (labeled as "e") shows a small GFP spot that exhibited one-step photobleaching in a photobleaching experiment, indicating the presence of a single GFP molecule in the spot. Fig. 9 f (marked with "f") displays Fig. 9 b and Fig. 9 Total intensity scale of the points in d: sub-plots Fig. 9 a and Fig. 9 c: 20 microns; sub-image Fig. 9 b and Fig. 9 d: 3 microns.

[0037] Fig.10 a-10d show pull-down of PKA complex in single cells. Fig.10 a (labeled as "a") shows that both PKA-C-eGFP (green) and PKA-R-mCherry (red) were pulled down in single cells by anti-GFP-coated magnetic nanobeads in the absence of cAMP, but Fig.10 As shown in b (labeled "b"), in the presence of cAMP, only PKA-C-eGFP (green) was pulled down. Fig.10c (labeled as "c") shows PKA-C-eGFP (green) and PKA-R-HA (red) in single cells pulled down by magnetic nanobeads coated with anti-GFP. PKA-R-HA was visualized by immunostaining with anti-HA antibody and Alexa-561-conjugated secondary antibody. Fig.10 d (labeled as "d") shows that when PKA-R-HA was replaced by PKA-R-GFP, few HA-positive spots were seen, indicating that Fig.10 The HA staining in c is HA specific. Fig.10 In a-10d, the lower images are magnifications of the small framed areas in the upper overlay images, and the right images are their corresponding surface plots; scale bars: upper left, 20 μm; lower left, 3 μm; right, 3 μm. DETAILED DESCRIPTION

[0038] This description of the exemplary embodiments is intended to be read in conjunction with the accompanying drawings, which are considered a part of the entire written specification. In addition, the section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0039] It has also been found that by employing the systems described herein, nanobead-based methods designed for single cell isolation and analysis can be used for cell populations and, more importantly, single cells. The methods described herein are much simpler and faster than conventional pull-down methods, and are also significantly more widely applicable to all cell types. These two key features enable the methods described herein to be generally applicable in common biological and clinical laboratories. By providing the application of single cell isolation and analysis in the form of single cell capture as described herein, the methods described herein represent a major technological breakthrough in the field of single cell research.

[0040] Conventional co-immunoprecipitation methods (co-IP / pull-down) commonly used to explore protein-protein interactions (PPIs) require large numbers of cells and are therefore not suitable for studying PPIs in rare cells such as sensory hair cells, circulating tumor cells, and embryonic stem cells; this greatly hinders the study of rare cells, thus requiring the development of single-cell pull-down techniques. Equally important, such single-cell pull-down techniques can enable the study of cell-to-cell variations in PPIs.

[0041] Described herein is a magnetic nanobead-based approach to SiMPull for cell populations and, more importantly, single cells, utilizing substrates containing multiple micron-sized features.

[0042] The systems and methods described herein are particularly useful for:

[0043] 1) Assessing cellular heterogeneity in complex tissue samples. For example, in one embodiment, the relative expression of EGFR and GRB2 in different cells can be measured using the methods and systems described herein.

[0044] 2) Measuring the presence and extent of a dynamic network of complex cellular protein interactions and signaling pathways. For example, in one embodiment, the methods and systems described herein can be used to measure the HIF repressor pathway and the mTOR signaling pathway in the tumor microenvironment.

[0045] 3) Explore cell-to-cell variation in viral infection. For example, in one embodiment, the methods and systems described herein can be used to determine the rate of cell infection facilitated by a population of influenza viruses or COVID-19 viruses.

[0046] 4) Profiling cell type percentages in complex tissues. For example, in one embodiment, the methods and systems described herein can be used to measure and quantify immune cell populations in the tumor microenvironment.

[0047] 5) The methods and systems described herein can be used to validate the protein information of single-cell RNA-seq data.

[0048] 6) The methods and systems described herein can be used to detect small molecules in blood (eg, hemoglobin cells) or lymph fluid.

[0049] definition

[0050] As used herein, the term "comprising" is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended, and does not exclude additional, unrecited elements or ingredients from a drug (or, in the case of a method, a step). The phrase "consisting of excludes any element, step, or ingredient not specified in the drug (or, in the case of a method, a step). The phrase "consisting essentially of refers to the specified materials as well as those materials that do not significantly affect the basic and novel characteristics of the drug (or, in the case of a method, a step).

[0051] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation (whether malignant or benign) and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorders," "proliferative disorders," and "tumor" are not mutually exclusive when referred to herein.

[0052] As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth and / or proliferation. Some cancers are composed of cells that divide rapidly, while other cancers are composed of cells that divide more slowly than normal cells. Examples of cancer types may or may not include, for example, carcinomas, lymphomas (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastomas, sarcomas, and leukemias. More specific examples of such cancers may or may not include, for example, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0053] As used herein, the term "contacting" generally refers to providing proximity of one component, reagent, analyte, or sample to another component, reagent, analyte. For example, contacting may involve mixing a solution comprising functionalized nanoparticles with a sample comprising cells. A solution comprising a component, reagent, analyte, or sample may also comprise another component or reagent, such as dimethyl sulfoxide (DMSO) or a detergent, which promotes mixing, interaction, uptake, or other physical or chemical phenomena that facilitate contact between components, reagents, analytes, and / or samples. In some embodiments of the invention, contacting involves adding a solution comprising functionalized nanoparticles to a sample comprising cells using a delivery device, such as a pipette-based device or a syringe-based device.

[0054] As used herein, the terms "subject" and the like (including "individual" and "patient", all of which are used interchangeably herein) refer to any mammal, including humans, domestic animals and farm animals, as well as zoo animals, safari animals, sports animals or pet animals, which may or may not include dogs, horses, cats, sheep, pigs, cattle, etc. Preferred mammals are humans, including adults, children, and the elderly. Preferred sports animals are horses and dogs. Preferred pet animals are dogs and cats. The subject may be, for example, an aquatic park animal, which may or may not include dolphins, whales, seals, or walruses. In certain embodiments, the subject, individual, or patient is a human.

[0055] As used herein, the term "sample" may refer to a solution, suspension, mixture or undiluted amount of an aqueous solution containing target cells. In some embodiments, the target cells are rare cells. In some embodiments, the rare cells are skin cells. In some embodiments, the rare cells are circulating cancer cells. In some embodiments, the aqueous solution further comprises a buffer, a stabilizer, a cell lysing agent, a preservative, EDTA, etc. In some embodiments, "sample" is a body fluid. As used herein, the term "body fluid" may refer to any fluid that can be separated from the subject's body, which may include or not include: whole blood, plasma, serum, bile, saliva, urine, tears, sweat, cerebrospinal fluid (CSF), semen, swab samples (such as cheek swabs, nasal swabs, throat swabs, etc.), mucus, sputum, menstrual blood, menstrual fluid, vaginal mucus, amniotic fluid, synovial fluid, breast milk, lochia, thin mucus, lymph, pus, etc. In some embodiments, body fluid may more specifically refer to whole blood, serum, urine, saliva, swab samples, mucus or semen. In certain embodiments, the body fluid may more specifically refer to whole blood, serum, urine, or saliva. In some embodiments, the body fluid may contain an analyte of interest (eg, a target prey biomolecule).

[0056] As used herein, the terms "specifically bind" and "specific binding" mean that an antibody or capture agent binds to a target, such as another antibody, a bait biomolecule, or a prey biomolecule, with a greater affinity than it binds to other molecules under the specified conditions of the invention. In various embodiments of the invention, "specifically binds" may mean that an antibody or capture agent binds to a molecule unrelated to the target molecule with at least about 10 6 times greater, preferably at least about 10 7 times greater, more preferably at least about 10 times greater 8 times greater and most preferably at least about 10 9 Typically, specific binding refers to binding with an affinity that is approximately 10 times greater than nonspecific binding. 6 times to about 10 9 In some embodiments, specific binding may be characterized by a 10-fold greater affinity than nonspecific binding. 9 Whenever a range appears herein, such as in "1-10 or one to ten", the range refers to but is not limited to each integer or measurement unit in the given range. Thus, 1-10 means each of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and any sub-ranges therebetween.

[0057] As used herein, the term "prey" or "prey biomolecule" or "target prey biomolecule" refers to a target protein, peptide, or polynucleic acid (eg, DNA, mRNA, tRNA, cDNA) present in a selected cell type to be analyzed.

[0058] As used herein, the term "bait" or "bait biomolecule" refers to an identified protein, peptide, or polynucleic acid that can specifically bind to the prey biomolecule. The binding of the bait biomolecule and the prey biomolecule forms the basis of the detection event. In some embodiments, the bait biomolecule specifically binds to the prey biomolecule with high affinity. In some embodiments, inhibition kinetic studies can be used to measure high binding affinity. In some embodiments, high binding affinity can be less than 10 -3 M (i.e., 0.001 M), 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M or less. In some embodiments, the bait biomolecule is an antigen and the prey biomolecule is an antibody. In some embodiments, the bait biomolecule is a first protein and the prey biomolecule is a second protein. In such embodiments, the present invention can be used to measure protein-protein interactions (PPI). In some embodiments, the first protein and the second protein can be independently selected from antibodies, single-chain antibodies, antigen-binding antibody fragments, antigens, receptors, ligands, aptamers, aptamer receptors, polynucleic acids, cell membrane proteins, cytokines, transporter proteins, cell signaling molecules, viral particle components, bacterial particle components or housekeeping proteins. In some embodiments, when the prey biomolecule is a polynucleic acid, the bait biomolecule is a polynucleic acid comprising a sequence, a portion of which has a reverse complement to a portion of the sequence of the prey biomolecule, so that a portion of the bait biomolecule polynucleic acid sequence hybridizes with a portion of the prey biomolecule. In such embodiments, the present invention can be used to detect or separate selected polynucleic acids from cells in a sample. In such embodiments, the prey biomolecule and the bait biomolecule can be independently connected to a biotin functional group so as to enable streptavidin-antibody binding by the methods described herein.

