Exosome analysis and cancer diagnosis methods

Abstract

The present invention relates to the quantification, separation and characterization of exosomes. Exosome immune complexes are constructed on beads to produce constructed immune complex beads, and target exosomes are digitally quantified by droplet microfluidics technology and exosomes are measured at the level of single exosomes by diluting and encapsulating the exosomes into a sufficient number of droplets. Quantified exosomes can be used to diagnose cancer in a subject.

Description

[0001] This application is a divisional application of Chinese patent application 201811166707.4, whose application date is October 8, 2018, whose applicant is the Hong Kong University of Science and Technology, and whose invention name is “Exosome Analysis and Cancer Diagnosis Methods”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 606,687, filed on October 5, 2017, the disclosure of which is incorporated by reference in its entirety, including any figures, tables, or drawings. Technical Field

[0004] The present application relates to the field of exosomes, and more specifically, to the capture and analysis of exosomes and their uses. Background Art

[0005] Exosomes are positioned as effective biomarkers for cancer diagnosis. Exosomes are non-uniform membranous particles with a diameter of 30-150 nm that are secreted from cells through the plasma membrane fusion of multivesicular bodies (MVBs). Exosomes shed from tumor tissues carry many biomarkers, such as transmembrane proteins and cytoplasmic proteins (CD9, CD63, CD81, etc.), lipids, DNA, and microRNAs. Special proteins such as Glypican-1 (GPC1), fibronectin (FN), prostate-specific membrane antigen (PSMA), and functional nucleic acids such as microRNA-145 are clinically significant in early cancer diagnosis. In addition, exosomes are widely present in human biological fluids such as serum, urine, amniotic fluid, cerebrospinal fluid, saliva, and even tears; therefore, they provide non-invasive unique features for cancer diagnosis. Therefore, exosomes are increasingly valued in the diagnosis of cancer, monitoring of liquid biopsies, and prognosis. Reliable methods and tools for the isolation, quantification, and characterization of cancer exosomes are essential to advance the development of this field.

[0006] Conventional methods for isolating exosomes include ultracentrifugation (UC), filtration, and density gradient separation. Among them, UC is considered the "gold standard" for exosome isolation. However, these traditional separation methods are mechanically based and time-consuming. At the same time, these methods lack the specificity to distinguish between tumorigenic and non-tumorigenic exosomes.

[0007] Exosomes are usually analyzed using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), or flow cytometry. NTA provides a rough value for the number of vesicles, but requires samples to be at high concentration levels (1 × 10 7 -10 9particles / mL). For early diagnosis of cancer, where exosomes are usually present at low concentration levels, NTA cannot provide accurate measurement of biomarkers for monitoring cancer progression. Western blot and ELISA analysis are considered the "gold standard" methods, but are still limited by poor sensitivity and large sample volumes. Flow cytometry can be used for high-throughput sorting of exosomes with fluorescent labels. However, this method is not efficient because exosomes are often bound to water beads and the weak light scattering of flow cytometry can cause quantitative losses.

[0008] Electrical methods include electrohydrodynamic systems and electrochemical biosensors, especially aptamer-based electrochemical sensors (aptamer sensors) have been used to detect exosomes. Electrohydrodynamic systems use surface shear forces to reduce nonspecific adsorption and improve specificity, but the limit of detection (LOD) is insufficient for many applications. Aptamer sensors have the advantages of electrochemical detection methods such as rapidity, sensitivity, low power consumption and continuous monitoring. However, due to the unpredictable secondary structure of aptamers, it is still difficult to obtain suitable aptamers, and effective aptamer selection methods have not yet been developed. Recently, new technologies such as surface plasmon resonance (SPR) and Raman scattering can read out target exosomes in real time and without labels. However, these methods are still challenging for clinical application in terms of throughput and cost.

[0009] Droplet- or microwell-based microfluidics have proven to be "miniaturized reactors" that have revolutionized biological and chemical analyses performed in traditional pipettes, beakers, tubes, or flasks. Shrinking the reaction volume in droplets or microwells brings a variety of unique features, such as high throughput, minimal reagent consumption, no contamination, fast response, miniaturized sample loss, and separation of parallel reactions. In the past decade, with the rapid development of microfluidics technology, droplet microfluidics technology has become a versatile platform for molecular detection, material synthesis, segmented reactions, or high-throughput screening in the fields of chemistry and biology. Summary of the invention

[0010] The present invention provides the application of microfluidics technology for the quantification, separation and characterization of exosomes. In certain embodiments, a) quantifying exosomes in a sample by contacting a sample containing a plurality of exosomes with i) capture beads comprising beads bound to a first binding agent, and ii) a second binding agent comprising a detectable label, wherein the first binding agent specifically binds to a first biomolecule present in a plurality of exosomes to produce a first complex comprising the capture beads and the first exosome, and the second binding agent specifically binds to a second biomolecule present in a plurality of exosomes to produce an exosome-second binding agent complex comprising a second binding agent and a second exosome, or a second complex comprising the capture beads, the first exosome and the second binding agent; b) separating the capture beads, the first complex and the second complex from the composition produced at the end of step a), c) separating the capture beads, the first complex and the second complex from each other from the composition produced at the end of step b), d) optionally, contacting the separated capture beads, the first complex and the second complex with a matrix, the matrix producing a detectable signal from the second binding agent present in the second complex, e) detecting the detectable signal from the second complex to quantify the exosomes in the sample. The relative proportion of beads in the second complex compared to the capture beads and the first complex can be used to quantify exosomes in the sample.

[0011] The first binding agent and the second binding agent can bind to one or more cancer biomarkers. Therefore, the methods disclosed herein can be used to isolate exosomes that indicate cancer. Accordingly, certain embodiments of the present invention provide methods for diagnosing cancer by quantifying exosomes containing cancer biomarkers in a sample obtained from a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 An exemplary procedure for preparing exosome immune complexes on beads.

[0013] Figure 2 Schematic diagram of digital quantification of exosomes with specific proteins using droplet- or microwell-based approaches.

[0014] Figure 3 Schematic diagram of the isolation of desired exosomes with specific biomarkers using droplet sorting.

[0015] Figure 4 Schematic diagram of the single exosome assay platform using droplet fusion and sorting technology.

[0016] Figure 5a to 5d are schematic diagrams of the droplet digital exosome enzyme-linked immunosorbent assay (ExoELISA) for exosome quantification. (a) Single exosome immune complex constructed on magnetic beads. (b) Co-encapsulation of matrix and beads in droplets. (c) Droplet digital ExoELISA chip. (d) Fluorescence readout for counting positive droplets with target exosomes.

[0017] Figure 6 a to 6c are characterizations of exosomes. (a) TEM shows exosomes with a diameter of about 30-150 nm and a double-walled lipid membrane layer. (b) Size distribution of MDA7 MB-231 exosomes analyzed by NTA. This band depicts three replicate experiments. (c) Expression of CD63 (exosome marker) and GPC-1 (diagnostic marker) in MDA-MB-231 exosomes and parental cells by Western blot analysis. Equal amounts of protein (20 μg) were loaded in exosomes and cells.

[0018] Figure 7 a to 7h show the generation of droplets. (a) The prepared beads and FDG matrix were co-encapsulated into 40 μm diameter droplets, which were spread as a monolayer in the device for detection. (b) Droplet digital ExoELISA calibration results show that the dynamic range of captured exosomes spans 5 orders of magnitude. The dashed line is 3 times the background plus the standard deviation, indicating the LOD (~10 exosomes / μL). (c) Negative control without target exosomes. (dh) Fluorescence reading gradient of exosome samples isolated from MDA-MB-231 by serial dilution. NanoSight is used as a benchmark measurement of exosome number concentration.

[0019] Figure 8 a to 8b show the specificity of the assay. Specificity of the assay. (a) Western blot analysis showing the differential expression of GPC-1 in MDA-MB-231 cells (positive control) and exosomes isolated from the culture medium of MDA-MB-231, HL-7702, RAW264.7, and hES cells. 20 μg of protein was loaded per lane. (b) Specificity of droplet digital ExoELISA of exosomes isolated from the culture medium of MDA-MB-231, HL-7702, RAW264.7, and hES cells. The case of magnetic beads without CD63 Ab and the detection sample solution without exosomes was used as negative control. Each sample solution contained 6.39×104 8 exosome particles per μL.

[0020] Fig. 9a to 9c show clinical analysis of GPC-1(+) exosomes by droplet digital ExoELISA. (a) Quantification of GPC-1(+) exosomes from serum samples of 5 healthy samples (HS), 5 patients with benign breast disease (BBD), and 12 patients with breast cancer (BC). (b) The scatter plot shows that GPC-1(+) exosomes in BC patients are significantly overexpressed compared with HS and BBD (****, p<0.0001). (c) Quantification of GPC-1(+) exosomes in 2 patients with breast cancer (BC) and breast cancer after surgery (BC-AS). Error bars represent the standard deviation of three independent experiments.

[0021] Fig.10 a to 10f show two-color super-resolution images of CD63 and GPC-1 in exosomes isolated from the culture medium of MDA-MB-231 cells. Stochastic optical reconstruction microscopy (STORM) images show (a) exosome membrane stained with PKH67; (b) CD63 labeled with Alexa Fluor 647; (c) merged image of (a) and (b); (d) exosome membrane stained with PKH67; (e) GPC-1 labeled with Alexa Fluor 647; (f) merged image of (d) and (e).

