EP4698321A2 - An immuno-janus particle (ijp) assay for exosome and virus detection - Google Patents

Abstract

Described herein are systems, methods, and devices for isolating exosomes and detecting colocalized exosomal proteins. In some embodiments, the disclosed systems, methods, and devices may comprise asymmetric nanopore membranes, magnetic or non-magnetic Janus particles, and capture and detector antibodies.

Description

AN IMMUNO-JANUS PARTICLE (UP) ASSAY FOR EXOSOME AND VIRUS DETECTION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 63/460,136, filed on April 18, 2023, which is incorporated by reference herein in its entirety.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant number CA241684 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Small extracellular vesicles (sEVs; also known as exosomes) with sizes 30 to 200 nm are the key mediators of intercellular communications originating from endosome-multivesicular body complexes. They are released to the extracellular environment including blood, urine, saliva, and breast milk. Exosomes secreted by parental or cancer cells are rich in proteins with a highly diverse molecular composition and their surface proteins bear characteristics of their tissue of origin. Circulating plasma exosomes, because of their abundance and broad origin, are thus of great interest as cancer biomarkers to assess cancer progression and monitor the response of therapy that otherwise requires invasive tissue biopsy that is often not feasible. Several studies suggest that multiple surface proteins of cancer-derived exosomes play key roles in different oncogenic processes such as intercellular signaling, immune response, and metastasis to promote tumorigenesis and progression. Growing evidence suggests the tetraspanin markers (CD63, CD81 , CD9) and cancer markers (CA125, GPC1, EGFR, EpCAM, CD24) are elevated in circulating exosomes of cancer patients compared to healthy controls. For example, activation of overexpressed Epidermal Growth Factor Receptor (EGFR) is known to be associated with tumorigenesis; about 26% of colorectal cancer is EGFR positive. Zhang et al. have observed that ovarian cancer-derived exosomes are highly overexpressed with CD24 and EpCAM markers Zhang et al., Nature Biomedical Engineering 3(6: 438-451 (2019). A study by Melo et al. has shown that GPC1 in exosomes is highly enriched in the serum of patients with pancreatic cancer Melo et al., Nature 523(7559): 177-182 (2015). Unfortunately, few of these biomarkers, if any, are specific to one cancer. Instead, they are pan-cancer markers and upregulated in multiple cancers. For example, studies show that GPC1 is upregulated in pancreatic, prostate, breast, colon, and ovarian cancers. The most promising strategy to identify specific cancer from circulating exosomes is to quantify multiple colocalized protein markers on single exosomes, with the hypothesis that the profile would be different for different cancers. CEA and NCAM, for example, are specific to the gastrointestinal tract and brain tissues, respectively.

One of the major challenges to accurate quantification and profiling of exosomal protein markers is high-yield isolation of the exosomes from plasma due to their small size. Although several exosome isolation technologies are available, none of them allow high-throughput and high-yield isolation from plasma, making the exosome analysis labor-intensive and slow. Existing exosome isolation technologies, including differential ultracentrifugation (DUC), precipitation (such as ExoQuickTM), size-exclusion chromatography (such as qEV) and immunocapture, often work well for conditioned cell media but not plasma. DUC is rather non-discriminating as exosomes and microvesicles are known to have overlapping density ranges. Further, DUC requires a large sample volume and complex gradient separation steps, making the process slow (6 to 10 hours) with low yield (5-23%). Precipitation methods, such as ExoQuickTM and Total Exosome IsolationTM, use organic solvents. They are attractive because of their simplicity and inexpensive equipment, but they require overnight incubation, and their yield is low because significant amount of protein impurities co-precipitate with the exosomes. It is hence difficult to isolate the precipitated exosomes intact. Similarly, size-exclusion chromatography techniques have low yield and require large samples. Immunocapture is membrane protein specific, but they also have rather a low yield and may require more than 1 day to achieve optimal recovery rates. Moreover, while exosomes do exhibit signature proteins like CD63, CD9, and CD81, these tetraspanins are shared by microvesicles and this differentiation cannot be achieved with immunocapture. Clearly, current commercial exosome isolation technologies are too inefficient (< 40% yield), slow (~1 day) and non-specific to be part of a plasma exosome diagnostic platform.