[0059] As used herein, the term "magnetic beads" refers to any beads having at least some magnetic characteristics (e.g., ferromagnetism, paramagnetism, and superparamagnetism). In some embodiments, the magnetic beads comprise iron, nickel, cobalt, iron oxide, barium iron oxide, manganese oxide, chromium oxide, cobalt oxide, nickel oxide, cobalt manganese phosphide, or a combination thereof. In some embodiments, the magnetic beads may be coated with polymers, hydrogels, polyethylene glycol (PEG) and / or functionalized silanes. In some embodiments, the magnetic beads may be coated with a layer of silica around the magnetic characteristic inducing element. In some embodiments, the magnetic beads may be functionalized with carboxylic acids, amines, biotin, streptavidin, avidin, neutravidin, NHS, or maleimide. Methods for preparing magnetic beads are well known in the art, as described in, for example, U.S. Patent Nos. 6,773,812 and 6,280,618, the entire contents of which are incorporated herein by reference. Magnetic beads are available from commercial suppliers (which may or may not include: Sera-Mag Streptavidin Speedbeads TM ,Sera-Mag NeutravidinSpeedbeads TM 、Streptavidin-Mag or Streptavidin-Mag Agarose TM (CytivaSciences (formerly GE Healthcare, formerly Amersham Biosciences)); AbraMag Streptavidin Magnetic Beads TM or AbraMag Biotin Magnetic Beads TM (Abraxis, ThomasScientific); Dynabeads TM (Thermo Fisher), Beads (Miltenyi Biotec) or Turbobeads TM (Turbobeads)).

[0060] As used herein, the term "primary antibody against a bait biomolecule" refers to an antibody, a single-chain antibody (which may or may not include ScFv, nanobodies, cameloid antibodies), an antigen-binding antibody fragment, an antigen, a receptor, a ligand, an aptamer, an aptamer receptor, or a polynucleic acid that specifically binds to a bait biomolecule. In some embodiments, the primary antibody against a bait biomolecule is a monoclonal antibody. In some embodiments, the primary antibody against a bait biomolecule is a polyclonal antibody.

[0061] As used herein, the term "secondary antibody directed against a primary antibody directed against a bait biomolecule" refers to an antibody, a single-chain antibody (which may or may not include ScFv, nanobodies or camelid antibodies), an antigen-binding antibody fragment, an antigen, a receptor, a ligand, an aptamer, an aptamer receptor or a polynucleic acid that specifically binds to a portion of a primary antibody directed against a bait biomolecule. The epitope site of the primary antibody directed against a bait biomolecule to which the secondary antibody binds does not disrupt the binding of the primary antibody directed against the bait biomolecule. In some embodiments, the secondary antibody directed against a primary antibody directed against a bait biomolecule is either directly conjugated to a functional group on the secondary antibody (which may or may not include a carboxylic acid, an amine or a cysteine ​​of an amino acid of the antibody, or synthetically added to the antibody), or indirectly connected to a magnetic bead through a biotin-streptavidin (or neutravidin or avidin) interaction. In some embodiments, the secondary antibody directed against a primary antibody directed against a bait biomolecule is a monoclonal antibody. In some embodiments, the secondary antibody directed against a primary antibody directed against a bait biomolecule is a polyclonal antibody.

[0062] As used herein, the term "primary antibody against a prey biomolecule" or "primary antibody against a target prey biomolecule" refers to an antibody, a single-chain antibody (which may or may not include ScFv, nanobodies, or camelid antibodies), an antigen-binding antibody fragment, an antigen, a receptor, a ligand, an aptamer, an aptamer receptor, or a polynucleic acid that specifically binds to a prey biomolecule. In some embodiments, the primary antibody against the prey biomolecule binds to an epitope site on the prey biomolecule while the prey biomolecule interacts with the bait biomolecule. In some embodiments, the prey biomolecule interacts with the bait molecule to form a complex, and a portion of the primary antibody against the prey biomolecule binds to the complex. In some embodiments, the primary antibody against the prey biomolecule is labeled with a fluorophore.

[0063] As used herein, the term "secondary antibody directed against a primary antibody directed against a prey biomolecule" refers to an antibody, a single chain antibody (which may or may not include ScFv, nanobody or camelid antibody), an antigen-binding antibody fragment, an antigen, a receptor, a ligand, an aptamer, an aptamer receptor or a polynucleic acid that specifically binds to a primary antibody directed against a prey biomolecule. In some embodiments, a secondary antibody directed against a primary antibody directed against a prey biomolecule is provided to a primary antibody directed against a prey biomolecule, and the primary antibody directed against a prey biomolecule binds to the prey molecule (prey sequential addition). In some embodiments, a secondary antibody directed against a primary antibody directed against a prey biomolecule first binds to an epitope site of the primary antibody directed against the prey biomolecule, and then the primary antibody directed against the prey biomolecule binds to the prey biomolecule (prey complex addition). The epitope of the primary antibody directed against the prey biomolecule is not part of the prey biomolecule-bait biomolecule interaction, and the secondary antibody directed against the primary antibody directed against the prey biomolecule binds to the epitope. In some embodiments, the secondary antibody directed against the primary antibody directed against the prey biomolecule is a monoclonal antibody. In some embodiments, the secondary antibody directed against the primary antibody directed against the prey biomolecule is a polyclonal antibody. In some embodiments, the secondary antibody directed against the primary antibody directed against the prey biomolecule is labeled with a fluorophore.

[0064] As used herein, the term "capture agent" refers to a biomolecule that can selectively bind to a target biomolecule. In some embodiments, the capture agent includes an antibody, a single-chain antibody (which may or may not include a ScFv, a nanobody, or a camelid antibody), an antigen-binding antibody fragment, an antigen, a receptor, a ligand, an aptamer, an aptamer receptor, or a polynucleic acid that specifically binds to a prey biomolecule.

[0065] As used herein, the term "fluorophore" refers to a moiety that emits a fluorescent signal in response to a light stimulation event. The fluorophore can be a small molecule compound or a fluorescent protein. In some embodiments, the fluorophore can be selected from a chemiluminescent fluorophore or a fluorescent fluorophore. In some embodiments, small molecule fluorophores may or may not include: FAM, Cy dyes, AlexaFluors, Pacific Blue, Coumarin, BODIPY, Dansyl, Lucifer Yellow, rhodamine, Texas Red-X, Pacific Green, Oregon Green, Texas Red, Tetramethylrhodamine, Pacific Orange, eFlours, PE-eFlours, PerCP-eFluors, Super Bright Fluors, DyLight Fluors, StarBright Fluors, DRAQ and CyTRAK probes, EverFluor fluors, Bella Fluors, Atto tags, Abberior dyes, MegaSTOKES dyes, DYfluors, HiLyte Fluors, SeTau Dytes, Quasar and Cal Fluors, SureLight dyes, APC (allophycocyanin), APCXL, RPE, BPE, YOYO-1, and other fluorophores described in the following references: Handbook of Molecular Probes, Fluorescence The Molecular Probes Handbook, A Guide to Fluorescent Probes and Labeling Technologies) , 11th edition, by Iain Johnson and Michelle TZSpence, published by Life Technologies in 2010, and Invitrogen TM Molecular Probes Handbook: Fluorescent Probes and Labeling Technical Guide (Invitrogen TM Molecular Probes Handbook:A Guide to Fluorescent Probes and Labeling Technologies) , 11th edition (manufacturer code Invitrogen TMH37126), both of which are hereby incorporated by reference in their entirety. Fluorophores can be any fluorophore understood in the art and can be attached to antibodies by methods well known in the art. In some embodiments, functionalization of the antibody with a fluorophore can be performed by NHS chemistry or maleimide chemistry, such that the fluorophore is covalently conjugated to the antibody via an amine or cysteine ​​residue, respectively. In some embodiments, fluorescent proteins are independently co-expressed with bait biomolecules, prey biomolecules, or both.In some embodiments, the fluorescent protein may or may not include: GFP (green fluorescent protein), YFP (yellow fluorescent protein), CFP (cyan fluorescent protein), RFP (red fluorescent protein), mCherry, mNeon Green, Sirius, Sandercyanin, shBFP-N158S, Azurite, EBFP2, mKalama1, mTagBFP2, TagBFP, shBFP, ECFP, Cerulean, mCerulean3, SCFP3A, CyPet, mTurquise, mTFP1, monomeric Midoriishi-Cyan, Aquamarine, TurboGFP, TagGFP2, mUKG, Superfolder GFP, Emerald, EGFP, monomeric Azami Green, mWasabi, Clover, NowGFP, mClover3, TagYFP, EYFP, Topaz, Venus, SYFP2, Citrine, Ypet, IanRFP-deltaS83, mPapaya1, mCyRFP1, monomer Kusabira-Orange, mOrange, mOrange2, MOKk, MKO2, TagRFP, TagRFP-T, RRvT, mRuby, mRuby2, mTangerine, mApple, mStrawberry, FusionRed, mNectarine, mRuby3, mScarlet, mScarlet-I, mKate2, HcRed-Tandem, mPlum, mRaspberry, mNeptune, NirFP, TagRFP657, TagRFP675, mCardinal, mStable, mMaroon1, mGarnet2, iFP1.4, iFP713, iFP670, iFP682, iFP702, iFP720, iFP2.0, mIFP, TDsmURFP, miRFP670, Sapphire, T-Sapphire, mAmetrine, mKeima, mBeRFP, LSS-mKate2, LSS-mKate1, LSSmOrange or CyOFP1.

[0066] As used herein, the term "lysis buffer" or "cell lysis buffer" refers to an agent that is capable of disrupting cell membranes. The lysis buffer may include a denaturing or non-denaturing detergent (also referred to herein as a surfactant). When the target biomolecule is a polynucleic acid, a denaturing detergent may be used. When the target biomolecule is a protein or polypeptide that needs to retain a tertiary structure for detection, a non-denaturing detergent is used. In some embodiments, the denaturing detergent is SDS (sodium dodecyl sulfate). In some embodiments, the non-denaturing detergent is selected from: CHAPS, deoxycholate, Triton TM X-100, NP40 and Tween 20. The lysis buffer may further include buffer salts well known in the art.