[0022] Fig.11 a to 11b are TEM images showing single exosomes captured by immunomagnetic. (a) PBS was used instead of MDA-MB-231 exosome solution as a negative control. (b) MDA-MB-231 exosomes were captured on CD63 antibody-bound beads. Arrows indicate single exosomes.

[0023] Fig.12 Figures a to 12b show bright field images captured under a microscope with a 20X objective, showing that the magnetic beads are well separated into droplets. The circles indicate the areas where the beads are located in the droplets. (a) When the average number of beads per droplet is set to ~0.1, all droplets contain 0 or 1 bead in the field of view. (b) When the average number of beads per droplet is set to ~0.3, only 1 / 85 of the droplets contain 2 magnetic beads, 5 / 85 of the droplets contain 1 magnetic bead, and the rest of the droplets are empty, which is consistent with Poisson statistics.

[0024] Fig.13 Optimization of incubation time for FDG-catalyzed reaction in microdroplets is shown. F and F 0 The mean fluorescence intensity of the signals from all droplets and the background, respectively. The normalized signal reached the highest value at 30 min. The error bars are the standard deviation of three experiments.

[0025] Fig.14a to 14c are representative NTA images showing the size distribution of exosomes isolated from (a) HL-7702, (b) RAW264.7, and (c) hES cell culture media, respectively. The bands depict three experiments. DETAILED DESCRIPTION

[0026] As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, where the terms "includes", "comprising", "having", "having", "and" or variations thereof are used in the specification and / or claims, these terms are intended to be inclusive in a manner similar to the term "comprising". The transitional terms / phrases (and any grammatical variations thereof) "comprises", "includes", "contains", include the phrases "consisting essentially of", "consisting essentially of", "consisting essentially of", and "consisting of".

[0027] The phrase "consisting essentially of" or "composed essentially of" means that the claim encompasses embodiments including the specified materials or steps as well as embodiments that do not materially affect the basic and novel characteristics of the claim.

[0028] The term "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Where particular values ​​are described in the specification and claims, the term "about" should be assumed to mean within an acceptable error range for the particular value unless otherwise indicated.

[0029] In this article, ranges are stated in abbreviated form to avoid having to specify and describe each value in the range. Where appropriate, any appropriate value in the range can be selected as the upper limit, lower limit or end point of the range. For example, a range of 1-10 represents the final values ​​of 1 and 10, and the intermediate values ​​of 2, 3, 4, 5, 6, 7, 8, 9, and all intermediate ranges contained in 1-10, such as 2-5, 2-8 and 7-10. In addition, when a range is used herein, it is intended to clearly include combinations and sub-combinations of ranges (e.g., sub-ranges within the disclosed ranges) and specific embodiments thereof.

[0030] The present invention provides microfluidic methods for quantification, separation and characterization of exosomes. The microfluidic methods include droplet or microwell microfluidic technologies, such as compartmentalization, separation and sorting. For digital quantification and separation of desired exosomes, enzyme-linked immunosorbent assays can be used to identify exosomes containing specific biomarkers. For example, through specific antigen-antibody binding, target exosomes are identified and fixed on capture beads to form enzyme-linked immune complexes. The immune complex solution is divided into a sufficient number of evenly separated compartments (e.g., droplets or microwells) so that each compartment contains one or no beads. If necessary, a matrix is ​​added to each compartment to produce a color or fluorescence or detectable signal from the beads. For those compartments containing beads, the attached enzyme triggers the matrix in the compartment to produce absorbance or fluorescence or electrochemical signals (e.g., current), which are measured to determine the presence and number of exosome immune complexes. Due to the random nature of bead preparation and distribution, the percentage of beads containing immune complexes and the percentage of partitions containing beads follow a Poisson distribution. Based on the dependent Poisson statistics of the partitions, target exosomes can be quantified to single copy accuracy. After identification of target exosomes by a detectable signal, the partitions (either droplets or microwells) can be further analyzed using droplet sorting techniques (e.g., in combination with flow cytometry) or using camera imaging (for microwell-based methods). The target exosomes can be recovered for further analysis of proteins, nucleic acids presented on the exosome membrane or within the exosome.

[0031] Accordingly, certain embodiments of the present invention provide a method for isolating or quantifying exosomes in a sample, comprising the following steps:

[0032] a) contacting a sample containing a plurality of exosomes with:

[0033] i) capture beads comprising beads bound to a first binding agent, and

[0034] ii) a second binding agent comprising a detectable label,

[0035] wherein the first binding agent specifically binds to a first biomolecule present in a plurality of exosomes to produce a first complex comprising a capture bead and the first exosome, and the second binding agent specifically binds to a second biomolecule present in a plurality of exosomes to produce an exosome-second binding agent complex comprising the second binding agent and the second exosome, or a second complex comprising the capture bead, the first exosome, and the second binding agent;

[0036] b) separating the capture beads, the first complex and the second complex from the composition produced at the end of step a),

[0037] c) separating each capture bead, the first complex and the second complex from each other from the composition produced at the end of step b),

[0038] d) optionally, contacting the separated capture beads, the first complex, and the second complex with a matrix and generating a detectable signal from the second binding agent present in the second complex,

[0039] e) detecting a detectable signal from the second complex.

[0040] The steps a) to e) listed above are used herein to refer to the specific steps of the method of the present invention. In addition, step d) as listed above can be performed before step c), but it is preferred that step d) is performed after step c).

[0041] Those skilled in the art will recognize that steps i) and ii) of contacting the sample with the capture beads and the second binding agent can be performed simultaneously or sequentially with each other. For example, the sample, the capture beads, and the second binding agent can be mixed together. Alternatively, the sample and the second binding agent can be mixed first, and then the capture beads can be added. In addition, the sample and the capture beads can be mixed first, and then the second binding agent can be added. Regardless of the order in which the different components are contacted, this step generally results in the formation of the following mixture: capture beads, the first complex, the second complex, and the exosome-second binding agent complex.

[0042] If the capture beads and the second binding agent are contacted with the sample sequentially, a washing step may be performed between the two contacting steps. For example, the mixture comprising the capture beads, the first complex, the exosomes and other components of the sample may be washed to remove unbound exosomes and / or other components in the sample. This washing separates the capture beads and the first complex, which may then be contacted with the second binding agent comprising a detectable label.

[0043] The step of contacting the sample with the capture beads and / or the second binding agent is performed under suitable conditions and for a suitable time to allow the generation of the corresponding binding complex. Typically, a majority of the exosomes containing the appropriate biomolecules present in the sample, for example, more than about 90% of the relevant exosomes present in the sample, are bound to the capture beads and / or the second binding agent. One of ordinary skill in the art can achieve appropriate conditions for maximum binding between binding partners.

[0044] The size range of beads used in the present invention is about 0.5 microns to about 20 microns, preferably about 1 to 15 microns, more preferably about 2 to 10 microns, even more preferably about 3 to 6 microns, and most preferably about 4 to 5 microns. The beads are typically made of an inert material, such as agarose or an inert polymer. The beads can also be superparamagnetic, that is, they exhibit magnetism in a magnetic field and have no residual magnetism once the magnetic field is removed. Exemplary superparamagnetic materials include ferrites or magnetite (Fe 3 O 4). Additional superparamagnetic materials suitable for use in beads are known to those skilled in the art, and such embodiments are within the scope of the present invention.

[0045] The beads may also have a core of superparamagnetic material covered with an inert material such as a polymer. Exemplary polymers include polystyrene. Other materials suitable for producing capture beads are known to those skilled in the art, and such embodiments are within the scope of the present invention.

[0046] The beads are bound to a first binding agent to produce capture beads. The first binding agent specifically binds to a first biomolecule present in the exosomes.

[0047] For purposes of the present invention, the phrase "specifically binds" or grammatical variations thereof refers to the ability of a binding agent to bind only to its binding partner with relatively little nonspecific affinity for other biomolecules. Specificity can be determined relatively by binding or competitive binding assays. Specificity can be calculated mathematically, for example, by an affinity / avidity ratio of about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1, or more, where the affinity / avidity ratio refers to the ratio of specific binding to a binding partner relative to nonspecific binding to other unrelated biomolecules. For example, the equilibrium dissociation constant (K) of an antibody that specifically binds to an antigen is D ) is less than about 10 -6 M, less than about 10 -9 M, or less than about 10 -12 M, for the binding between antibodies and corresponding antigens.

[0048] On the other hand, "non-specific binding" refers to binding that is not based on specific interactions between a binding agent and its binding partner. Non-specific binding may be caused by non-specific interactions, such as van der Waals forces. For example, the K of binding between an antibody and a non-specific antigen is D Usually above about 10 -6 M, above about 10 -4 M or above about 10 -2 M.

[0049] The first binding agent can be an antibody, an antigen binding fragment of an antibody, an aptamer, a protein binding partner, or a nucleic acid binding partner of a first biomolecule present in an exosome. In a preferred embodiment, the first binding agent binds to a first biomolecule present in an exosome that is a cancer biomarker. Certain such biomolecules include CD9, CD63, CD81, GPC1, FN, PSMA, or microRNA-145. Accordingly, the first binding agent can specifically bind to CD9, CD63, CD81, GPC1, FN, PSMA, or microRNA-145. Other examples of biomolecules that are cancer biomarkers present in exosomes are known in the art, and these embodiments are within the scope of the present invention.