After purification, the proteins of the vesicles are traditionally lysed and analyzed with western blot, enzyme- linked immunosorbent assays (ELISA), and mass spectrometry. These analytical tools have advanced the understanding of exosome biology, but they are not sensitive enough for clinical samples. More importantly, by lysing the exosomes, profiling of colocalized proteins on the exosomes becomes impossible. Characterization of exosomal proteins with intact exosomes has been the focus of recent technological research. There are several breakthroughs. For example, the nano-plasmonic exosome (nPLEX) assay has a wider dynamic range and better sensitivity as high as 100 to 1000-fold than conventional enzyme-linked immunosorbent assay (ELISA). Beckman Coulter and other companies are developing next-generation flow cytometry instrumentation that can analyze individual exosomes down to 70-80 nm through enhanced fluorescence techniques. In addition, a number of novel methods for the detection of exosomes have been reported based on fluorescence, surface plasmon resonance (SPR), nuclear magnetic resonance, electrochemical, and field-effect transistor methods. Except for advanced flow cytometry and some advanced fluorescence techniques based on plasmonics and interference, these methods do not allow profiling of multiple colocalized exosomal proteins. Importantly, optical technologies, including flow cytometry and plasmonics/interferometry, suffer from resolution limit for exosomes below 70 nm, which represent a significant fraction of the exosome population. Electrochemical/charge sensing technologies, on the other hand, suffer from sensitivity to sample pH and ionic strength that require tedious buffer exchange. Most batch (non-flow) platforms also have dynamic range issues and can only sample a small number of exosomes.

What is needed are devices and methods for high yield (>85%), throughput, and purity isolation of exosomes in plasma by size integrated with a highly sensitive and specific magnetic Janus particle (MJP) immunocapture technology for profiling colocalized proteins.

SUMMARY

One embodiment described herein is a system for isolating exosomes and detecting colocalized exosomal proteins from a sample, the system comprising: a device for isolating exosomes from a sample, the device comprising: a first chamber; a second chamber; a third chamber; a sample comprising exosomes positioned within the first chamber; a filter positioned between the first chamber and the second chamber, the filter comprising a first filter surface facing and at least partially defining the first chamber, a second filter surface facing and at least partially defining the second chamber, and a plurality of filter pores extending between the first and second filter surfaces; an asymmetric nanopore membrane positioned between the second chamber and the third chamber, the asymmetric nanopore membrane comprising a first membrane surface facing and at least partially defining the second chamber, a second membrane surface facing and at least partially defining the third chamber, and a plurality of asymmetrically-shaped nanopores extending between the first and second membrane surfaces, wherein each nanopore includes a first nanopore opening at the first membrane surface having a first diameter, and a second nanopore opening at the second membrane surface having a second diameter that is greater than the first diameter; and a source for inducing fluid crossflow through the asymmetric nanopore membrane from the second chamber to the third chamber by pressure driven flow, electroosmotic flow, centrifugal force, or a combination thereof; magnetic or non-magnetic Janus particles conjugated to one or more capture antibodies having specific binding affinity to one or more exosomal proteins; one or more fluorophore-conjugated detector antibodies having specific binding affinity to one or more exosomal proteins; optionally, a magnet; and an image recording device to detect and measure changes in the rotational frequency of the magnetic or nonmagnetic Janus particles. In one aspect, the sample comprises whole blood, plasma, serum, urine, saliva, breast milk, or cell media. In another aspect, the first chamber comprises one or more baffles on a wall opposite of the first filter surface. In another aspect, the exosomal proteins comprise exosomal surface proteins. In another aspect, the system isolates exosomes ranging in size from about 30 nm to >200 nm. In another aspect, each filter pore of the plurality of filter pores has a diameter of about 200 nm to about 5 pm. In another aspect, the first diameter of the first nanopore opening is between about 10 nm and about 200 nm. In another aspect, the second diameter of the second nanopore opening is less than about 2 pm. In another aspect, the asymmetric nanopore membrane is formed from one or more materials comprising one or more of a polyethylene terephthalate (PET), a polycarbonate (PC), a polypropylene (PP), a polyimides (PI), or a polyethersulphone (PES). In another aspect, the magnetic or non-magnetic Janus particles are about 500 nm to about 1 pm in diameter size. In another aspect, the one or more capture antibodies are conjugated to a gold-coated portion of the magnetic or non-magnetic Janus particles. In another aspect, the source for inducing fluid crossflow generates a pressure less than about 1 atm. In another aspect, the source for inducing fluid crossflow comprises a syringe pump, an electroosmotic pump, a micropump, a centrifuge, or a combination thereof. In another aspect, the image recording device comprises a smartphone. In another aspect, the system achieves an isolated exosome yield of at least > 85% from the sample.