[0067] In some embodiments, the prey biomolecule and the bait biomolecule are independently selected from cell surface proteins or cytosolic proteins. In some embodiments, the prey biomolecule and the bait biomolecule are independently selected from any protein in the Human Protein Atlas (https: / / www.proteinatlas.org / , accessed on June 26, 2020). In some embodiments, the prey biomolecule and the bait biomolecule are independently selected from the group consisting of: HLA-DR, kappa, lambda, Pax-5, BCL-2, Ki-67, ZAP-70, MPO, TdT, FMC-7, HER2, NEU, prostate stem cell antigen (PSCA), epithelial specific antigen (ESA), epithelial cell adhesion molecule (EpCAM), α2β1, VEGFR-1, VEGFR-2, CD133, AC133 antigen, CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD34, CD38, CD41, C43, CD45, CD56, CD57, CD58, CD61, CD64, C71, CD79a, CD99, CD103, CD117, CD123, CD138, CD138, CD163, CD235a, HLA-DR, κ, λ, Pax-5, BCL-2, Ki-67, ZAP-70, MPO, TdT, FMC-7, Pro2PSA, ROMA (HE4+CA-125), OVA1 (multiple proteins), HE4, fibrin / fibrinogen degradation products (DR-70), AFP-L3%, circulating tumor cells (EpCAM, CD45, cytokeratin 8, cytokeratin 18+, cytokeratin 19+), HER2, NEU, prostate stem cell antigen (PSCA), epithelial specific antigen (ESA), epithelial cell adhesion molecule (EpCAM), α2β1, VEGFR-1, VEGFR-2, CD133, AC133 antigen, p63 protein, c-Kit, CA19-9, estrogen receptor (ER), progesterone receptor (PR), Pro2PSA, HER-2 / neu, CA-125, CA15-3, CA27.29, free PSA, thyroglobulin, nuclear mitotic apparatus protein (NuMA, NMP22), α-fetoprotein (AFP) b, ROMA (HE4+CA-125), OVAL HE4, DR-70, p63 protein, c-Kit, CA19-9, total PSA, α-methylacyl-CoA racemase / AMACR, CA125 / MUC16, ERα / NR3A1, ERβ / NR3A2, thymidine kinase 1, AG-2, BRCA1, BRCA2, CA15-3 / MUC-1, caveolin-1, CD117 / c-kit, CEACAM- 5 / CD66e, cytokeratin 14, EGF R / ErbB1, HIN-1 / SCGB3A1, Ki-67 / MKI67, MKP-3, Nestin, NGF R / TNFRSF16, NM23-H1, PARP, PP4, serine protease inhibitor E1 / PAI-1, 14-3-3β, 14-3-3σ, 14-3-3ζ, 15-PGDH / HPGD, 5T4, A33, ABCBS, ABCB6, ABCG2, ACE / CD143, ACLP, ACP6, filamentous actin binding protein (Afadin) / AF-6, Afamin, AG-2, AG-3, Akt, aldehyde-keto reductase 1C3 / AKR1C3, α1B-glycoprotein, α1-microglobulin, αB crystallin / CRYAB, α-fetoprotein / AFP, α-methylacyl-CoA racemase / AMACR, AMFR / gp78, annexin A3, annexin A8 / ANXA8, APC, apolipoprotein AI / ApoA1, apolipoprotein A-II / ApoA2, apolipoprotein E / ApoE, APRIL / TNFSF13, ASCL1 / Mash1, ATBF1 / ZFHX3, attractant, Aurora kinase A (Aurora A), BAP1, Bcl-2, Bcl-6, β2-microglobulin, β-1,3-glucuronyltransferase 1 / B3GAT1, β-catenin, β-III tubulin, Bikunin, BMI-1, B-Raf, BRCA1, BRCA2, Brk, C4.4A / LYPD3, CA15-3 / MUC-1, c-Ab1, cadherin-13, calmodulin binding protein / CALD1, calmodulin 1, calretinin, carbonic anhydrase IX / CA9, catalase, cathepsin D, caveolin-1, caveolin-2, CBFB, CCR7, CCR9, CEACAM-19, CEACAM-20, CEACAM-4, CHD1L, chitinase 3-like 1, cholecystokinin-BR / CCKBR, chorionic gonadotropin alpha chain (αHCG), Human chorionic gonadotropin α / β (HCG), CKAP4 / p63, claudin-18, clusterin, c-Maf, c-Myc, Coactosin-like protein 1 / CotL1, COMMD1, Cornulin, cortical actinProtein, COX-2, CRISP-3, CTCF, CTL1 / SLC44A1, CXCL17 / VCC-1, CXCL8 / IL- 8, CXCL9 / MIG, CXCR4, cyclin A1, cyclin A2, cyclin D2, cyclin D3, CYLD, Cyr61 / CCN1, cytokeratin 14, cytokeratin 18, cytokeratin 19, DAB2, DCBLD2 / ESDN, DC-LAMP, Dkk-1, DLL3, DMBT1, DNMT1, DPPA2, DPPA4, E6, E-cadherin, ECM-1, EGF, EGF R / ErbB1, ELF3, ELTD1, EMMPRIN / CD147, EMP2, Endosialin / CD105, Endosialin / CD248, Enolase 2 / Neuron-specific Enolase, EpCAM / TROP1, Eps15, ERα / NR3A1, ERβ / NR3A2, ErbB3 / Her3, ErbB4 / Her4, ERCC1, ERK1, ERK5 / BMK1, Ets-1, Exostosin 1, EZH2, Ezrin, FABP5 / E-FABP, Fascin, FATP3, FCRLA, Fetuin A / AHSG, Acidic FGF, Basic FGF, FGF R3, FGF R4, fibrinogen, fibroblast activation protein α / FAP, follistatin-like 1 / FSTL1, FOLR1, FOLR2, FOLR3, FOLR4, FosB / GOS3, FoxMl, FoxO3, FRAT2, FXYD5 / antiadhesin, GABA-A Rα1, GADD153, GADD45α, galectin-3, galectin-3BP / MAC-2BP, γ-glutamyl cyclotransferase / CRF21, Gasl, gastrin-releasing peptide R / GRPR, motilin 1, gelsolin / GSN, GFAP, GLI-2, glutathione peroxidase 3 / GPX3, phosphatidylinositol proteoglycan 3, Golgi glycoprotein 1 / GLG1, gp96 / HSP90B1, GPR10, GPR110, GPR18, GPR31, GPR87, GPRC5A, GPRC6A, GRP78 / HSPA5, HE4 / WFDC2, heparanase / HPSE, serine protease (Hepsin), Her2, HGF R / c-MET, HIF-2α / EPAS1, HIN-1 / SCGB3A1, HLA-DR, HOXB13, HOXB7, HSP70 / HSPA1A, HSP90, hyaluronidase 1 / HYAL1, ID1, IgE, IGFBP-2, IGFBP-3. IGFBP-4, IGFBP-6, IGF-I, IGF-I R, IGF-II, IGFL-3, IGFLR1, IL-1β / IL-1F2, IL-17E / IL-25, IL-2, IL-6, IMP dehydrogenase 1 / IMPDH1, importin α2 / KPNA2, ING1, integrin β1 / CD29, integrin β3 / CD61, IQGAP1, isocitrate dehydrogenase 1 / IDH1, ITIH4, ITM2C, Jagged 1, JNK, JunB, JunD, kallikrein 2, kallikrein 6 / calcium glycerophosphate (Neurosin), KCC2 / SLC12A5, Ki-67 / MKI67, KiSS1R / GPR54, KLF10, KLF17, L1CAM, lactate dehydrogenase A / LDHA, lamin B1, LEF1, leptin / OB, LIN-28A, LIN-28B, lipocalin-2 / NGAL, LKB1 / STK11, LPAR3 / LPA3 / EDG-7, LRMP, LRP-1B, LRRC3B, LRRC4, LRRN1 / NLRR-1, LRRN3 / NLRR-3, Ly6K, LYPD1, LYPD8, MAP2, matriptase / ST14, MCAM / CD146, M-CSF, MDM2 / HDM2, Melan-A / MART-1, melanocortin-1R / MC1R, melanotransferrin / CD228, melatonin, Mer, mesothelin, metadherin, Metastin / KiSS1, methionine aminopeptidase, methionine aminopeptidase 2 / METAP2, MFAP3L, MGMT, MIA, MIF, MINA, Mind Bomb 2 / MIB2, Mindin, MITF, MKK4, MKP-1, MKP-3, MMP-1, MMP-10, MMP-13, MMP-2, MMP-3, MMP-8, MMP-9, MRP1, MRP4 / ABCC4, MS4A12, MSH2, MSP R / Ron, MSX2, MUC-4, Musashi-1, NAC1, Napsin A, NCAM- 1 / CD56, NCOA3, NDRG1, NEK2, NELL1, NELL2, Nesfatin-1 / Nuclear nectin-2, Nestin, NFkB2, NF-L, NG2 / MCSP, NGF R / TNFRSF16, Nicotinamide N-methyltransferase / NNMT, NKX2.2, NKX3.1, NM23-H1,NM23-H2, Notch-3, NPDC-1, NTS1 / NTSR1, NTS2 / NTSR2, OGR1, Olig2, Osteopontin / OPN, Ovastacin, OXGR1 / GPR80 / P2Y15, p130Cas, p15INK4b / CDKN2B, p16INK4a / CDKN2A, p18INK4c / CDKN2C, p21 / CIP1 / CDKN1A, p27 / Kip1, P2X5 / P2RX5, p53, PARP, PAUF / ZG16B, PBEF / Visfatin, PDCD4, PDCD5, PDGF Rα, PDGF Rβ, PDZD2, PEA-15, Pepsinogen A5 / PGA5, Peptidase Inhibitor 16 / PI16, Peroxiredoxin 2), PGCP, PI 3-kinase p85α, PIWIL2, PKM2, PLK1, PLRP1, PP4, P-Rex1, PRMT1, inhibitory protein 1, progesterone RB / NR3C3, progesterone R / NR3C3, progranulin / PGRN, prolactin, prostaglandin E synthase 2 / PTGES2, PSAP, PSCA, PSMA / FOLH1 / NAALADase 1, PSMA1, PSMA2, PSMB7, PSP94 / MSMB, PTEN, PTEN, PTH1R / PTHR1, PTK7 / CCK4, PTPβ / ζ / PTPRZ, Rab25, RARRES1, RARRES3, Ras, Reg4, Ret, RNF2, RNF43, S100A1, S100A10, S100A16, S100A2, S100A4, S100A6, S100A7, S100A9, S100B, S100P, SART1, SCUBE3, secretin R, serpin A9 / Centerin, serpin E1 / PAI-1, serum amyloid A1, serum amyloid A4, SEZ6L, SEZ6L2 / BSRP-A, Skp2, SLC16A3, SLC45A3 / Prostein, SLC5A5, SLC5A8 / SMCT1, SLC7A7, Smad4, SMAGP, SOCS-1, SOCS-2, SOCS-6, SOD2 / Mn-SOD, Soggy-1 / DkkL1,SOX11, SOX17, SOX2, SPARC, SPARC-like 1 / SPARCL1, SPINK1, Src, STEAP1, STEAP2, STEAP3 / TSAP6, STRO-1, STYK1, survivin, synaptotagmin-1, synaptotagmin-1 / CD138, syntaxin 4, synuclein-γ, tankyrase 1, T, TCF-3 / E2A, TCL1A, TCL1B, TEM7 / PLXDC1, TEM8 / ANTXR1, tenascin C, TFF1, TGF-β1, TGF-β1, 2, 3, TGF-β1 / 1.2, TGF-β2 / 1.2, TGF-βRI / ALK-5, THRSP, thymidine kinase 1, thymosin β10, thymosin β4, thyroglobulin, TIMP assay kit, TIMP-1, TIMP-2, TIMP-3, TIMP-4, TLE1, TLE2, TLR2, TM4SF1 / L6, TMEFF2 / Tomoregulin-2, TMEM219, TMEM87A, TNF-α, TOP2A, TopBP1, t-plasminogen activator / tPA, TRA-1-60(R), TRA-1-85 / CD147, TRAF-4, transaminase / TAGLN, trypsin 2 / PRSS2, tryptase α / TPS1, TSPAN1, UBE2S, uPAR, u-plasminogen activator / urokinase, tenosynovin-II R, VAP-1 / AOC3, VCAM-1 / CD106, VEGF, VEGF R1 / Flt-1, VEGF R2 / KDR / Flk-1, VEGF / P1GF heterodimer, VSIG1, VSIG3, YAP1, ZAG, ZAP70, ZMIZ1 / Zimp10 and carcinoembryonic antigen.