[0050] The second binding agent specifically binds to a second biomolecule present in the exosomes. The first binding agent and the second binding agent can bind to the same biomolecule or different biomolecules. If the first binding agent and the second binding agent bind to the same biomolecule, it is preferred that they bind to different binding sites on the same biomarker. Typically, the second biomolecule is different from the first biomolecule. Thus, the second binding agent specifically binds to a second biomolecule that is different from the first biomolecule bound by the first binding agent.

[0051] For the purposes of the present invention, the phrase "biomolecule present in exosomes" means that the biomolecule can be present on the surface of the exosomes or in the lumen of the exosomes. Preferably, the biomolecule is present on the surface of the exosomes to provide a binding agent with easier access to the biomolecule.

[0052] The second binding agent can be an antibody, an antigen binding fragment of an antibody, an aptamer, a protein binding partner, or a nucleic acid binding partner of a second biomolecule present in the exosomes. In a preferred embodiment, the second binding agent binds to a second biomolecule present in the exosomes that is a cancer biomarker. Certain such biomolecules include CD9, CD63, CD81, GPC1, FN, PSMA, or microRNA-145. Accordingly, in certain embodiments, the second binding agent binds to CD9, CD63, CD81, GPC1, FN, PSMA, or microRNA-145. Other examples of biomolecules that are cancer biomarkers present in exosomes are known in the art, and these embodiments are within the scope of the present invention. The references of Li et al. and Nedaeinia et al. are incorporated herein by reference in their entirety.

[0053] The capture beads, the first complex and the second complex are separated from the composition produced at the end of step a). In certain embodiments, the beads may be washed with a suitable buffer to remove the exosome-second binding agent complex and other components that may be from the sample and other reagents.

[0054] Washing the beads can be performed by methods known in the art and suitable for the particular beads. For example, the beads can be centrifuged after repeated washings to separate the beads from the remaining components. If the beads are magnetic or superparamagnetic, a magnetic field can be used to capture the beads, and the remaining components can be washed away with an appropriate buffer. One of ordinary skill in the art can design a suitable washing method to separate the captured beads, the first complex and the second complex from the composition produced at the end of step a).

[0055] After step b), the capture beads, the first complex and the second complex are separated from each other. Thus, the composition produced at the end of step b) is divided into a plurality of compartments, each compartment containing no beads, one capture bead, one first complex or one second complex.

[0056] In certain embodiments, droplet generation is used to separate the steps of capturing beads, the first complex and the second complex. In droplet generation, the composition comprising capture beads, the first complex, and the second complex (the composition produced at the end of step b)) is divided into droplets, wherein each droplet encapsulates a capture bead, a first complex or a second complex. For the method disclosed herein that works as expected, less than about 5%, preferably less than about 4%, more preferably less than about 3%, even more preferably less than about 2%, and most preferably less than about 1% of the compartments contain two or more beads. Ideally, no compartment contains two or more beads.

[0057] In an exemplary embodiment, two immiscible phases are used for droplet generation; a continuous phase (the composition that separates into droplets) and a dispersed phase (the phase that forms the droplets). The size of the droplets can be controlled by adjusting various parameters, such as the flow rate ratio of the continuous and dispersed phases, the interfacial tension between the two phases, and the geometry of the channel used for droplet generation.

[0058] Droplet generation can be active or passive. In active droplet generation, an external energy input, such as electrical, magnetic, centrifugal energy, is provided for droplet manipulation. Passive droplet generation can be performed using certain microfluidic geometries, i.e., cross-flow, flow focusing, and co-flow.

[0059] Cross flow involves the continuous phase and the dispersed phase running at an angle to each other. Typically, these phases are perpendicular to each other, i.e., in a T-junction, the dispersed phase crosses the continuous phase. Other configurations, such as a Y-junction, may also be implemented. The dispersed phase extends into the continuous phase and is stretched until shear forces break the droplets. In a T-junction, the flow rate ratio and the capillary number control the droplet size and formation rate. The capillary number depends on aspects such as the viscosity of the continuous phase, the apparent velocity of the continuous phase, and the interfacial tension. Other details about cross flow droplet generation are well known to those of ordinary skill in the art, and these embodiments are within the scope of the present invention.

[0060] Flow focusing involves the dispersed phase flowing and intersecting the continuous phase, usually at an angle (non-parallel flow). The dispersed phase is then subjected to a constraint to generate droplets. The constraint is typically a narrow channel, which generates droplets by symmetrical shearing. The slower the flow rate, the larger the droplet size, and vice versa. Other details about flow focusing droplet generation are well known to those of ordinary skill in the art, and these embodiments are within the scope of the present invention.

[0061] In co-flow, the dispersed phase channel is enclosed in the continuous phase channel, and at the end of the dispersed phase channel, the fluid is stretched until it breaks to form droplets by dripping or spraying. When capillary forces are dominant in the system, dripping occurs, and droplets are generated at the channel end points, and when the continuous phase moves slowly, spraying occurs by widening or stretching, and fluid is produced from the dispersed phase channel opening. In the widened form, the dispersed phase moves faster than the continuous phase, causing the dispersed phase to slow down, making the droplets widen and increase in diameter. In the stretched form, viscous drag dominates, causing the fluid to narrow and produce smaller droplets. Droplet size depends on the phase flow rate and the stretched or widened form. Other details about co-flow droplet generation are well known to those of ordinary skill in the art, and these embodiments are within the scope of the present invention.

[0062] Typically, the composition produced at the end of step b) is used as the droplet phase and provides a continuous phase, for example, comprising oil or an emulsion. The specific details of the droplet generation step depend on the expected size of the droplets, the type of sample tested, the content of biomarkers in the exosomes, etc., and a person of ordinary skill in the art can determine such conditions as needed, and these embodiments are within the scope of the present invention. Some such embodiments are described in Examples 1-4 below.

[0063] As described above, the composition produced at the end of step b) is divided into multiple compartments, each compartment containing no beads, one capture bead, one first complex or one second complex. In certain embodiments, the step of separating the capture beads, the first complex and the second complex is performed using a micropore. For example, the composition produced at the end of step b) can be introduced onto a carrier comprising a micropore.

[0064] "Microwell" refers to a hole with a volume of 1 fl to 1000 nl, preferably 50 nl to 900 nl, more preferably 150 nl to 700 nl, even more preferably 250 nl to 600 nl, and most preferably about 500 nl. The size of the microwell on the chip is such that only one capture bead, only one first complex, or only one second complex can fit into one microwell. Therefore, the size of the microwell can be selected according to the size of the capture bead.

[0065] An example of a carrier comprising micropores is a glass substrate combined with a silicon grid, which forms micropores. The carrier comprising micropores can also be made of poly (dimethylsiloxane) polymers or plastics. Other materials suitable for preparing the carrier comprising micropores are known to those skilled in the art, and these embodiments are within the scope of the present invention.

[0066] Once the capture beads, first complexes, and second complexes are separated from one another, the number and / or amount of the second complexes can be determined based on the detectable signal provided by the second binding agent.

[0067] In the method of the present invention, one capture bead may contain thousands of molecules of a first binding agent capable of capturing exosomes. By controlling the ratio of beads to exosomes, it can be ensured that one capture bead binds to no more than one exosome. Each exosome can then bind to one or more molecules of a second binding agent. For example, one capture bead can bind to one exosome, and each exosome can bind to several molecules of a second binding agent. Thus, more molecules of the second binding agent will produce a relatively stronger signal. Therefore, quantification of exosomes in a sample can be performed based on the number of capture beads and the intensity of the signal generated by each capture bead.

[0068] As described above, the second binding agent contains a detectable label.Thus, the second complex can be distinguished from the capture beads and the first complex based on the presence or absence of a detectable signal.

[0069] The detectable label can produce a detectable signal with or without a substrate. For example, if the detectable label is a fluorescent, radioactive or chemiluminescent molecule, the second binding agent can produce a detectable signal without a substrate. On the other hand, if the detectable label is an enzyme that acts on a substrate to produce a detectable signal, the substrate is provided to produce a detectable signal, and then the detectable signal is detected to detect the second complex.

[0070] Detectable labels suitable for the methods disclosed herein include, but are not limited to, fluorescent moieties, chemiluminescent and bioluminescent agents, enzymes, and radioisotopes. Fluorescent moieties include, but are not limited to, fluorescein, fluorescein isothiocyanate, cascade blue, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, Texas Red, Oregon Green, cyanine (e.g., CY2, CY3, and CY5), umbelliferone, allophycocyanin, or phycoerythrin. Examples of luminescent materials include luminol. Examples of bioluminescent materials include, but are not limited to, fluorescein, green fluorescent protein (GFP), enhanced GFP, and aequorin. Applicable enzymes include, but are not limited to, luciferase, β-galactosidase, acetylcholinesterase, horseradish peroxidase, glucose-6-phosphate dehydrogenase, and alkaline phosphatase.

[0071] When the detectable label is an enzyme, a suitable substrate is provided to the enzyme to produce a detectable signal. For example, if the detectable label is peroxidase, the substrate may be hydrogen peroxide (H 2 O 2 ) and 3-3' diaminobenzidine or 4-chloro-1-naphthol. Other substrates suitable for other enzymes are well known in the art.

[0072] Suitable isotopes include but are not limited to 125 I. 14 C. 35 S and 3 H.

[0073] If the second binding agent requires a matrix to produce a detectable signal, the separated capture beads containing the first binding agent, the first complex, and the second complex are contacted with the matrix, thereby producing a detectable signal from the second binding agent. The step of contacting the matrix with the separated beads can be performed in various ways, depending on the method used to separate the beads.