Another embodiment described herein is a method for isolating exosomes and detecting colocalized exosomal proteins from a sample, the method comprising: introducing a sample comprising exosomes into a device for isolating exosomes, the device comprising: a first chamber, wherein the sample comprising exosomes is positioned within the first chamber; a second chamber; a third chamber; a filter positioned between the first chamber and the second chamber, the filter comprising a first filter surface facing and at least partially defining the first chamber, a second filter surface facing and at least partially defining the second chamber, and a plurality of filter pores extending between the first and second filter surfaces; an asymmetric nanopore membrane positioned between the second chamber and the third chamber, the asymmetric nanopore membrane comprising a first membrane surface facing and at least partially defining the second chamber, a second membrane surface facing and at least partially defining the third chamber, and a plurality of asymmetrically-shaped nanopores extending between the first and second membrane surfaces, wherein each nanopore includes a first nanopore opening at the first membrane surface having a first diameter, and a second nanopore opening at the second membrane surface having a second diameter that is greater than the first diameter; and a source for inducing fluid crossflow through the asymmetric nanopore membrane from the second chamber to the third chamber by pressure driven flow, electroosmotic flow, centrifugal force, or a combination thereof; inducing fluid crossflow through the asymmetric nanopore membrane from the second chamber to the third chamber, whereupon the exosomes are isolated in the third chamber; mixing the isolated exosomes with magnetic or non-magnetic Janus particles conjugated to one or more capture antibodies having specific binding affinity to one or more exosomal proteins to generate exosome-bound magnetic or non-magnetic Janus particles; optionally, isolating the exosome-bound magnetic or non-magnetic Janus particles using a magnetic pulldown, and removing unbound exosomes; mixing the exosome-bound magnetic or non-magnetic Janus particles with one or more fluorophore-conjugated detector antibodies having specific binding affinity to one or more exosomal proteins to generate magnetic or nonmagnetic Janus particles bound to exosomes and fluorophore-conjugated detector antibodies; optionally, isolating the magnetic or non-magnetic Janus particles bound to exosomes and fluorophore-conjugated detector antibodies using a magnetic pulldown, and removing unbound fluorophore-conjugated detector antibodies; detecting and measuring changes in the rotational frequency of the magnetic or non-magnetic Janus particles bound to exosomes and fluorophore- conjugated detector antibodies using an image recording device, thereby detecting colocalized exosomal proteins; and tracking individual magnetic or non-magnetic Janus particle with an image analysis software to isolate the Janus particle rotation from translation. In one aspect, the sample comprises whole blood, plasma, serum, urine, saliva, breast milk, or cell media. In another aspect, inducing fluid crossflow through the asymmetric nanopore membrane from the second chamber to the third chamber comprises a flow rate between about 0.01 mL/hour to about 1000 mL/hour. In another aspect, the method achieves an isolated exosome yield of at least > 85% from the sample. In another aspect, the method is performed in about < 60 minutes. In another aspect, the method has an about 100-exosome particle detection sensitivity and a 4-log dynamic range.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the Magnetic Janus Particle Assay Strategy. FIG. 1 shows a schematic of ANM-based isolation of exosomes from cell media or plasma samples and of the assay steps. FIG. 1 also shows pictures of the ANM isolation module and experimental setup.

FIGS. 2A-B show schematics of particle behavior around (FIG. 2A) symmetric and (FIG. 2B) asymmetric nanopore membrane. FIGS.3A-B show the ANM. FIG. 3A shows a schematic diagram of the ANM fabrication process. FIG 3B shows SEM images of a single conic nanopore and the membrane on both tip and base side of 10 nm unetched ion track nanopore membranes, 10 nm ANMs, and 20 nm ANMs.