[0068] In some embodiments, the prey biomolecule is selected from a biomolecule expressed by a kidney cell, an infectious agent or a parasitic agent, a solid tumor cell, a circulating tumor cell, or any other cell that can be used for diagnosis or prognosis. In some embodiments, the prey biomolecule is selected from a biomolecule expressed on the surface of a kidney cell, an infectious agent (e.g., a bacterium or a virus), a solid tumor cell, or a circulating tumor cell or within these cells. In some embodiments, the prey biomolecule expressed on the surface of a kidney cell or within a kidney cell may or may not include: KIM-1, albumin, beta-2 microglobulin, cystatin C, clusterin, apolipoprotein AI / ApoA1, CXCL8 / IL-8, ERCC1, Ki-67 / MKI67, MMP-9, or trefoil factor-3.

[0069] In some embodiments, the prey biomolecule is one or more biomarkers for a particular type of cancer. In some embodiments, the prey biomolecule for breast cancer may include or exclude her2-neu, ER, PR, Ki-67 and p53. In some embodiments, the prey biomolecule for lung cancer may include or exclude TTF-1, new aspartic proteinase A, CK 5 / 6, p40 / 63 and synaptophysin (Synaptophosmin). In some embodiments, the prey biomolecule for prostate cancer may include or exclude AMACR, PSA, CEA and p63. In some embodiments, the prey biomolecule for colorectal cancer may include or exclude MLH1, MSH2, PMS2, MSH6, c-Kit, p16 and BRAFV600E. In some embodiments, the prey biomolecule for tumor infiltrating lymphocytes may include or exclude CD4, CD8, CD14, CD20, CD45RO, FoxP3, PD-L and PD-L1. In some embodiments, prey biomolecules for urological (bladder, kidney, urethra) cancer may or may not include CK7, p63, CK20, p53, Ki-67, PSA, vimentin, and PAX8.

[0070] In some embodiments, the prey biomolecule is cell-specific. The cell may be in a healthy state (normal) or a diseased state (abnormal). Monocytes and macrophages may present prey biomolecules including or excluding CD14 and CD16 biomolecules. Lymphocyte B cells may present prey biomolecules including or excluding CD20 biomolecules. Lymphocyte NK cells may present prey biomolecules including or excluding CD56 biomolecules. Lymphocyte T cells may present prey biomolecules including or excluding CD3 biomolecules. T Reg cells may present prey biomolecules including or excluding CD4, CD25, and FoxP3 biomolecules. Cytotoxic T cells may present prey biomolecules including or excluding CD8 biomolecules. Helper T cells may present prey biomolecules including or excluding CD4 biomolecules. Naive T cells may present prey biomolecules including or excluding CD45RA biomolecules. Memory T cells may present prey biomolecules including or excluding CD45RO biomolecules. Tth cells may present prey biomolecules including or not including CXR5 biomolecules. Th17 cells may present prey biomolecules including or not including CCR6 biomolecules. Th2 cells may present prey biomolecules including or not including CCR4 biomolecules. Th1 cells may present prey biomolecules including or not including CXCR3 biomolecules. Tumor cells may present prey biomolecules including or not including PanCK biomolecules.

[0071] As used herein, the term "antibody" refers to a protein that can specifically bind to an antigen. In some embodiments, an antibody may or may not include any recombinant or naturally occurring immunoglobulin molecule, which may or may not include members of the IgG class (e.g., IgG1), IgM class, IgY class, IgD class, IgE class; and any antigen-binding immunoglobulin fragment, which may or may not include Fv, Fab and F(ab')2 fragments, antibody fragments, ScFv (single chain variable fragment, i.e., immunoglobulin heavy chain (V H ) and light chain (V L ) of the variable region of the heavy chain (V H ) and light chain (V L ) connected to a short linker peptide of 10 to about 25 amino acids) or a single domain antibody (nanobody), and any derivatives thereof. In an embodiment, the antibody may be a monoclonal antibody or a polyclonal antibody.

[0072] The term "antibody fragment" as used herein refers to a part of a complete antibody, wherein the part retains at least one and up to most or all functions that are usually associated with the part when it is present in a complete antibody. In some embodiments, the antibody fragment comprises the antigen binding site of the complete antibody, thus retaining the ability to bind antigens. In some embodiments, an antibody fragment (e.g., an antibody fragment comprising an Fc region) retains at least one biological function that is usually associated with the Fc region when it is present in a complete antibody, and the function may include or not include FcRn binding, antibody half-life regulation, ADCC function, and complement binding. In some embodiments, an antibody fragment is a monovalent antibody with a half-life in vivo that is substantially similar to a complete antibody. For example, such an antibody fragment may include an antigen binding arm that is connected to an Fc sequence that can confer stability in vivo on the fragment.

[0073] "Polyclonal antibodies" or "PAbs" are heterogeneous populations of antibody molecules derived from the serum of animals immunized with an antigen or an antigenic functional derivative thereof. To produce polyclonal antibodies, host animals, which may or may not include rabbits, mice, and goats, may be immunized by injection with an antigen or antigen conjugate optionally supplemented with an adjuvant. Polyclonal antibodies may be unpurified, purified, or partially purified in antisera from other species. Techniques for preparing and purifying polyclonal antibodies are described in various general and more specific references, including but not limited to Kabat and Mayer, Experimental Immunochemistry, 2nd Edition, (Thomas, Springfield, 111. (1961)); Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988)); and Weir, Handbook of Experimental Immunology, 5th Edition (Blackwell Science, Cambridge, Mass. (1996)).

[0074] A "monoclonal antibody" or "MAb" is a homogeneous population of antibodies that bind a specific antigen and can be obtained by any technique that provides for the production of antibody molecules, which may or may not include continuous culture of cell lines. Such techniques include, but are not limited to and Milstein's hybridoma technology, "Continuous cultures of fused cells secreting antibiody of predefined specificity", Nature, 256:495-497 (1975); and U.S. Pat. No. 4,376,110), human B cell hybridoma technology (Kozbor et al., "The Production of Monoclonal Antibodies From Human Lymphocytes", Immunology Today, 4:72-79 (1983); Cote, RJ et al., "Generation of human monoclonal antibodies reactive with cellular antigens", Proc. Natl. Acad. Sci. USA, 80:2026-30 (1983)), and EBV-hybridoma technology (Cole et al., Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., New York, pp. 77-96 (1985). Such antibodies may belong to any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. Hybridomas producing the MAbs of the invention may be cultured in vitro or in vivo. The production of high titer MAbs in vivo makes this the currently preferred method of production.

[0075] Alternatively, techniques described for the production of single-chain antibodies (U.S. Pat. No. 4,946,778; Bird et al., "Single-chain Antigen-Binding Proteins", Science, 242:423-426 (1988); Huston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia Coli", Proc. Natl. Acad. Sci. USA, 85:5879-83 (1988); and Ward., "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted from Escherichia coli". Coli), 334: 544-546 (1989)) can be modified to produce a gene-single-chain antibody suitable for use in the present invention. Single-chain antibodies are usually formed by connecting the heavy chain fragment and the light chain fragment of the Fv region via an amino acid bridge to produce a single-chain polypeptide.

[0076] Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, such fragments include, but are not limited to, F(ab')2 fragments that can be generated by pepsin digestion of antibody molecules, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab')2 fragments. Alternatively, Fab expression libraries (Huse et al., "Generation of a large combinatorial library of theimmunoglobulin repertoire in phage lambda", Science, 246: 1275-1281 (1989)) can be constructed to allow rapid and easy identification of monoclonal Fab fragments with desired specificity.

[0077] As used herein, the terms "polypeptide," "peptide mimetic," and "mimetic" include synthetic or genetically engineered chemical compounds that may have substantially the same structural and functional characteristics as the protein region that they mimic.

[0078] In some embodiments, the polypeptide can be modified or unmodified. Generally, a peptide mimetic is a structural or functional mimetic that is identical or similar to an exemplary polypeptide (i.e., a polypeptide having a biological or pharmacological function or activity), but may also have one or more peptide bonds that are optionally substituted by a bond selected from, for example, a group consisting of: -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-. A mimetic may be composed entirely of natural amino acids, synthetic chemical compounds, non-natural analogs of amino acids, or a chimeric molecule that is partly a natural peptide amino acid and partly a non-natural analog of an amino acid. A mimetic may also contain any amount of conservative substitutions of natural amino acids, as long as such substitutions do not significantly change the mimetic activity.

[0079] As used herein, the term "about" is intended to quantify the numerical value it modifies, indicating such values ​​that are variable within a margin of error. When no specific margin of error (such as, for example, standard deviation relative to the mean) is recited, the term "about" means plus or minus 10% of the numerical value of the number used. For example, "about 50%" means within a range of 45% to 55%.