[0074] For example, if a support comprising micropores is used to separate beads, the substrate is introduced into the micropores and incubated for an appropriate period of time under appropriate conditions to produce a detectable signal. The substrate can be introduced in the form of a suitable composition, such as a buffer. Depending on the type of enzyme used as a detectable label, excess substrate can be washed off before detecting the signal.

[0075] If droplet generation is performed to separate beads, the matrix can be incorporated into the continuous phase or the droplet phase. Figure 4 )

[0076] If the second binding agent does not require a matrix to produce a detectable signal, the detectable signal of the separated capture beads, the first complex and the second complex is tested to identify and quantify the second complex. The step of detecting the signal depends on the type of signal to be detected. For example, if the detectable signal is a fluorescent emission, a fluorescent camera can be used. Other methods for detecting specific detection signals are well known in the art and can be easily identified by a person of ordinary skill in the art. These embodiments are within the scope of the present invention.

[0077] Detecting the signal from the second complex can be used to distinguish the second complex from capture beads containing the first binding agent and the first complex. Such detection can be performed in a variety of ways, depending on the method used to separate the beads.

[0078] For example, if a support comprising microwells is used to separate beads, a camera can be used to image the microwells and identify the number of microwells containing capture beads and the first complex and the second complex. If droplet generation is used to separate beads, flow cytometry can be performed to identify the number of droplets containing capture beads and the first complex and the second complex.

[0079] The relative amount of the second complex compared to the capture beads and the first complex, and the intensity of the detectable signal from each second complex can be used to quantify the second complex, thereby quantifying the exosomes in the sample. A standard curve can be used with a control sample containing a known amount of exosomes to further facilitate the quantification of exosomes in the sample. A person skilled in the art can design a suitable standard curve for such quantification, and these embodiments are within the scope of the present invention.

[0080] Exosomes can be used as biomarkers for cancer diagnosis. Exosomes are shed from tumor tissues and carry many cancer biomarkers, such as transmembrane proteins and cytoplasmic proteins (CD9, CD63, CD81, etc.), lipids, DNA, and microRNA. Special proteins such as GPC1, FN, PSMA, and functional nucleic acids such as microRNA-145 can be used for early cancer diagnosis. In addition, exosomes are widely present in human biological fluids, such as serum, urine, amniotic fluid, cerebrospinal fluid, saliva, and even tears; therefore, they provide a unique non-invasive feature for cancer diagnosis. Therefore, the detection and quantification of exosomes according to the methods described herein can be used for cancer diagnosis, monitoring, and prognosis.

[0081] Accordingly, certain embodiments of the present invention provide a method for detecting cancer in a subject, the method comprising:

[0082] (I) determining the level of exosomes containing one or more cancer biomarkers in the following samples:

[0083] i) a test sample obtained from a subject,

[0084] ii) optionally, a control sample;

[0085] (II) arbitrarily obtaining a reference value corresponding to the level of exosomes containing one or more cancer biomarkers,

[0086] (III) The subject is identified as:

[0087] i) having cancer based on the level of exosomes containing one or more cancer biomarkers in the test sample compared to the level in a control sample or a reference value, or

[0088] ii) not having cancer based on the level of exosomes containing one or more cancer biomarkers in the test sample compared to the level in a control sample or a reference value.

[0089] If the subject is identified as having cancer, the method may further include administering a therapeutic method to the subject to treat and / or manage the cancer. If the subject is identified as not having cancer, the method may further include stopping the therapeutic method to the subject to treat and / or manage the cancer.

[0090] Cancer treatment methods can be selected from radiation therapy, chemotherapy, surgery, immunotherapy, such as monoclonal antibody therapy (e.g., bevacizumab or cetuximab), or any combination thereof. The therapy administered to the subject depends on the type of cancer, the age of the subject, the stage of the cancer, and other such individualized parameters.

[0091] In a preferred embodiment, the above disclosed method for quantifying exosomes in a sample is used to determine the level of exosomes containing one or more cancer biomarkers in a test sample obtained from a subject and any control sample. Therefore, certain embodiments of the present invention provide a method for determining the level of exosomes containing one or more cancer biomarkers in a sample, comprising the following steps:

[0092] a) contacting the sample with:

[0093] i) capture beads comprising beads bound to a first binding agent, and

[0094] ii) a second binding agent comprising a detectable label,

[0095] wherein the first binding agent specifically binds to a first cancer biomarker present in the exosomes to produce a first complex comprising the capture bead and the first exosome, and the second binding agent specifically binds to a second cancer biomarker present in the exosomes to produce an exosome-second binding agent complex comprising the second binding agent and the second exosome, or a second complex comprising the capture bead, the first exosome, and the second binding agent;

[0096] b) separating the capture beads, the first complex and the second complex from the composition produced at the end of step a),

[0097] c) separating each capture bead, the first complex and the second complex from each other from the composition produced at the end of step b),

[0098] d) optionally, contacting the separated capture beads, the first complex, and the second complex with a matrix and generating a detectable signal from the second binding agent present in the second complex,

[0099] e) detecting a detectable signal from the second complex to quantify the exosomes in the sample.

[0100] The first binding agent and the second binding agent can be, independently of each other, an antibody, an antigen-binding fragment of an antibody, an aptamer, a protein binding partner, or a nucleic acid binding partner of a first cancer biomarker present in an exosome. Certain such cancer biomarkers include CD9, CD63, CD81, GPC1, FN, PSMA, or microRNA-145. Accordingly, in certain embodiments, the first binding agent binds CD9, CD63, CD81, GPC1, FN, PSMA, or microRNA-145. Other examples of cancer biomarkers present in exosomes are known in the art, and these embodiments are within the scope of the present invention.

[0101] The first binding agent and the second binding agent may bind to the same cancer biomarker or different cancer biomarkers. If the first binding agent and the second binding agent bind to the same cancer biomarker, preferably they bind to different binding sites on the same cancer biomarker.

[0102] The details of the method for quantifying exosomes in a sample discussed above are also applicable to the cancer diagnosis method described herein. For example, the specific binding agents, beads, detectable labels, substrates, methods for separating beads, methods for detecting detectable signals, methods for quantifying second complexes, etc. discussed above are also applicable to cancer diagnosis methods, and these embodiments are within the scope of the present invention.

[0103] To implement the methods described herein for identifying a subject as having cancer, a control sample may be obtained from one or more of the following:

[0104] a) an individual of the same species as the subject who does not have cancer,

[0105] b) an individual who is of the same species as the subject and is known to have a low risk or no risk of developing the cancer, or

[0106] c) Subjects before developing cancer.

[0107] Other examples of control samples are known to those of ordinary skill in the art, and such embodiments are within the scope of the present invention.

[0108] In certain embodiments, control sample and test sample are obtained from the organ or tissue of the same type. The non-limiting examples of the organ or tissue that can be used as sample are placenta, brain, eye, pineal gland, pituitary gland, thyroid gland, parathyroid gland, thorax, heart, lung, esophagus, thymus gland, pleura, adrenal gland, appendix, gallbladder, bladder, large intestine, small intestine, kidney, liver, pancreas, spleen, stomata, ovary, uterus, testis, skin, blood or the buffy coat sample of blood. Other examples of organ and tissue are well known to those of ordinary skill in the art, and these embodiments are within the scope of the present invention.

[0109] In certain other embodiments, the control sample and the test sample are obtained from the same type of body fluid. Non-limiting examples of body fluids that can be used as samples include amniotic fluid, aqueous humor, vitreous humor, bile, blood, cerebrospinal fluid, chyle, endolymph, perilymph, female ejaculation, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rhubarb, saliva, sputum, synovial fluid, vaginal secretions, semen, blood, serum or plasma. Other examples of body fluids are well known to those of ordinary skill in the art, and these embodiments are within the scope of the present invention.

[0110] The methods described herein can be used to identify a subject as having cancer. In certain embodiments, the subject is a mammal. Non-limiting examples of mammals include humans, apes, dogs, pigs, cows, rodents, or felines.