FIGS. 4A-G show the ANM filtration system. FIG. 4A shows the extracted amount of EVs (as determined by Nanoparticle Tracking Analysis (NTA)) as a function of the degree of asymmetry of ANMs. The corresponding pressure for filtration is also indicated. FIG. 4B shows the protein concentration in the flow-through effluent as a function of the volume of washing buffer. FIG. 4C shows representative western blot images of CD63 and TSG101 proteins from the isolated small EV fraction. FIG. 4D shows the NTA-based EV characterization before isolation (left) and after isolation (middle: purified materials after isolation with 200 nm ANM as indicated as 1 in FIG. 1 ; and right: purified materials after isolation with 30 nm ANM as indicated as 2 in FIG. 1). The inset of the figure shows the TEM of the isolated small EVs. Scale bar 100 nm. FIG. 4E shows isolation purity of ANM-isolates compared to other techniques using human plasma. FIG. 4F shows the extraction yield comparison between ANM with the cylindrical membrane, ultracentrifugation (UC), precipitation technology (Exoquick), and size-exclusion (qEV) using human plasma. FIG. 4G shows the extraction yield comparison between ANM with the cylindrical membrane and ultracentrifuge (UC) technique using human plasma. The use of crossflow increases the isolation yield by 10-fold compared to the cylindrical.

FIGS. 5A-B show a reproducibility study for 6 mL of 30* diluted 200 pL plasma. FIG. 5A shows triplicate ANM isolations of sEV from the same diluted plasma sample with less than 2% variation in the yield and less than 10% in the change in the mode (peak) sEV size. The numbers correspond to the sEV number for the 200 pL plasma. FIG. 5B shows the sEV recovery rate for 3 plasma samples and 2 cell media is 85% with less than 10% variation.

FIGS. 6A-G show a schematic of a Janus particle consisting of Au coated with fluorescent polystyrene microbeads. FIG. 6A shows a SEM image of the Janus particles recorded with EDM and CBS detectors. The CD63 antibody is functionalized to the gold side. FIG. 6B shows the blinking of a Janus bead imaged by a fluorescent camera. FIG. 6C shows a schematic of the simple assay steps compatible for POC. FIG. 6D shows the conversion of the single-particle video image into a time series for the rotation angle. FIG. 6E shows the estimated rotation frequency distribution over all JPs at each time interval. FIG. 6F shows the time-averaged distribution of the rotation period for all Janus particles with or without EV, showing a significant increase in rotation periods. FIG. 6G shows a standard curve of the rotation period as a function of the EV concentration, with a 10 fM limit of detection (LoD) for a 2 micro-liter sample volume. FIG. 7A shows a schematic of exosome profiling of two surface proteins showing MJP with (w/) exosome and without (w/o) exosome. FIG. 7B shows the blinking frequency with the increased exosome number per MJP.

FIG. 8A shows a sketch of smartphone imaging of MJPs in a solution. FIG. 8B shows a snapshot of blinking micron sized IJPs in suspension in a well as imaged by a smartphone camera.

FIG. 9 shows a schematic of a baffle chip design.

FIGS. 10A-E show a schematic for tracking the blinking of the particles. FIG. 10A shows that even at the lower resolutions and in an automated fashion, the algorithm uses the concept of dilation and normalization which produces reasonable circles from even few pixels. Once dilated, a disk detection method using accumulation points and Circular Hough Transform can be used for the detection of the particles in the image. FIG. 10B shows the detected centers are then mapped onto the original image and are tracked frame-by-frame. The meaningful information, such as the intensity of the particle, can then be used to perform wavelet analysis and get its frequency of blinking. The larger aggregates have a much lower blinking frequency and thus can be removed by only considering the range of frequencies in which the Janus Particle can lie. FIG. 10C shows empirical mode decomposition of the particle. FIG. 10D shows autocorrelation of the sample. FIG. 10E shows the wavelet of the sample.