[0080] system

[0081] In some embodiments, the present disclosure provides systems for isolating single cells and analyzing selected cellular components. In some embodiments, the systems described herein can quantitatively analyze selected cellular components with a signal-to-noise ratio (SN) of greater than 10, greater than 12, greater than 15, or greater than 20.

[0082] The combination of single cell isolation, single cell lysis, antibody / capture agent coated beads and TIRF or confocal microscopy allows sensitive detection of selected cellular components. In addition, the design of targeting bait biomolecules, followed by complex formation between bait biomolecules and prey biomolecules, followed by imaging by the methods described herein allows capture and analysis of bait-prey biomolecule interactions (protein-protein interactions, PPIs) that would otherwise not be detectable at such high SN levels.

[0083] Substrate

[0084] The invention described herein includes the use of substrates containing multiple micron-sized features for analyzing isolated single cells and / or selected components thereof. In some embodiments, the cover slip may be made of a transparent solid medium. The transparent solid medium may be selected from glass (e.g., pyrex, borosilicate, soda lime, optical glass, quartz), cycloolefin polymers (e.g., Zeonor, Zeonex) or polyacrylates. In some embodiments, the substrate may be a cover slip of type 0 to 4 (corresponding to a thickness of about 0.085 mm to about 0.64 mm). In some embodiments, the substrate may be coated. The coating may or may not include silane, polysilane or polymer. In some embodiments, the substrate is coated with polydimethyldichlorosilane. Micron-sized features may be etched into the substrate or provided on top of the substrate by a hardened polymer. In some embodiments, the hardened polymer may or may not include PDMS or SU8. SU-8 contains bisphenol A novolac epoxy monomers, which are dissolved in an organic solvent (γ-butyrolactone GBL or cyclopentanone, depending on the formulation) and may or may not include triaryl groups. In some embodiments, the hardened polymer can be made from a solid negative master template generated by conventional photolithography methods. The hardened polymer and the precursor of the crosslinking agent are contacted with the negative master template, the crosslinking reaction is initiated (e.g., heat and / or light), and the master is removed to generate micron-sized features.

[0085] The diameter of the features can be configured to allow one cell to enter each feature. Figure 66a and 6b, the size of the feature can be configured to selectively separate single cells per feature. This single cell separation especially allows the separation of cellular components of separated cells. With the functionalized beads co-located with the separated cells and the platform (e.g., TIRF or confocal microscopy) combination for realizing high-sensitivity fluorescence detection, the components of one or more separated cells can be analyzed, including protein-protein interactions. The separation of single cells also allows the analysis of rare cells, which may or may not include hair cells (e.g., cochlear cells), circulating tumor cells or circulating embryonic cells. The features of multiple micron sizes in the substrate may have circular features. The features of multiple micron sizes in the substrate may include a series of features, and the features independently have a diameter of 8 microns to 15 microns, 15 microns to 45 microns, 45 microns to 80 microns, 80 microns to 100 microns, 100 microns to 150 microns or the size of any range between the above ranges. In some embodiments, the feature diameter may be configured to separate selected cells (e.g., small cells in large cell / small cell population). In some embodiments, the diameter of the features can be 15 microns, 16 microns, 17 microns, 18 microns, 19 microns, 20 microns, 21 microns, 22 microns, 23 microns, 24 microns, 25 microns, 26 microns, 27 microns, 28 microns, 29 microns, 30 microns, 31 microns, 32 microns, 33 microns, 34 microns, 35 microns, 36 microns, 37 microns, 38 microns, 39 microns, 40 microns, 41 microns, 42 microns, 43 microns, 44 microns, or 45 microns. The features can be spaced far enough apart that the fluorescent bloom from a fluorophore in any one feature will not overlap with the fluorescent bloom from a fluorophore in another feature. In some embodiments, the distance between the centers of the features can range from about 10 microns to about 5000 microns. The distance between the centers of the features may be in the range of 10 microns to 100 microns, 100 microns to 200 microns, 200 microns to 500 microns, 500 microns to 1000 microns, 1000 microns to 2000 microns, 2000 microns to 5000 microns, or any range therebetween. The depth of the features may be sufficient to separate and maintain single cells within the features while preventing two or more cells from occupying the same feature. In some embodiments, the depth of the features may be in the range of about 15 microns to about 150 microns. In some embodiments, the depth of the features may be in the range of about 15 microns to about 40 microns, about 40 microns to about 60 microns, about 60 microns to about 75 microns, about 75 microns to about 90 microns, about 90 microns to about 120 microns, about 120 microns to about 150 microns, or any range therebetween.

[0086] Beads

[0087] The invention described herein relates to the use of nano-sized functionalized beads. The size of the beads is such that multiple beads will enter the features (holes) of the substrate. The size of nano-sized beads can be 5 nanometers to 1000 nanometers. In some embodiments, the diameter of the beads can be 5 nanometers to 20 nanometers, 20 nanometers to 50 nanometers, 50 nanometers to 100 nanometers, 100 nanometers to 500 nanometers, 500 nanometers to 1000 nanometers, or any of the above ranges. The beads can be coated with a polymer, silica or hydrogel layer. The beads can be functionalized to bind antibodies or capture agents. The beads can be functionalized with carboxylic acids, amines, biotin, streptavidin, avidin, neutravidin, NHS or maleimide. Functionalized beads can be coated with biomolecules via direct covalent bonds (e.g., NHS-functionalized beads with polynucleic acids functionalized with lysine residues or 5' amino groups on polypeptides or antibodies), or by indirect means (e.g., NTA-Ni-functionalized beads bound to hexahistidine-modified proteins).

[0088] In some embodiments, nano-sized functionalized beads can be functionalized by using streptavidin-biotin binding. In some embodiments, nano-sized particles coated with carboxylic acid functional groups can be activated with EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxy-succinimide) and then washed to produce EDC-functionalized nanoparticles. Other amide coupling agents can be used instead of EDC, for example, DCC (dicyclohexylcarbodiimide), EDAC.HCl, (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide.HCl), HOBt (1-hydroxybenzotriazole), HOOBt (HODhbt) (hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine), HOAt (1-hydroxy-7-aza-1H-benzotriazole), DMAP (4-(N,N-dimethylamino)pyridine), BOP (benzotriazol-1-yloxy-tris(dimethylamino)- Hexafluorophosphate), PyBOP (benzotriazol-1-yloxy-tripyrrolidino Hexafluorophosphate), PyOxim (cyano(hydroxyimino)acetoxy-O2)-tris-(1-pyrrolidinyl)- ethyl hexafluorophosphate), PyBrOP (7-aza-benzotriazol-1-yloxy-tripyrrolidino hexafluorophosphate), DEPBT (3-(diethoxy-phosphoryloxy)-1,2,3-benzo[d]triazine-4(3H)-one), TBTU / HBTU (2-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethylamine Tetrafluoroborate / hexafluorophosphate), HCTU ((2-(6-chloro-1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethylamine Hexafluorophosphate), HDMC (N-[(5-chloro-1H-benzotriazol-1-yl)-dimethylamino-morpholino]-urea Hexafluorophosphate N-oxide), HATU (2-(7-aza-1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethylamine hexafluorophosphate), COMU (1-[1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholino]-urea Hexafluorophosphate), TOTT ((2-(1-oxy-pyridin-2-yl)-1,1,3,3-tetramethylisothiourea tetrafluoroborate), TFFH (tetramethylfluoroformamidine hexafluorophosphate), EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline), T3P (2-propanephosphonic anhydride), DMTMM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine Salt) or CDI (1,1′-carbonyldiimidazole). In some alternative embodiments, the coupling can be carried out in the presence of a base. The base can be organic or inorganic. The inorganic base may or may not include, for example, a carbonate buffer or a phosphate buffer. The organic base may be triethylamine, diisopropylethylamine (DIPEA) or N-methylmorpholine (NMM).

[0089] Washing can be a mild pH wash so as not to hydrolyze the NHS moiety. Mild pH washes can be performed with PBS buffer (phosphate buffered saline, pH about 7.4). Next, streptavidin can be reacted with EDC-functionalized nanoparticles to produce streptavidin-functionalized nanoparticles. Other avidin-like molecules can be used instead of streptavidin, for example: avidin, neutravidin, superavidin, and streptavidin to which one, two, or three biotins have been bound. In some embodiments, the capture agent / antibody can be functionalized with biotin. The antibody can be reacted with a cross-linking agent such as sulfo-SMCC (Pierce) and then with thiol-conjugated biotin to produce a biotinylated antibody. In an alternative embodiment, the antibody can be reacted with NHS-conjugated biotin, wherein the NHS-conjugated biotin can react with any free amine on the antibody (preferably a free amine from a lysine residue) to produce a biotinylated antibody. In alternative embodiments, the antibody can be reacted with DTT (dithioerythritol) to break the dithiol cysteine ​​bond to produce a free sulfhydryl group (sulfuryl hydryl). The sulfhydryl group can be reacted with maleimide-conjugated biotin to produce a biotinylated antibody. In some embodiments, nanoparticles with functional groups selected from the following can be purchased: carboxylic acid, NHS, streptavidin, amine, alkyne or aldehyde. The biotinylated antibody can be reacted with streptavidin functionalized nanoparticles to produce directly attached antibody functionalized nanoparticles.

[0090] Each nanometer-sized functionalized bead is homogeneously functionalized with the same biomolecule. However, in some embodiments, multiple types of homogeneously functionalized nanometer-sized beads can be added to the micrometer-sized substrate features to enable capture and analysis of multiple prey biomolecules within the same cell. In some embodiments, relative amounts of each of the multiple types of homogeneously functionalized nanometer-sized beads can be added to the micrometer-sized substrate features to enable relative quantification of various cellular components in the same isolated cells.

[0091] In some embodiments, nanometer-sized functionalized beads are functionalized with a single capture agent or antibody (primary antibody that binds to a bait protein). In some embodiments, nanometer-sized functionalized beads are functionalized with a secondary antibody, and the primary antibody binds to a portion of the secondary antibody. In some embodiments, the secondary antibody is directed to a bait biomolecule. Such configurations allow programmability of the functionalized beads to the bait biomolecule by adding the identified antibodies to the primary antibody functionalized beads.