[0111] The method of diagnosing cancer can be used to diagnose cancer types, including but not limited to: acanthoma, acinar cell carcinoma, acoustic neuroma, acromelanoma, acrospira, acute eosinophilic leukemia, acute lymphocytic leukemia, acute megakaryocytic leukemia, acute monocytic leukemia, mature acute myeloid leukemia, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adamantoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatous odontogenic tumor, adrenal cortical carcinoma, adult T cell leukemia, aggressive NK cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft tissue sarcoma, ameloblastoma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastoma Immunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, adnexal carcinoma, astrocytoma, atypical teratoma rhabdoid tumor, basal cell carcinoma, basaloid carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, embryoblastoma, bone cancer, bone tumor, brain stem tumor, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioalveolar carcinoma, brown tumor, Burkitt lymphoma, carcinoma of unknown primary site, carcinoid tumor, carcinoma, carcinoma in situ, penile cancer, carcinoma of unknown primary site, carcinosarcoma, Castleman disease, embryonal tumor of the central nervous system, cerebellar astrocytoma, brain astrocytoma, cervical cancer, bile duct cancer, enchondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic Leukemia, chronic monocytic leukemia, chronic myeloproliferative disease, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos' disease, cutaneous fibrosarcoma, dermoid cyst, hyperplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial carcinoma, endometrial carcinoma, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, estrogen blastoma, Ewing's tumor family, Ewing's family sarcoma, Ewing's sarcoma, extracranial germ cell tumor, extrahepatic germ cell tumor, extrahepatic bile duct cancer, breast extrahepatic Jegs disease, fallopian tube cancer, fetus in fetus, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer, glioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, germ cell tumor, gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, gliomatosis cerebri, glomus tumor, glucagonoma, gonadoblastoma, granular cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, heart cancer, hemangioblastoma, hemangiopericytoma, angiosarcoma, hematological malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast cancer, Hodgkin lymphoma,Hypopharyngeal cancer, Hypothalamic glioma, Inflammatory breast cancer, Intraocular melanoma, Islet cell carcinoma, Islet cell tumor, Juvenile myelomonocytic leukemia, Sarcoma, Kaposi's sarcoma, Kidney cancer, Kratzkin tumor, Krukenberg tumor, Laryngeal cancer, Malignant melanoma, Leukemia, Leukemia, Lip and oral cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphocytic leukemia, Lymphoma, Macroglobulinemia, Malignant fibrous histiocytoma, Malignant fibrous histiocytoma of bone, Malignant glioma, Malignant mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant tritonoma, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid carcinoma, Neuroblastoma medulloblastoma, medulloblastoma, melanoma, melanoma, meningioma, merkel cell carcinoma, mesothelioma, occult squamous neck cancer, occult primary, metastatic urothelial carcinoma, mixed mullerian tumor, monocytic leukemia, oral cancer, myxoma, multiple endocrine neoplasms syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic syndrome, myeloid leukemia, myeloma, myeloproliferative disorders, myxoma, nasal cancer, nasopharyngeal carcinoma, nasopharyngeal carcinoma, tumor, schwannoma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, ocular oncology, oligolymphocytic tumor, oligodendroglioma, philadelphic acid cell tumor, optic nerve sheath meningioma, oral cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, Paget's disease of the breast, superior sulcus tumor, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, intermediately differentiated pineal parenchymal tumor, pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasma cell tumor, pleuropulmonary blastoma, polyembryonic tumor, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prodromal prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, respiratory tract cancer involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyomas, rhabdomyosarcomas, Richter's lesions, sacrococcygeal teratomas, salivary gland cancer, sarcomas, schwannomatosis, sebaceous gland carcinoma, secondary tumors, seminoma, serous tumors, Sertoli cell-stromal tumors, sex cord-stromal tumors, Cesari syndrome, signet ring cell carcinoma, skin cancer, small blue round cell tumors, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestinal cancer, soft tissue sarcomas, somatostatinomas, smoke warts, spinal cord tumors, spinal tumors, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumors, surface epithelial-stromal tumors, synovial sarcoma,T cell acute lymphoblastic leukemia, T cell large granular lymphocytic leukemia, T-cell leukemia, T cell lymphoma, T cell lymphocytic leukemia, teratoma, advanced lymphoma, testicular cancer, theca cell tumor, laryngeal cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal carcinoma, urethral cancer, genitourinary tumors, uterine sarcoma, uveal melanoma, vaginal cancer, Verner-Morrison syndrome, verrucous carcinoma, optic pathway glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, or any combination thereof. In a preferred embodiment, the method for diagnosing cancer according to the present invention can be used to diagnose brain tumors, breast cancer, gastrointestinal cancer, colorectal cancer, lung cancer or prostate cancer. ,

[0112] All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0113] The following are examples illustrating the implementation of the method of the present invention. These examples should not be construed as limiting. Unless otherwise indicated, all percentages are by weight and all solvent mixture ratios are by volume.

[0114] Example 1 - Construction of exosome immune complexes on beads

[0115] Digital ELISA has been demonstrated in various microfluidic platforms. Exosome solutions are obtained from biofluids and prepared by ultracentrifugation, ultrafiltration, density gradient separation, and immunoaffinity capture methods. Since antigens are present on the surface of exosomes, they can be recognized by specific antibodies. A pair of antibodies for identifying exosomes is constructed on beads in the form of immune complexes. The construction of immune complexes on beads is shown in Figure 2. Figure 1 As shown. Antibodies that can recognize biomarkers on the surface of exosomes (e.g., CD63) are coupled to beads (e.g., Dynabeads TM or agarose beads). The beads are then incubated with the exosome solution. After incubation, the beads are collected by magnetic force or centrifugation. After thorough washing, the target exosomes bound to the beads are purified from the sample solution. Next, the exosomes are detected using a second antibody that can recognize the same (e.g., CD63) or different biomarkers (e.g., GPC-1) on the exosomes. The detection antibody is typically bound to a tag (e.g., biotin) that can recognize an enzyme (e.g., streptavidin-bound β-galactosidase). The methods disclosed herein for quantification and separation of exosomes are not limited to specific biomarkers. Different exosome biomarkers with corresponding antigen-antibody pairs that have been found on the exosome membrane are applicable.

[0116] Example 2-Digital Quantification of Target Exosomes

[0117] Digital quantification of immune complex beads bound to target exosomes by specific protein biomarkers. The immune complex constructed bead solution flows into a channel to mix the solution with the matrix (e.g., FDG) flow of another channel and form droplets of the mixture. In addition to using droplets as compartments, microwells made on a flat chip can also be used to divide the sample solution. The sample with beads can first be dropped on the chip and scraped into the wells. The matrix (e.g., FDG) solution is then added to each compartment. The microwell chip is then sealed at the top to isolate each separate space for reaction. Microfluidic workflow as shown Figure 2 As shown. After incubation, the droplets / wells with beads constructed with immune complexes emit color or fluorescence or electrochemical signals for detection. The signals can be detected by fluorescence microscopy or electrochemical sensor arrays. By counting the number of positive and negative droplets / wells, the number of target exosomes can be calculated according to the following formula:

[0118]

[0119] Where N is the absolute number of captured molecules, N b is the total number of beads, V s is the total test sample volume, V d is the droplet / well volume and p is the ratio of the number of positive droplets / well to the total number of droplets / well.

[0120] Example 3 - Exosome Isolation

[0121] By constructing immune complexes on beads and encapsulating them into droplets, signals from labeled fluorescein or chemiluminescence can be used as a trigger for droplet sorting. Droplets containing target exosomes can be isolated by droplet sorting techniques, including electrical sorting, mechanical sorting, or acoustic sorting. Figure 3 Schematic diagram of the isolation of fluorescent exosomes with desired information.

[0122] Example 4 - Exosome Characterization

[0123] Droplet microfluidics can be used for high-throughput assays because exosomes shed from tumor tissue carry many biomarkers (e.g., proteins, DNA, or microRNA, etc.) to characterize and analyze the content of each exosome individually. Figure 3 Schematic diagram of characterizing exosomes by droplet fusion, sorting or other droplet manipulation techniques. By diluting and encapsulating the exosomes into a sufficient number of droplets, exosome assays can be performed at the level of a single exosome. By adding reagents to the droplets with exosomes, the information contained in a single exosome can be studied. The reagents added to the droplets can be exosome lysis buffer, PCR mixture, RT mixture, etc.

[0124] Example 5 - Single exosome counting immunoassay for cancer diagnosis

[0125] Exosomes shed by tumor cells are considered promising biomarkers for cancer diagnosis due to their unique composition and function. Quantification of low-concentration specific exosomes present in very small amounts of clinical samples can be used for non-invasive cancer diagnosis and prognosis. The present invention provides an immunosorbent assay for digital quantification of target exosomes using droplet microfluidics. Exosomes are immobilized on magnetic microspheres via sandwich ELISA complexes, which are labeled with enzyme reporters that produce fluorescent signals. The constructed beads are further separated and encapsulated into a sufficient number of droplets to ensure that only a single bead is encapsulated in the droplet. The droplet-based single exosome counting enzyme-linked immunosorbent assay (droplet digital ExoELISA) method enables absolute counting of cancer-specific exosomes, achieving unprecedented accuracy. The limit of detection (LOD) reached 10 enzyme-labeled exosome complexes per microliter (~10 -17 M). The application of the droplet digital ExoELISA platform in the quantitative detection of exosomes in plasma samples directly from breast cancer patients was demonstrated. The use of the methods disclosed herein can achieve early diagnosis of cancer and accelerated discovery of cancer exosome biomarkers for clinical diagnosis.

[0126] There is evidence that exosomal molecular material shed from tumor tissue can be identified as a potential non-invasive cancer diagnostic biomarker because it reflects genetic or signaling alterations in the parent tumor. For example, through immunoblotting analysis, it was found that the expression of exosomal membrane protein glypican-1 (GPC-1) on cancerous exosomes is much higher than that on non-cancer exosomes, revealing its clinical value as an exosomal biomarker in the early diagnosis of pancreatic, breast and colorectal cancer.