FIG. 11 shows the fractional change in the rotational time period of the Janus particle after incubating with a known quantity of exosomes (through NTA). A limit of detection of 10⁷ exosomes/mL was observed using only 1-10 pL of the sample. The sensor has about 3 log dynamic range and saturates at 10¹⁰ exosomes per mL.

FIGS. 12A-C show how the signal changes when exosomes of equivalent amount are in pure form (suspended in buffer) versus when they are in biofluid with sEV-free part. Even a change in sEV-free fraction of human plasma by a factor of 100 did not change the signal producedfrom the plasma or the pure exosomes. FIG. 12A is a schematic showing the sEV fraction and sEV-free fraction. FIG. 12B is a typical NTA spectra showing the size distribution of the sEV in plasma, in membrane isolated sEV, and in sEV-free flow through (FT) solution of the filtration membrane. FIG. 12C is a graph showing the predicted concentration of sEVs/mL of plasma.

FIG. 13 is a graph showing results from isotype control experiments that were performed to determine whether a signal is produced with a non-target antibody. No signal was observed from the controls showing the selectivity of the signal from the target and the negligible role of physisorption in changing the signal. FIG. 14A is a diagram showing colocalization of proteins on exosomes. FIG. 14B is a diagram showing the use of magnetic Janus particles for pulling down and analyzing the proteins associated with the exosomes. FIG. 14C is a schematic showing magnetic core Janus particles for studying colocalization of different proteins on exosomes. The fluorophores will blink at the same rate as Janus particles so it can be known which fluorophores lie on the Janus particles allowing differentiation from the unbound fluorophore-conjugated antibodies. This can be enhanced by removing the unbound using a magnetic pulldown of the Janus particles.

FIG. 15A is a schematic of a Janus particle. FIG. 15B are images of Janus particles showing surface topography and surface composition of the particles.

FIGS. 16A-C show a calibration curve, control experiments, source capture, and size based. FIG. 16A is a calibration curve showing that the detected change in IJP rotational frequency correlates to the sEV concentration as measured by the Nanoparticle Tracking Analyzer (NTA). FIG. 16B is a schematic and graph showing the isotype control data from the calibration curve of FIG. 12B from the diluted plasma sample with anti-CD63 on the IJP, with another antibody on the IJP and with lysed sEV with anti-CD63. CD63 is a known sEV marker. FIG. 16C is a graph showing additional experiments with sEVs spiked into the sEV-free FT solution.

FIGS. 17A-H show data from diseased patients. FIG. 17A is a flowchart showing the process for determining the type of disease a patient has. FIG. 17B is a graph showing the concentration of aEGFR-positive sEVs in a sample from healthy and diseased patients. FIG. 17C is a graph showing the concentration of CEA-positive sEVs in a sample from healthy and diseased patients. FIG. 17D is a graph showing the concentration of GPC1-positive sEVs in a sample from healthy and diseased patients. FIG. 17E is a graph showing the concentration of pTau181- positive sEVs in a sample from healthy and diseased patients. FIG. 17F is a graph showing the concentration of aEGFR-positive sEVs in a sample from healthy and various diseased patients. FIG. 17G is a graph showing the concentration of GPC1-positive sEVs in a sample from healthy and various diseased patients. FIG. 17H is a graph showing the concentration of CEA-positive sEVs in a sample from healthy and various diseased patients.

FIG. 18 is a heat map showing the expression of various markers in diseased patients in various types of extracellular vesicles.

FIG. 19A is maps showing the expression of markers in various types of extracellular vesicles. FIG. 19B is graphs showing the concentration of particles per micro-gram of protein for various markers and extracellular vesicles. DETAILED DESCRIPTION

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

As used herein, the terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting essentially of,” and “consisting of’ the embodiments or elements presented herein, whether explicitly set forth or not.

As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.

As used herein, the term “or” can be conjunctive or disjunctive.

As used herein, the term “and/or” refers to both the conjuctive and disjunctive.

As used herein, the term “substantially” means to a great or significant extent, but not completely.

As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or 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, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol means “about” or “approximately.”

All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1 , 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points.

As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.