[0092] In some embodiments, capture agents / antibodies may be added to nano-sized functionalized beads before they are provided to a substrate comprising a plurality of micron-sized features, or after they are provided to micron-sized features. In some embodiments, a plurality of antibody-coated beads may include a plurality of secondary antibodies to a primary antibody directed to a target bait biomolecule. A plurality of antibody-coated beads may include a plurality of primary antibodies directed to a target bait biomolecule. In some aspects, a plurality of antibody-coated beads include secondary antibodies that bind to a plurality of primary antibodies that bind to bait biomolecules. In some embodiments, capture agents / antibodies may be added to a solution comprising cells before the cells are provided to a substrate comprising a plurality of micron-sized features. In such embodiments, the antibodies may associate with cell surface cellular components in free solution before the antibodies are attached to the nano-sized functionalized beads, which may increase the reaction kinetics.

[0093] Nano-sized functionalized beads (further functionalized with capture agents / antibodies) may be added to a substrate comprising multiple micron-sized features before or after multiple cells are provided to the substrate. In some embodiments, the beads may be magnetic so that the introduction of a magnetic field pulls the beads to the surface of the substrate to allow more efficient sensitivity of fluorescence detection when using TIRF or confocal microscopy. In some embodiments, gravity (e.g., allowing sedimentation time, or subjecting the substrate to high gravity in a centrifuge configured to accept a planar substrate) may be used to pull the beads down to the substrate surface. In some embodiments, magnetic forces are applied before or after cells are provided to a substrate comprising multiple micron-sized features. In some embodiments, capture agents / antibodies are added to nano-sized functionalized beads, which are first added to a substrate comprising multiple micron-sized features.

[0094] In some embodiments, the system may further comprise one or more types of cells. The type of cells may be a rare cell, which may or may not include hair cells (e.g., cochlear cells), circulating tumor cells, or circulating embryonic cells. Cochlear cells cannot be measured by standard cell sampling methods because most cell analysis methods do not allow for analysis of very small numbers of cochlear cells (10 4, from the whole human cochlea). The high SN (signal-to-noise ratio) single cell component analysis system and method of the present disclosure also allow the detection of circulating tumor cells in the early stages of cancer, at which stage tumor cells are usually not detectable. In some embodiments, the high SN single cell component analysis system and method of the present disclosure also allow the detection of circulating embryonic cells in pregnant or pregnant females. Circulating embryonic cells can be microchimeric fetal cells. Microchimeric cells are often found in females after birth and can be attributed to an enhanced immune system (Dawe et al., "Cell Migration from Baby to Mother", Cell Adh Migr. 2007 Jan-Mar; 1(1): 19–27). ]

[0095] In some embodiments, the cell may be from a subject. The cell from the subject may contain biomolecules that can be used to help identify the cell state, identity, growth rate, lineage, mutation, variant, expression level, cancer stage or remission state and / or latent or active infection. Such cells may or may not include, for example, mammalian cells, immunomodulatory cells, lymphocytes, monocytes, polymorphs, T cells, tumor cells, yeast cells, bacterial cells, infectious agents, parasites, plant cells, transfected cells, such as NSO cells, CHO cells, COS cells, 293 cells.

[0096] In some embodiments, the cells can be alive, dead, fixed and / or substantially intact. In some embodiments, the cells can be of the same type or of different types. When the cells are of different types, the cells can be from different tissues or tumor sources, exhibit different pathologies, express different or mutated biomolecules, express different levels of biomolecules, express biomolecules with different post-translational modifications, or exhibit different morphologies. In some embodiments, the capture agent / antibody is capable of distinguishing between mutant biomolecules and wild-type biomolecules. When the cells are from different tumor sources, the cells can be from a tumor, which may or may not include, for example, breast cancer, lung cancer, prostate cancer, bone cancer, colorectal cancer, liver cancer, pancreatic cancer, thyroid cancer, bladder cancer, or other types of cancer.

[0097] In some embodiments, the cells may contain one or more types of target prey biomolecules. When multiple types of target prey biomolecules are to be detected, multiple types of nano-sized functionalized beads may be used, the beads having multiple types of antibodies corresponding to baits corresponding to the prey biomolecules.

[0098] In some embodiments, a plurality of target prey biomolecules may include biomolecules that can be found in the cell membrane of the cell. In some embodiments, a plurality of target prey biomolecules may include biomolecules that can be found in the cytoplasm of the cell. In some embodiments, bait biomolecules and prey biomolecules may be independently selected from: proteins, DNA, RNA, mRNA, tRNA, cRNA, antibodies, antibody fragments, ScFv, peptides and small molecules. The DNA or RNA may be further modified with biotin. The systems and methods described herein may enable single-cell sequencing, wherein the prey biomolecule is DNA or RNA from isolated cells. In some embodiments, the captured DNA or RNA as a prey biomolecule may be dehybridized from the corresponding bait polynucleotide sequence using heating or a denaturant (e.g., low salt, DMSO, urea, formamide, etc.), and the dehybridized DNA or RNA may be further analyzed by a DNA analysis method to determine part or all of the sequence of the DNA or RNA. In some embodiments, the DNA analysis method may include or not include: DNA sequencing, qPCR, SSCP, STR or genotyping by microarray or mass spectrometry. DNA sequencing methods may or may not include the use of an Illumina HiSeq, MiSeq, iSeq, or NovaSeq; a ThermoFisher IonTorrent system; a Qiagen GeneReader system; or a BGIDNBSEQ-T7, DNBSEQ-G400, DNBSEQ-50 system, using protocols and instructions provided by the manufacturer.

[0099] In some embodiments, the system further comprises a cell membrane disrupting agent to release cell components. The cell membrane disrupting agent can be a lysis buffer. The lysis buffer may or may not include a denaturing detergent (when analyzing polynucleotides) or a non-denaturing detergent (when analyzing proteins, antibodies, and polypeptides). In some embodiments, the denaturing detergent is SDS (sodium dodecyl sulfate). In some embodiments, the non-denaturing detergent is selected from: CHAPS, deoxycholate, Triton TM X-100, NP40 and Tween 20. The lysis buffer may further include buffer salts well known in the art.

[0100] In some embodiments, the system and the glass substrate used therein can be configured to be measured using TIRF (total internal reflection microscopy). TIRF illumination can eliminate or reduce scattering from other light scattering elements on the substrate surface. TIRF illumination provides the advantage that illumination and fluorescence do not interact and are affected by surface debris. TIRF microscopy in the present disclosure reduces background fluorescence from outside the focal plane and can significantly improve the signal-to-noise ratio of fluorescence emitted from fluorophores attached to prey biomolecules-bait biomolecules / antibody complexes. TIRF microscopy utilizes induced evanescent waves in a limited substrate area adjacent to an interface between two media with different refractive indices. In some embodiments, the TIRF interface utilized can be the contact area between the substrate and the glass cover. In some embodiments, the illumination system can include an optical fiber to deliver light to the edge of the slide. In some embodiments, the illumination system can include a Darklite vertical illuminator (Micro Video Instruments, Avon, Mass.).

[0101] method

[0102] The present invention provides, inter alia, methods for identifying biomolecule-biomolecule interactions, comprising the steps of: (a) contacting one or more cell types with a system as described herein, (b) providing a lysis buffer to the system, and (c) imaging the system by fluorescence microscopy. In some embodiments, the biomolecule is a low copy number protein in a cell. In some embodiments, the biomolecule is selected from a bait biomolecule and a prey biomolecule. A bait biomolecule is a biomolecule known or suspected to have an interaction with another biomolecule (prey biomolecule) within or on a selected cell. The prey biomolecule is then complexed with a primary antibody or a primary-secondary antibody complex, wherein one or more of the prey biomolecule, primary antibody, or secondary antibody is fluorescently labeled with a fluorophore. In some embodiments, imaging can be performed by fluorescence microscopy. Fluorescence microscopy can be selected from TIRF or confocal microscopy. The primary antibody can be added to the prey biomolecule-bait biomolecule complex alone or as a complex with a secondary antibody. In some embodiments, the primary antibody is first added to the prey biomolecule-bait biomolecule complex, followed by the addition of the secondary antibody (directed to the primary prey antibody).

[0103] The present invention particularly provides a method for identifying biomolecule-biomolecule interactions, the method comprising the following steps: (a) contacting a substrate comprising a plurality of micron-sized features with one or more cell types, wherein a portion or all of the one or more cell types enter a portion or all of the plurality of micron-sized features; (b) providing a plurality of antibody-coated magnetic beads to the substrate, wherein the antibody-coated magnetic beads comprise antibodies that bind to bait biomolecules; (c) optionally, applying a magnetic field to the antibody-coated beads; (d) contacting the substrate with a lysis buffer; (e) forming a complex, the complex being selected from the antibodies and bait biomolecules of the antibody-coated magnetic beads, the bait biomolecules and prey biomolecules, or all of the bait biomolecules and the prey biomolecules bound to the antibodies of the antibody-coated magnetic beads; (f) providing an antibody or antibody complex bound to the prey biomolecule; and (g) imaging the antibody or antibody complex bound to the prey biomolecule by fluorescence microscopy. In some embodiments, the cell may comprise a bait biomolecule and / or a prey biomolecule. In some embodiments, the antibody or antibody complex that binds to the prey biomolecule is fluorescently labeled. In some embodiments, the antibody complex that binds to the prey biomolecule may comprise a primary antibody that binds to the prey biomolecule, and a secondary antibody that binds to the primary antibody that binds to the prey biomolecule, wherein the secondary antibody is fluorescently labeled. In some embodiments, the prey biomolecule and the bait biomolecule may be independently selected from the group consisting of proteins, DNA, RNA, mRNA, tRNA, cRNA, antibodies, antibody fragments, ScFv, peptides, and small molecules.

[0104] In some embodiments, step (g) of imaging the antibody or antibody complex bound to the prey biomolecule by fluorescence microscopy can be performed by TIRF or confocal microscopy.

[0105] In some embodiments, each nanometer-sized functionalized bead isolates a single prey biomolecule.

[0106] Example

[0107] The work described in these Examples evaluates and demonstrates the positive effects of using antibody-coated magnetic nanobeads and substrates containing multiple micron-sized features for single cell isolation and analysis of their composition.