[0127] Exosomes secreted by nucleated cells are widely present in human biological fluids, and various exosome subpopulations exist. Recently, tumor-derived exosome subpopulations have been found to be valuable for clinical diagnosis. Accurately quantifying and classifying tumor-derived exosomes from biological fluids has potential significance for cancer diagnosis, prognosis, and monitoring response to treatment. Conventional methods such as nanoparticle tracking analysis (NTA), western blotting, ELISA, and flow cytometry have been widely adopted in research laboratories for quantitative measurement of exosomes. However, NTA is only effective at high concentration levels (1×10 7 -10 9Particles / mL) provide an estimated number of exosomes and lack specificity. Western blot, ELISA, and flow cytometry all require large sample inputs and have limited sensitivity. Unfortunately, in the early stages of cancer, it is difficult to detect the limited tumor-derived exosomes in peripheral blood circulation using these traditional quantitative methods. Researchers have made many efforts to improve the sensitivity of detection methods, including miniature microfluidic platforms, aptamer-based electrochemical sensors, surface plasmon resonance (SPR), and Raman scattering. However, these detection methods are performed in bulk solutions, which can hardly achieve absolute quantification or classification. Since the concentration of cancer biomarkers present in the early stage of liquid biopsy ranges from 10 -12 Up to 10 -16 To quantify such low-abundance markers, the sensitivity required for detection needs to reach the single-molecule level. Recently, single extracellular vesicle analysis (SEA) based on photon counting technology has been applied to multiplex analysis of single extracellular vesicles using ELISA. Due to the low signal-to-noise ratio of single vesicles, careful buffer washing and complex imaging procedures are required to distinguish them from protein complexes or other clusters, and the detection limit is still very high (e.g., with 10 2 However, these methods remain impractical for widespread adoption due to throughput and cost. Reliable platforms for exosome quantification with high sensitivity and specificity are still lacking.

[0128] In recent years, digital PCR and digital ELISA platforms have revolutionized the detection technology of absolute quantification of nucleic acids and proteins. Compared with traditional biological and chemical analyses performed in large quantities in pipettes, beakers, tubes or flasks, the basic principle of molecular digital quantification is to divide the sample evenly into a large number of small compartments (in microwells or in droplets). By doing so, single molecules are confined to a small volume, where the signal can be amplified and concentrated for detection. Partitioning technology ensures that the molecules in each compartment are separated to follow a Poisson distribution, which is the core of the success of digital quantification. Droplet microfluidics produces uniform droplets at a high throughput (in kHz) at a scale of picoliters to nanoliters, enabling many single-molecule analyses to be performed in parallel. In recent years, great progress has been made in the development of droplet-based platforms for forming and manipulating monodisperse droplets and a series of related applications based on fluorescence technology for high-throughput and high-sensitivity droplet content analysis.

[0129] A droplet-based single exosome counting immunoassay was developed for digital quantification of exosomes. Exosomes with target membrane protein biomarkers were identified using an exosome enzyme-linked immunosorbent assay (ExoELISA). This method is also referred to as droplet digital ExoELISA in this article, and its process is described in Figure 5As shown in a-5d. Magnetic beads are used as a medium to capture and separate target exosomes. First, the exosome suspension is mixed with a sufficient number of magnetic beads bound to capture antibodies that can selectively bind to specific proteins on the exosome membrane. After effective magnetic separation and washing, a target exosome is fixed and captured on the magnetic beads. The detection antibody labeled with an enzyme reporter further recognizes the antigen on the captured exosome, forming a single enzyme-linked immunosorbent complex (ELIC) on the beads. Figure 5 a). Second, the prepared beads and enzymatic matrix were co-encapsulated into a sufficient number of droplets using a microfluidic chip to ensure that most droplets contained no more than one bead ( Figure 5 b-5c). Third, for those droplets containing beads with exosome immune complexes, the matrix is ​​enzymatically catalyzed to emit fluorescein within the droplet ( Figure 5 d). Based on the statistics of fluorescent droplets, the concentration of targeted exosomes can be calculated. The droplet digital ExoELISA method is able to detect as few as ~5 exosomes per μL. In addition to high sensitivity, the droplet digital ExoELISA provides high specificity and absolute quantification for targeting exosomes with specific protein biomarkers. For clinical validation, GPC-1(+) exosomes from breast cancer patients and results with different GPC-1(+) expression levels before and after surgery were generated, indicating the great potential of the droplet digital ExoELISA platform for cancer diagnosis.

[0130] Following our previous work, exosomes were purified and isolated from a breast tumor cell line (MDA-MB-231) by multi-step ultracentrifugation. Standard characterization of exosomes was performed using transmission electron microscopy (TEM), NTA, and Western blotting, respectively. Figure 6 As shown in a, TEM images show that after ultracentrifugation, the lipid bilayer structure remains intact on the purified exosomes, and the size of the exosomes ranges from 50 nm to 150 nm in diameter. The size distribution and concentration of the exosomes were determined by NTA analysis ( Figure 6 b). The average diameter of the prepared exosomes was 104.2±3.9 nm, and the corresponding concentration was 6.39×10 8 ±4.90×10 6 Particles / mL. CD63 protein is a member of the transmembrane 4 superfamily and was selected as a protein biomarker for exosome capture because it is a membrane-rich protein in exosomes and, according to the literature, is commonly used for exosome capture. Western blot analysis showed that the exosome marker CD63 on exosomes isolated from the culture medium of MDA-MB-231 was consistent with the CD63 protein extracted from the same cell line as the positive control, indicating the presence of CD63 in these samples ( Figure 6 c, top row). In addition, two-color super-resolution microscopy was used to confirm the localization of CD63 on the exosome membrane ( Fig.10 ac). GPC-1 protein was selected as a breast cancer reporter gene. Western blot analysis ( Figure 6 c, bottom row) and two-color super-resolution microscopy ( Fig.10 df) confirmed the high expression of GPC-1 on exosomes of MDA-MB-231 cell line and the location of GPC-1 on the exosome membrane. Therefore, the isolated breast cancer exosomes can be further used for the construction of exosome immune complexes on magnetic beads using ExoELISA.

[0131] A protocol for constructing single exosome immune complexes on beads was established. First, magnetic beads conjugated to CD63 antibodies were prepared. The functionalized beads were then used to capture exosomes. The probability of the number of exosomes bound to one bead follows the Poisson statistic. Therefore, when the average number of exosomes captured per bead was less than 0.1, most beads (>99.53%) captured at most one target exosome. Therefore, 10 times more beads than the expected exosomes were added to ensure single exosome capture. To demonstrate the successful capture of exosomes by CD63 antibody-antigen conjugated beads, TEM experiments were performed. Magnetic beads coated with CD63 capture antibodies were exposed to two samples: one with MDA-MB-231 exosomes and the other without exosomes as a control group. Fig.11 a shows naked beads without exosomes on the surface, while Fig.11 b clearly shows that one exosome was constructed on the magnetic beads. These results demonstrate that the functionalized magnetic beads are able to specifically bind exosomes in a single complex by ExoELISA. After capturing a single exosome on the beads, anti-GPC-1 previously biotinylated with a biotin tag was used as a detection antibody to bind the GPC-1 protein marker on the membrane of the target exosome. After the formation of the immune complex on the beads, the detection antibody was further conjugated to the enzyme reporter β-galactosidase, which catalyzed the fluorescein-di-β-D-galactopyranoside (FDG) substrate to produce a fluorescent signal for detection in a droplet microfluidic system.

[0132] A flow focusing droplet generation device with two sample inlets, one for the prepared bead sample and the other for the FDG matrix solution, was used to generate droplets with a diameter of 40 μm in mineral oil ( Figure 7 a). Similarly, the encapsulation of beads in droplets is also based on a Poisson distribution. The average number of beads per droplet was set to <0.3 to ensure that most droplets contained no or one bead (see Fig.12(Bright image of an array of bead-encapsulated droplets captured in ). Importantly, positive droplets containing at least one target exosome can be counted from the ratio of target molecules to magnetic beads and the ratio of magnetic beads to droplets after 1 analysis of two related Poisson distributions. These two ratios were set low enough to allow the linear dynamic range of Poisson statistics to count target exosomes. Therefore, almost all positive droplets contain only one target exosome. Moreover, direct “digital” counting of target exosomes is feasible, by simply counting fluorescent droplets, without the need for highly sensitive detection methods or complex image processing to measure the true number of magnetic beads.

[0133] The generated droplets were spread in a monolayer configuration in the droplet reservoir and incubated before observation. The fluorescence signal rise time was several minutes, indicating that the effect of premixing in the microchannel before droplet generation was negligible. The FDG-catalyzed reaction was studied to optimize the assay incubation time ( Fig.13 ). 30 minutes was chosen as the optimal incubation time for 40 μm diameter droplets, but the incubation time can be shortened if smaller droplets are used. Once the incubation is complete, an endpoint count of fluorescent droplets (positive copies) is performed. The number of fluorescent droplets represents the number of target exosomes.

[0134] The droplet digital ExoELISA was calibrated using the MDA-MB-231 exosomes described above. 8 The samples were serially diluted 10-fold with 10 exosomes / mL. Figure 7 b. The detected GPC-1 exosomes have a good linear relationship with the total particles measured in NanoSight. Error bars represent the standard deviation of three repeated experiments. Due to the size of picoliter droplets, the LOD of our droplet digital ExoELISA, determined by the background (negative control) signal plus 3 times the standard deviation (SD) of the background signal, is approximately 10 exosomes / μL. Compared with the reported exosome detection methods (Table 1), the method disclosed in this article achieves the lowest LOD. Because in the sample discretization, a sufficient number of beads are mixed with the exosomes and the beads are divided into a sufficient number of droplets to achieve one fluorescent droplet representing one target exosome with a confidence level of more than 99%. Figure 7 c shows the background of the assay, which may be caused by nonspecific binding to the bead surface or free reporter enzyme carried over into the encapsulated droplets. Figure 7(dh) is an image of fluorescent droplets in a chamber through a 10-fold serial dilution. It is noteworthy that among the fluorescent droplets, some droplets emit stronger fluorescence signals than others. This difference may be caused by different expression of GPC-1 on individual exosomes or heterogeneity of single enzyme catalysis. One million droplets were generated and the dynamic range was allowed to reach a 5log range in the linear regime. The dynamic range can be further extended by adopting two correlated Poisson statistics.

[0135] Table 1. Comparison of limits of detection (LOD) and loaded serum sample volumes of current test methods for detecting exosomes.

[0136]

[0137] The diversity of exosome subpopulation protein biomarkers significantly complicates exosome counting. The differentiation of exosome subpopulations is based on immunoassays, which have excellent specificity. To examine the specificity of GPC-1(+) exosome detection in breast cancer exosomes (MDA-MB-231exo), three non-cancerous exosomes, including human normal liver exosomes (HL-7702exo), mouse normal macrophage exosomes (RAW264.7 exo), and human embryonic stem exosomes (hES exo), were used for control experiments. Western blot analysis was used to identify the expression levels of GPC-1 in MDA-MB-231exo, HL-7702exo, RAW264.7 exo, and hES exo, and it was found that the expression of GPC-1 in MDA-MB-231exo was slightly higher than that in the other three groups ( Figure 8 a) Due to the limited detection capacity of western blot, if the sample contains a small amount of GPC-1(+) exosomes, other proteins on the exosomes in the sample may interfere with GPC-1(+) in western blot analysis. In addition, western blot analysis can only qualitatively indicate whether GPC-1 is expressed in the sample because it cannot measure the specific number of GPC-1(+) exosomes. Next, the specificity of droplet digital ExoELISA for the detection of GPC-1(+) exosomes was measured in four selected exosomes and two negative controls: samples using magnetic beads without CD63 Ab and samples without exosomes ( Figure 8 b) NTA analysis was used to estimate the number concentration of exosomes. The measured values ​​for HL-7702 exo, RAW264.7 exo, and hES exo were 4.22×10 8 , 2.86×10 8 and 2.85×10 8 Particles / mL( Fig.14ac). After appropriate dilution, each sample contained 6.39×104 15 exosomes per μL. Among these samples, only MDA-MB-231exo showed a significantly high number of GPC-1(+) exosomes (40141 exosomes per μL). For the negative control examples, very few fluorescent droplets were observed in each experiment (~5 detectable copies per μL), confirming that the background of the assay was mainly due to low enzyme nonspecific binding to the magnetic beads.

[0138] To demonstrate the clinically relevant application of our method, a droplet digital ExoELISA for the detection of GPC-1(+) exosomes was performed using clinical samples of sera from 5 healthy individuals (HS), 5 patients with benign breast disease (BBD), 12 breast cancer patients (BC), and 2 breast cancer patients after surgery (BC-AS). Fig. 9 ). Serum samples obtained from HS were used as controls for this study. Even in healthy human serum samples, about 0.3%-4.7% (average 2.3%) GPC-1(+) exosomes are present, and about 10 per ml of blood 9 A vesicle. Fig. 9 a shows that the HS group had an average of 5448 GPC-1(+) exosomes per microliter, similar GPC-1(+) exosomes in the BBD group (~6914 exosomes / μL), while the average GPC-1(+) exosomes in the BC group increased by 5 to 7 times. Therefore, the expression of GPC-1 was significantly increased on tumor-derived exosomes compared with normal and benign breast disease samples. This increase may be the result of tumor cells shedding higher levels of GPC-1(+) exosomes than normal cells. Fig. 9 b shows that BC patients overexpress GPC-1(+) exosomes and can be well distinguished from HS and BBD groups (p<0.0001). It is worth noting that for BC1-AS and BC2-AS, the measured values ​​of GPC-1(+) exosomes in two samples of patients BC1 and BC2 after surgery were significantly lower in BC1-AS and BC2-AS than in BC1 and BC2, respectively ( Figure 5 c), but relatively higher than HS and BBD ( Figure 5a). Therefore, these data not only confirm that GPC-1 can be considered as an exosomal biomarker to distinguish non-BC patients from breast cancer patients, but also indicate that the method disclosed in this article is suitable for detecting GPC-1(+) exosomes for preoperative and postoperative monitoring. Droplet digital ExoELISA has been demonstrated to be a reliable method for quantifying target exosomes of HS, BBD, and BC-AC from BC clinical samples. In the early stages of the disease (especially cancer), some of the exosome subpopulations secreted only by tumor cells are very small, and compared with other reported methods, droplet digital ExoELISA is very valuable for detecting exosomes of extremely low abundance (Table 1). Therefore, droplet digital ExoELISA can be used for early cancer diagnosis and postoperative monitoring in clinical studies.

[0139] This paper describes a method for single molecule / copy detection using droplet microfluidics. The standard ExoELISA technology was extended to detect ultra-low-speed ambulance exosomes with specific target proteins. The digital ExoELISA method enables unprecedented accuracy and high specificity in exosome quantification, and the target protein expression level on individual exosomes can be distinguished by the level of fluorescence signal in the droplet. The droplet digital ExoELISA can detect target exosomes over a dynamic range of 5log, and the detection limit can be as low as 10 exosomes per μL. High specificity was also demonstrated by quantifying exosomes with the target GPC-1 biomarker from multiple exosomal subpopulation protein biomarkers. The method disclosed in this paper can be used to absolutely quantify exosomes in serum samples from breast cancer patients. Therefore, the droplet digital ExoELISA method can promote the discovery of cancer exosome biomarkers.

[0140] method

[0141] Microfluidic device fabrication and ExoELISA assay in microdroplets

[0142] The droplet digital ExoELISA device was made of polydimethylsiloxane (PDMS) using standard soft lithography procedures. Sylgard-184 PDMS (Dow Corning) was cast on top of the master mold with a base and crosslinker mixing ratio of 10:1, degassed in vacuum and cured in an oven at 70 °C for 2 hours. Then, the cured PDMS was removed from the mold and cut into individual chips. The holes for the liquid inlet and outlet were punched with a Pan's needle. The PDMS replica and the glass slide (SAIL BRAND) were placed on a 40-mm-diameter plate with an O-ring. 2The device was plasma treated and bonded together. The device was baked on a hot plate at 100 °C for 8 h to restore the surface hydrophobicity. The magnetic beads and fluorescein-di-β-D-galactopyranoside (FDG) matrix solution were encapsulated into droplets with a diameter of 40 μm using mineral oil, which contained 3 wt.% of ABIL EM 90 and 0.1 wt.% of Triton X-100 stabilizing surfactant ( Figure 7 a). For device operation, the flow rates of the bead suspension and FDG phases were kept the same at 0.7 μL / min, while the flow rate of the oil phase was controlled at 2.3 μL / min using a syringe pump (PHDULTRA, Harvard Apparatus). After the droplet generation was completed, the droplets were incubated in situ for 30 minutes.

[0143] Fluorescence image acquisition and data analysis

[0144] After the incubation was completed, the device was placed on an inverted fluorescence microscope (EclipseTi-U, Nikon) with a fiber illuminator (Nikon Intensilight C-HGFI) with an intensity of 50mW through a filter cube of FITC 18 dye (Ex: 490nm, Em: 525nm). In order to reduce the complexity and duration of the droplet imaging process, the entire droplet storage chamber was scanned on an automatic XY motorized stage, and images were taken using a CCD camera (EXi Blue, QImaging) combined with a 2X objective to obtain a wider image window for counting more droplets in one frame. After taking out all the droplet images in the storage chamber, a custom program was used to merge and analyze the fluorescence and total droplets. By setting the intensity threshold, two different droplet populations with different intensities were obtained, and the number of positive droplets was counted. In each experiment, one million droplets were counted for data analysis.

[0145] Cell culture and exosome isolation

[0146] All cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China. MDA-MB-231 and HL-7702 were cultured in 5RPMI-1640 medium containing 10% (v / v) fetal bovine serum (FBS, System Biosciences) and 61% (v / v) penicillin-streptomycin. RAW264.7 was cultured in DMEM cell culture medium supplemented with 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin. All cell lines were cultured under 5% CO 2Incubate at 37°C in a humidified atmosphere. To isolate exosomes from the three cell lines, cells were cultured to 60–70% confluence in medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin, washed twice with phosphate-buffered saline (PBS), and then maintained in serum-free basal medium for 12 h, then washed once with PBS, and then in medium containing 2% (v / v) Exo-FBS. TM hES (human embryonic stem) cell lines were cultured in PSCeasy medium (Cellapybio) at 37°C in the presence of 5% CO 2 The cells were cultured in an incubator until 90-100% confluence. The supernatants were collected from the four cell lines and centrifuged at 2000 g for 20 min to eliminate cells and debris, and at 10000 g for 30 min to eliminate microvesicles. The exosomes were then ultracentrifuged twice at 135000 g for 70 min using a W32Ti rotor (L-80XP, Beckman Coulter), resuspended in PBS, and stored at -80 °C until further use.

[0147] Nanoparticle Tracking Analysis (NTA)

[0148] The concentration and size of exosomes were measured using NanoSight NS300 and NTA 3.2 software (Malvern). The samples were diluted to an appropriate concentration of ∼1 × 10 7 -10 9 Particles / mL were injected into a detection chamber equipped with a 405 nm laser. Three sets of measurements were performed, each lasting 60 seconds.