[0108] Example 1

[0109] General representative method for nanobead-based single-cell isolation and analysis of cell populations

[0110] Magnetic nanobeads coated with streptavidin are modified by immobilizing a primary antibody on their surface for capturing target proteins (bait proteins) (as described in the Examples herein). Cells are lysed and magnetic nanobeads are added to the lysate and incubated for 30 minutes, and after capturing the prey protein by the beads, the protein is detected using a specific primary antibody and a fluorescently labeled secondary antibody. The nanobeads are washed three times to remove non-specifically bound proteins, transferred to a glass slide and covered with a cover slip, and imaged using a TIRF microscope. In some embodiments, the bait or prey protein is fluorescently labeled for direct visualization. ( Figure 1 )

[0111] Example 2

[0112] Nanobead-based SiMPull for green fluorescent protein (GFP) pull-down in cell populations

[0113] The nanobead-based SiMPull method was first used to validate protein pull-down by pulling down GFP, because GFP can be directly visualized without immunostaining, which simplifies validation, and more importantly, because GFP can be used in photobleaching experiments to assess the single-molecule state of the pulled-down fluorescent spots.

[0114] Pulldown of ectopic GFP in HEK293T cells using magnetic nanobeads Figure 2 a). In contrast, in the negative control experiment, very few GFP molecules were pulled down ( Figure 2 a), and the calculated assay signal-to-noise (SN) ratio was about 10-20 ( Figure 2 b) This SN ratio is comparable or better than the original SiMPull method of Jain et al. By applying the algorithm previously used by Jain et al. Figure 2 About 95% of the fluorescent spots in a show Gaussian distribution. Photobleaching experiments were performed on these fluorescent spots, and the results showed that 58% of the spots were single fluorophores (i.e., GFP monomers), 18% were two fluorophores, and 22%>2 fluorophores. Spots containing ≥2 fluorophores may represent ≥2 GFP molecules within a distance of 60nm (this number needs to be explained, one pixel = 60nm) located on a single nanobead. 5% of the fluorescent spots that do not show Gaussian distribution may be bundles of nanobeads stuck together. These results confirm that our bead-based method enables the capture of target proteins at the single molecule level with a very high SN ratio.

[0115] Example 3

[0116] Pulldown of cAMP-dependent protein kinase A (PKA) complexes in cell populations

[0117] The holoenzyme or complex of PKA, one of the most extensively studied protein kinases, exists as a heterotetramer comprising a dimer of regulatory (R) subunits and two catalytic (C) subunits ( Figure 3 a); cAMP can bind to the R subunit at physiological levels and release the C subunit from the PKA complex ( Figure 3 a). We used the well-characterized interaction between PKA-R subunits and PKA-C subunits to validate the nanobead-based SiMPull assay for studying PPIs.

[0118] The expression vectors used in this study were pEGFP-n1 (Addgene catalog number 6085-1), pcDNA3-mouse PKA-C-α-mEGFP (Addgene catalog number 45521), and pcDNA3-mouse PKA-RII-α-mEGFP (Addgene catalog number 45527) for GFP expression. pcDNA3-PKA-C-α-mCherry was constructed by replacing the GFP sequence in pcDNA3-PKA-C-α-mEGFP with the mCherry sequence.

[0119] The interaction of PKA-C-eGFP and PKA-R-mCherry was examined in HEK293T cells because these fluorescent fusion proteins allow direct visualization of PKA-C subunits and PKA-R subunits without immunostaining and facilitate the assessment of pull-down efficiency and specificity ( Figure 3 b). When magnetic nanobeads pre-coated with anti-GFP antibodies were used, both PKA-C-eGFP and PKA-R-mCherry were pulled down. If mCherry was used instead of PKA-R-mCherry in these experiments, very few mCherry molecules co-precipitated with PKA-C-eGFP ( Figure 3 c); This indicates the interaction between PKA-C-eGFP and PKA-R-mCherry ( Figure 3 b) specifically depends on PKA-R. In addition, the cAMP analog cpt-cAMP disrupted the interaction between PKA-C-eGFP and PKA-R-mCherry ( Fig.10 ), indicating that the interaction is physiological.

[0120] The majority of GFP and mCherry signals clearly colocalize ( Figure 3b), although a small portion does not. This incomplete colocalization of GFP and mCherry may be due to several reasons: (1) only 75% of GFP molecules are properly folded and fluoresce (Waldo, GS et al., "Rapid Protein-Folding Assay Using Green Fluorescent Protein", Nature Biotechnology, 1999; 17(7): 691-695); (2) the fluorescence of GFP or mCherry molecules in the complex is unevenly quenched; and (3) unbalanced expression of PKA-C and PKA-R.

[0121] Example 4

[0122] Schematic diagram and fabrication of microwell arrays

[0123] As a representative example of a substrate containing multiple micrometer-sized features, a microwell array chip ( Figure 4 ) (Huang, L. et al., "Centrifugation-Assisted Single-Cell Trapping in a Truncated Cone-Shaped Microwell Array Chip for the Real-Time Observation of Cellular Apoptosis", Analytical Chemistry, 2015; 87(24): 12169-12176). Depending on the size of the target cells, the diameter of the microwells on each chip varies (range: 15 μm to 50 μm), and the microwell depth is 70 μm, which is designed to capture cells and minimize lysate diffusion ( Figure 4 , Figure 5 a and Figure 5 b).

[0124] The fabrication of the cell capture microwell array was achieved by generating a porous PDMS membrane with microwell patterning on a glass cover slip. Briefly, a silicon mold featuring a micropillar pattern was first fabricated using the Bosch deep reactive ion etching (DRIE) process. The diameter (15 μm to 50 μm range) and spacing (4 times the diameter of the pillar, edge to edge) of the pillars were controlled using standard photolithography techniques. The etching depth of the pillars was controlled to 75 μm. The fabricated silicon mold was then cut into pieces of 1.0 × 1.0 cm in size. 2The chip was silanized overnight with dimethyldichlorosilane (Sigma-Aldrich) vapor in a vacuum container, followed by washing with acetone and deionized water in sequence, and dried with N2 before use. Then, 5 microliters of pre-degassed PDMS prepolymer (10:1, weight ratio of base to curing agent) was gently poured into the silanized silicon mold, and a glass cover slip pretreated with oxygen plasma and washed with acetone (BDH1101), 2-propanol (BDH1133) and DI water in a desktop ultrasonic cleaner (Bransonic) was placed in close contact with the chip to spread the prepolymer evenly over the entire chip. Subsequently, a 200 g weight was placed on the cover slip to ensure that the microfabricated columns completely penetrated the prepolymer layer and contacted the glass cover slip. The entire assembly was then placed in a 60 ° C oven for 3 h. After removing the weight, the cover slip was slowly peeled off from the mold forming the cell capture device together with a porous PDMS membrane characterized by micropores of various sizes. In some embodiments, the PDMS is a curable polydimethylsiloxane. In some embodiments, the PDMS is a SYLGARD 184 silicone elastomer kit (Dow). The PDMS may have a curable component of a cross-linked polydimethylsiloxane polymer. In some embodiments, the cross-linking agent may be a dimethyl, methylhydrogensiloxane copolymer with a platinum catalyst (e.g., hexachloroplatinate). In some embodiments, the pre-curing viscosity of the polydimethylsiloxane may be 5100 cP. In some embodiments, the kit viscosity (PDMS with cross-linking agent) may be 3500 cP. In some embodiments, the curing time of the PDMS at 100°C may be 35 minutes or less, or the curing time at 150°C may be 10 minutes or less.

[0125] Example 5

[0126] Schematic diagram of single-cell pull-down assay

[0127] First, single cells are trapped in microwells. Next, magnetic nanobeads pre-coated with bait antibodies are applied. In the absence of a magnet, the nanobeads are slowly settled by gravity alone. A magnetic field is applied via an external magnet for 3 minutes to pull the nanobeads down to the bottom of the microwell ( Figure 7 a and Figure 7 b). Subsequently, lysis buffer is added to the chip to lyse the cells in situ. The bait antibodies immobilized on the nanobeads capture the bait protein and prey protein in the lysate in the microwells. During the entire experiment, the magnetic beads remain fixed to the glass bottom in the presence of a magnet. Prey proteins labeled with fluorescent or immunostaining are visualized and identified using TIRF or confocal microscopy ( Figure 8 a and Figure 8 b).

[0128] As previously described (Alsina et al., 2017) and slightly modified, an Olympus IX-73 inverted fluorescence microscope (Olympus) equipped with an oil immersion objective (NA = 1.49, 100 ×, UAPON, Olympus) was used to capture fluorescence images from this and all examples. Images were collected using a sCMOS camera (Zyla-4.2P-Cl10, Andor Technology Co., Ltd.). GFP was excited using a 488nm laser (Coherent, USA) with an exposure time of 300ms. Total internal reflection illumination was used to excite only GFP near the cover slip surface. ZT488 / 561rpc (Chroma) was used as a dichroic mirror, and the emission signal was collected by an ET525 / 50m (Chroma) emission filter to collect fluorescence signals from GFP. The laser was focused on the rear focusing plane of the objective. A 15X beam expander (Edmond Optics, Singapore) and a focusing lens were used to evenly illuminate the sample. Total internal reflection illumination was generated by moving the illumination beam away from the center of the lens. All experiments were performed at room temperature.

[0129] The single cell occupancy rate (single cells per well) reached about 60% ( Figure 6 ), which depends on the cell density applied to the chip.

[0130] Example 6

[0131] Single-cell GFP pull-down

[0132] like Fig. 9 In a-9f, the nanobead-based single-cell SiMPull assay was evaluated by pulling down GFP from single cells. After suspension by trypsinization, HEK293T cells expressing GFP were added to a blank microwell chip and subjected to SiMPull by magnetic nanobeads applied to the chip surface. GFP was pulled down by magnetic nanobeads coated with anti-GFP antibody by biotinylated secondary antibody ( Fig. 9 a and Fig. 9 b) but not pulled down by nanobeads coated with the second antibody alone ( Fig. 9 c and Fig. 9 d). These results indicate minimal nonspecific binding of GFP to the nanobeads and that the SN ratio in these experiments was approximately 12 ( Fig. 9 e) Furthermore, photobleaching revealed that these spots displayed one-step bleaching (Figure 9f), indicating the presence of single GFP monomers.