[0149] Two-color super-resolution imaging

[0150] 50 μL of exosome sample solution was fixed on coverslips (SALD BRAND) coated with poly-L-lysine (Sigma-Aldrich), incubated at room temperature for 30 minutes, and then washed three times with PBS. Exosome membranes were stained using the PKH67 Green Fluorescent Cell Connector Micro Kit (Sigma Aldrich). 50 μL of the diluted PKH67 solution was quickly applied to the sample and mixed by pipetting. The mixture was incubated at room temperature for 4 minutes with regular mixing, and then 100 μL of 1% BSA was added for 2 minutes to prevent the binding of excess dye. After rinsing three times with PBS, the coverslips were immediately placed in the primary antibody solution (1:400 anti-CD63 or 1:400 anti-GPC-1) at room temperature for 1 hour, and then washed three times with PBS. In the last step, AlexaFluor 647-conjugated secondary antibody (1:2000 Bioss, bs-0295G-AF647) was applied, followed by incubation at room temperature for 30 minutes. The final samples were washed three times with PBS and stored in PBS for further super-resolution imaging of exosomes.

[0151] Images were captured using a Nikon N-STROM (Stochastic Optical Reconstruction Microscope) super-resolution microscope system by total internal reflection fluorescence 14 (TIRF) illumination with 488 and 647 nm. During imaging, exosomes were immersed in imaging buffer consisting of 0.56 mg / mL glucose oxidase (Sigma-Aldrich), 0.3 mg / mL catalase (Sigma-Aldrich), and 10 mM cysteamine (Sigma-Aldrich) in PBS. PKH67 and AlexaFluor 647 bound to the secondary antibody were excited for imaging of exosome membranes and proteins (CD63 or GPC-1), respectively. A series of 20,000 images were acquired with an iXon3 DU-897E electron multiplying charge-coupled device (EMCCD) camera (Andor Technology) through a Plan Apochromat TIRF 100× oil immersion lens with a numerical aperture of 1.49.

[0152] Transmission Electron Microscopy (TEM)

[0153] The isolated exosomes were stained with 2% phosphotungstic acid (PTA) at a concentration ratio of 4:1 for 10 minutes. The mixture was then loaded onto a copper grid and dried at room temperature. The grid was observed using a transmission electron microscope (HITACHI H-7650). For TEM analysis of immunomagnetically captured exosomes, single exosome-bead complexes were prepared using CD63-coated magnetic beads according to a Poisson distribution. The mixture was then stained with 2% PTA for 10 minutes and placed on a copper grid. After further drying, the grid was imaged by TEM. CD63-coated magnetic beads that were not mixed with exosomes were used as a negative control.

[0154] Western blot analysis

[0155] Total protein from MDA-MB-231 cells was extracted by RIPA lysis buffer (Beyotime Institute of Biotechnology). Cellular proteins or exosome supernatants were denatured in 5× sodium dodecyl sulfate (SDS) buffer. 20 μg of protein per lane was separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica), blocked in 5% skim milk for 2 h at room temperature, and then washed three times with TBS-Tween 20 (TBST) buffer (137 mM NaCl, 25 mM Tris-HCl, pH 7.6, 0.1% Tween 20). The membranes were probed with 1:1000 anti-CD63 (ab134045, Abcam) or 1:1000 anti-GPC-1 (ab199343, Abcam) overnight at 4°C. After washing with TBST buffer, the blots were incubated with fluorescent secondary antibodies (Cell Signaling Technology) for 1 h at room temperature, followed by chemiluminescence measurement using the Bio-Rad ChemiDoc XRS Imaging System (Bio-Rad Laboratories).

[0156] Preparation of magnetic beads conjugated to CD63 antibody

[0157] According to the manufacturer's instructions, MyOne TMAntibody-bound magnetic beads were prepared with N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, Thermo Scientific). Briefly, the carboxylic acid groups on the magnetic beads were activated with N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, Thermo Scientific), and then a volume of 50 μL of magnetic beads was mixed with 10 μL of CD63 antibody. The beads were blocked with 0.1% bovine serum albumin (BSA, Sigma-Aldrich), washed several times with PBS, and then resuspended in 100 μL of PBS before use. Depending on the initial concentration, the final concentration of CD63-coated magnetic beads was estimated to be 3.5-6.0 × 10 6 beads / μL.

[0158] Modification of GPC-1 antibody with biotin tag

[0159] Biotinylation of anti-GPC-1 was performed using the EZ-Link Micro Sulfo-NHS-LC-Biotinylation Kit (Thermo Scientific). 10 μL of anti-GPC-1 was mixed with 0.24 μL of 9 mM sulfo-NHS-LC-biotin at room temperature for 1 hour. Excess biotin was then removed using a Zeba desalting column (Thermo Scientific), resulting in 400 μL of 1:40 biotinylated anti-GPC-1 for the next study.

[0160] Exosome capture, magnetic separation, and enzyme binding

[0161] CD63-functionalized magnetic beads were mixed with MDA-MB-231 exosomes (various concentrations of 6.39, 63.9, 639, 6390, 63900 particles / μL). Incubate in a sample mixer (Invitrogen, LifeTechnology) for 1 hour, mixing regularly at room temperature to allow the antibody to capture the exosome target. The beads were separated by a magnet for 2 minutes and washed three times with PBS. Next, 40 μL of 1:400 biotinylated anti-GPC-16 was added, and the resulting mixture was incubated in a mixer for 1 hour at room temperature, then separated by a magnet for 2 minutes and washed three times with PBS. In the last step, 40 μL of 2 ng / μL β-galactosidase (Invitrogen, Life Technology) was mixed with the immunomagnetically captured exosomes and incubated at room temperature for 30 minutes, then washed three times with PBS and resuspended in 15 μL PBS for further application on the chip.

[0162] Clinical sample preparation

[0163] A total of 24 clinical serum samples (5 HS, 5 22 BBD patients, 12 BC patients, and 2 BC-AS patients) were obtained from the Department of Experimental Medicine, Nanfang Hospital, Southern Medical University, Guangzhou. The diagnosis of BBD and BC was confirmed by histological examination of tissue biopsies. Serum samples were centrifuged twice at 2000g for 5 minutes to eliminate cells and debris, and then centrifuged at 16100g for 20 minutes to remove microvesicles. The supernatant was carefully collected and stored at -80°C before use. The clinical serum samples involved were approved by the Ethics Committee of Nanfang Hospital, Southern Medical University, and written consent was obtained from all patients and healthy individuals.

[0164] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes inspired by those skilled in the art are included in the spirit and scope of the present application and in the scope of the appended claims. In addition, any element or limitation of any invention disclosed herein or its embodiment may be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or embodiment thereof, all of which are within the scope of the present invention, but are not limited thereto.

[0165] References

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[0169] 4.Zhao,Z.;Yang,Y.;Zeng,Y.;He,M.,A microfluidic ExoSearch chip formultiplexed exosome detection towards blood-based ovarian cancerdiagnosis.Lab Chip 2016,16,489-496.

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[0174] 9.Rissin,D.M.;Kan,C.W.;Campbell,T.G.;Howes,S.C.;Fournier,D.R.;Song,L.;Piech,T.;Patel,P.P.;Chang,L.;Rivnak,A.J.;Ferrell,E.P.;Randall,J.D.;Provuncher,G.K.;Walt,D.R.;Duffy,D.C.,Single-molecule enzyme-linkedimmunosorbent assay detects serum proteins at subfemtomolar concentrations.Nat.Biotechnol.2010,28,595-599.

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Claims

1. A method for digitally quantifying target exosomes, the method comprising the following steps: (1) constructing exosome immune complexes to produce constructed immune complex beads, comprising: Obtain exosome solutions from biological fluids and prepare exosome solutions by ultracentrifugation, ultrafiltration, density gradient separation, and immunoaffinity capture methods, binding an antibody capable of recognizing a biomarker on the surface of the exosome to the beads; incubating the beads with the exosome solution; After incubation, the beads are collected magnetically or by centrifugation; After thorough washing, the target exosomes bound to the beads are purified from the sample solution; and Detecting the exosomes using a second antibody, i.e., a detection antibody, that can recognize the same or a different biomarker on the exosomes, wherein the detection antibody is conjugated to a tag that can recognize the enzyme; (2) flowing the solution of immune complex beads constructed in step (1) into a channel to mix the solution with a matrix flow of another channel and form droplets of the mixture; or The sample with the constructed immune complex beads is dropped on the flat chip and scraped into the wells, followed by adding the matrix solution into each compartment, and then the microwell chip is sealed at the top to isolate each individual space for reaction; (3) after incubation, detecting the color, fluorescence or electrochemical signal emitted by the droplet / well having the constructed immune complex beads; (4) Detecting signals by fluorescence microscopy or electrochemical sensor array; (5) Calculate the number of target exosomes by counting the number of positive and negative droplets / wells according to the following formula: Where N is the absolute number of captured molecules, N b is the total number of beads, V s is the total test sample volume, V d is the droplet / well volume and p is the ratio of the number of positive droplets / well to the total number of droplets / well.

2. The method according to claim 1, wherein the substrate is fluorescein-di-β-D-galactopyranoside (FDG).

3. The method according to claim 1 or 2, wherein the biomarker on the surface of the exosomes is CD63.

4. The method according to claim 1 or 2, wherein the beads are Dynabeads™ or agarose beads.

5. The method of claim 1 or 2, wherein the different biomarker is GPC-1.

6. The method according to claim 1 or 2, wherein the enzyme is streptavidin-bound β-galactosidase. The method according to claim 1 , wherein the tag is biotin.

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