[0133] Example 7

[0134] Single-cell pull-down of PKA complexes

[0135] like Figure 3 In a-3c, the interaction between PKA-R subunits and PKA-C subunits was studied to validate the disclosed method for studying PPI. The interaction between PKA-C-eGFP and PKA-R-mCherry in single HEK293T cells was studied ( Fig.10 a). When anti-GFP coated magnetic nanobeads were used, both PKA-C-eGFP and PKA-R-mCherry were pulled down, and notably, very little PKA-R-mCherry was pulled down in the presence of the cAMP analog cpt-cAMP ( Fig.10 b); This suggests that the pull-down specificity of PKA-R-mCherry depends on its physiological interaction with PKA-C-eGFP rather than on nonspecific binding to the beads or the bottom of the microwell. Figure 3 Similar to the results shown in b, most of the GFP and mCherry signals clearly colocalize, although a small portion does not ( Fig.10 a).

[0136] Next, a more general application of nanobead-based SiMPull was tested: the prey protein (PKA-R) was not fluorescently tagged and needed to be visualized by immunostaining. Single HEK293T cells co-expressing PKA-C-eGFP and PKA-R-HA were subjected to SiMPull using anti-GFP coated nanobeads. After cell lysis, a mixture of anti-HA antibody and Alexa-561 coupled secondary antibody was added to the chip surface to visualize PKA-R-HA. PKA-R-HA was clearly visible after immunostaining, and about 80% of GFP spots co-localized with Alexa-561 spots ( Fig.10 c). If PKA-RH is replaced by PKA-R-GFP in these experiments, few Alexa-561 spots are visible ( Fig.10 d), indicating Fig.10 The Alexa-561 signal in c is specific for the HA-tag. Figure 3 A similar reason for the incomplete colocalization of GFP and mCherry spots in b may be Fig.10 The reason for the incomplete colocalization of GFP and Alexa spots in c.

[0137] The invention described and claimed herein has many attributes and embodiments, including but not limited to those stated or described or mentioned in the specific embodiments. But this is not intended to be all-inclusive, and the invention described and claimed herein is not limited to the features or embodiments determined in the specific embodiments, or is not limited thereto, and the purpose of including the specific embodiments is only to illustrate rather than to limit. It will be readily appreciated by those of ordinary skill in the art that many components and parameters may be changed or modified to a certain extent, or may be replaced by known equivalents, without departing from the scope of the present invention. It should be understood that such modifications and equivalents are incorporated herein as if stated separately. The present invention also includes all steps, features, compositions and compounds mentioned or indicated individually or jointly in this specification, as well as any and all combinations of any two or more of the steps or features.

[0138] All patents, publications, scientific articles, websites and other documents and materials cited or mentioned herein indicate the technical level of technicians in the field to which the present invention belongs, and each such cited document and material is hereby incorporated by reference to the same extent as if it has been individually incorporated by reference in its entirety or stated in its entirety herein. The applicant reserves the right to physically incorporate any and all materials and information from any such patents, publications, scientific articles, websites, electronically available information and other reference materials or documents into this specification. The reference to any application, patent and publication in this specification is not, and should not be considered as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0139] The specific methods and compositions described herein represent preferred embodiments and are exemplary, and are not intended to be limiting the scope of the invention. Other objects, aspects and embodiments will occur to those skilled in the art after considering this specification, and these objects, aspects and embodiments are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to those skilled in the art that different substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein may be suitably implemented in the absence of any one or more elements, or one or more restrictions, which are not explicitly disclosed herein as necessary. Thus, for example, in each case herein, and in embodiments or examples of the present invention, any one of the terms "comprising", "consisting essentially of" and "consisting of" may be substituted with any of the other two terms in the specification. The methods and processes illustratively described herein may be appropriately implemented in different step orders, and they are not necessarily limited to the step orders indicated herein or in the claims. As used herein and in the appended claims, unless the context otherwise clearly indicates, the singular forms "a / an" and "said" also include plural references. In any case, this patent should not be interpreted as being limited to the specific embodiments or embodiments or methods specifically disclosed herein. In addition, names, titles, etc. are provided to enhance the reader's understanding of this document and should not be interpreted as limiting the scope of the invention. Any examples of aspects, embodiments, or components of the invention mentioned herein should be considered non-limiting.

[0140] The terms and expressions that have been adopted are used in terms of description rather than limitation, and the use of these terms and expressions is not intended to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications can be made within the scope of the invention as claimed. Therefore, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be adopted by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

[0141] The invention has been described broadly and generally herein. Each narrower class and sub-general combination falling within this general disclosure also forms part of the present invention. This includes describing the invention generally with a proviso or negative limitation that removes any subject matter from the generality, regardless of whether the removed content is explicitly stated herein.

[0142] Other embodiments are within the following claims.In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

1. A system comprising: a magnet, a transparent glass substrate comprising a plurality of micrometer-sized features, a plurality of antibody-coated magnetic nanobeads, a plurality of primary antibodies directed against a bait biomolecule, a plurality of bait molecules, a plurality of target prey biomolecules, a plurality of primary antibodies directed against said target prey biomolecules, a cell lysis buffer, and a secondary antibody bound to said primary antibody bound to said prey biomolecule, wherein said secondary antibody is fluorescently labeled; wherein the plurality of antibody-coated magnetic nanobeads comprises a plurality of secondary antibodies directed against the primary antibody directed against the bait biomolecule, or secondary antibodies that bind to the primary antibody bound to the bait biomolecule; wherein the plurality of micrometer-sized features of the transparent glass substrate are a plurality of micropores, each of the micropores having a diameter of 15 micrometers to 45 micrometers, a distance between centers of the micropores ranging from 100 micrometers to 5000 micrometers, and a depth of each micropore ranging from 25 micrometers to 150 micrometers; The size of the plurality of antibody-coated magnetic nanobeads is 5 nanometers to 500 nanometers; The transparent glass substrate is a cover glass having a thickness of 0.085 mm to 0.64 mm, and the glass substrate is configured to be measurable by total internal reflection microscopy.

2. The system of claim 1, wherein the diameter of the microwells is 30 micrometers and the depth of each microwell is 70 micrometers.

3. The system according to claim 1 or 2, further comprising one or more types of cells.

4. The system of claim 3, wherein the cells comprise one or more types of target prey biomolecules.

5. The system of claim 3, wherein the plurality of types of cells are located in the plurality of microwells.

6. The system of claim 1 or 2, wherein the cell lysis buffer comprises a non-denaturing detergent.

7. The system of claim 1 or 2, wherein the distance between the centers of the microwells is 150 microns.

8. The system of claim 1, wherein the depth of each microwell is about 75 microns, wherein "about" means plus or minus 10% of the numerical value of the number used.

9. The system according to claim 1 or 2, wherein the cells are cells having an average diameter smaller than an average diameter of the pores.

10. The system of claim 9, wherein the cells have an average diameter of 30 microns.

11. The system of claim 1 or 2, wherein the antibody-coated magnetic nanobeads comprise an iron oxide core.

12. The system of claim 3, wherein the plurality of target prey biomolecules comprises molecules found in a cell membrane of the cell.

13. The system of claim 3, wherein the plurality of target prey biomolecules comprises molecules found in the cytoplasm of the cell.

14. The system of claim 1 or 2, wherein the plurality of microwells are etched directly into the glass substrate.

15. The system of claim 1 or 2, wherein the plurality of micropores are formed of polydimethylsiloxane.

16. The system of claim 14, wherein the plurality of microwells are formed of polydimethylsiloxane.

17. The system of claim 15, wherein the substrate comprises cured PDMS.

18. The system of claim 16, wherein the substrate comprises cured PDMS.

19. The system of claim 1 or 2, wherein the bait biomolecule and prey biomolecule are independently selected from the group consisting of: protein, DNA, and RNA.

20. The system of claim 1 or 2, wherein the bait biomolecule and prey biomolecule are independently small molecules.

21. The system of claim 1 or 2, wherein the bait biomolecule and prey biomolecule are independently peptides.

22. The system of claim 19, wherein the bait biomolecule and prey biomolecule are independently selected from: mRNA, tRNA, cRNA, antibodies.

23. The system of claim 19, wherein the bait biomolecule and prey biomolecule are independently antibody fragments.

24. The system of claim 19, wherein the bait biomolecule and prey biomolecule are independently ScFv.

25. The system of claim 19, wherein the DNA or RNA is further modified with biotin.

26. A method for identifying biomolecule-biomolecule interactions using the system according to claim 1 or 2, the method comprising the steps of: (a) contacting a substrate comprising the plurality of microwells with one or more cell types, wherein a portion or all of the one or more cell types enter a portion or all of the plurality of microwells; (b) providing a plurality of antibody-coated magnetic nanobeads to the substrate, wherein the antibody-coated magnetic nanobeads comprise antibodies that can bind to bait biomolecules; (c) applying a magnetic field to the antibody-coated magnetic nanobeads; (d) contacting the substrate with a lysis buffer; (e) forming a complex selected from the antibody of the antibody-coated magnetic nanobead and the bait biomolecule, the bait biomolecule and the prey biomolecule, or all of the bait biomolecule and the prey biomolecule bound to the antibody of the antibody-coated magnetic nanobead; (f) providing an antibody or antibody complex that binds to the prey biomolecule; (g) After cell lysis and protein capture, the magnetic nanobeads are dragged to the bottom of the microwell by a magnet; (h) imaging the antibody or antibody complex bound to the prey biomolecule by fluorescence microscopy; wherein the cell comprises a bait biomolecule and a prey biomolecule, and The antibody or antibody complex that binds to the prey biomolecule is fluorescently labeled.

27. The method of claim 26, wherein the antibody complex bound to the prey biomolecule comprises a primary antibody bound to the prey biomolecule, and a secondary antibody bound to the primary antibody bound to the prey biomolecule, wherein the secondary antibody is fluorescently labeled.

28. The method of claim 26, wherein the prey biomolecule and the bait biomolecule are each selected from the group consisting of: protein, DNA, and RNA.

29. The method of claim 26, wherein the bait biomolecule and prey biomolecule are each small molecules.

30. The method of claim 26, wherein the bait biomolecule and prey biomolecule are each a peptide.

31. The method of claim 26, wherein the bait biomolecule and prey biomolecule are each selected from the group consisting of: mRNA, tRNA, cRNA, and antibodies.

32. The method of claim 26, wherein the bait biomolecule and prey biomolecule are each an antibody fragment.

33. The method of claim 26, wherein the bait biomolecule and prey biomolecule are ScFv, respectively.

34. The method of claim 26, wherein step (g) imaging the antibody or antibody complex bound to the prey biomolecule by fluorescence microscopy is performed by total internal reflection microscopy or confocal microscopy.

35. The method of claim 26, wherein the signal-to-noise ratio is higher than 10.

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