The term “exosome” as used herein refers to cell-derived vesicles having a diameter of between about 20-250 nm, such as between 40 and 210 nm, for example, a diameter of about 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 mm, 110 nm, 120 nm, 130 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm. Exosomes may be isolated from any suitable biological sample from a mammal, including but not limited to, whole blood, serum, plasma, urine, saliva, breast milk, cerebrospinal fluid, amniotic fluid, ascitic fluid, bone marrow and cultured mammalian cells (e.g., immature dendritic cells (wild-type or immortalized), induced and non-induced pluripotent stem cells, fibroblasts, platelets, immune cells, reticulocytes, tumor cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige pre-adipocytes and the like). As one of skill in the art will appreciate, cultured cell samples will be in the cell- appropriate culture media (using exosome-free serum). Exosomes include specific surface markers not present in other vesicles, including surface markers such as tetraspanins, e.g., CD9, CD37, CD44, CD53, CD63, CD81 , CD82 and CD151 ; targeting or adhesion markers such as integrins, ICAM-1 , EpCAM and CD31; membrane fusion markers such as annexins, TSG101 , ALIX; and other exosome transmembrane proteins such as Rab5b, HLA-G, HSP70, LAMP2 (lysosome-associated membrane protein) and LIMP (lysosomal integral membrane protein). Exosomes may also be obtained from a non-mammal or from cultured non-mammalian cells. As the molecular machinery involved in exosome biogenesis is believed to be evolutionarily conserved, exosomes from non-mammalian sources include surface markers which are isoforms of mammalian surface markers, such as isoforms of CD9 and CD63, which distinguish them from other cellular vesicles. The term “non-mammal” is meant to encompass, for example, exosomes from microorganisms such as bacteria, flies, worms, plants, fruit/vegetables (e.g., corn, pomegranate), and yeast. As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), nonhuman primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human.

As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.

As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

One embodiment described herein is a system for isolating exosomes and detecting colocalized exosomal proteins from a sample, the system comprising: a device for isolating exosomes from a sample, the device comprising: a first chamber; a second chamber; a third chamber; a sample comprising exosomes positioned within the first chamber; a filter positioned between the first chamber and the second chamber, the filter comprising a first filter surface facing and at least partially defining the first chamber, a second filter surface facing and at least partially defining the second chamber, and a plurality of filter pores extending between the first and second filter surfaces; an asymmetric nanopore membrane positioned between the second chamber and the third chamber, the asymmetric nanopore membrane comprising a first membrane surface facing and at least partially defining the second chamber, a second membrane surface facing and at least partially defining the third chamber, and a plurality of asymmetrically-shaped nanopores extending between the first and second membrane surfaces, wherein each nanopore includes a first nanopore opening at the first membrane surface having a first diameter, and a second nanopore opening at the second membrane surface having a second diameter that is greater than the first diameter; and a source for inducing fluid crossflow through the asymmetric nanopore membrane from the second chamber to the third chamber by pressure driven flow, electroosmotic flow, centrifugal force, or a combination thereof; magnetic or non-magnetic Janus particles conjugated to one or more capture antibodies having specific binding affinity to one or more exosomal proteins, wherein the Janus particles are coated with a scattering material; one or more fluorophore-conjugated detector antibodies having specific binding affinity to one or more exosomal proteins; optionally, a magnet; and an image recording device to detect and measure changes in the rotational frequency of the magnetic or non-magnetic Janus particles.

In one aspect, the sample comprises whole blood, plasma, serum, urine, saliva, breast milk, or cell media. In another aspect, the first chamber comprises one or more baffles on a wall opposite of the first filter surface. In some embodiments, there may be at least 1, at least 2, at least 3, at least 4, or at least 5 baffles. In other embodiments, there may be at most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 400, at most 300, at most 200, at most 100, at most 50, or at most 25 baffles. The baffles may be made of fiberglass, plastic, a composite, or another material. In some embodiments, the baffles may be made of polycarbonate (PC), polystyrene (PS), polyethylene terephthalate (PET), polyvinylchloride (PVC), SU-8 photoresist and polyimide (PI), polydimethylsiloxane (PDMS), silicon, glass, or polymethyl methacrylate (PMMA). The baffles can be shaped like cubes, triangular prisms, rectangles, cones, or panels that are curved, zigzagged, corrugated, or L-shaped, have a combination of these shapes, or are otherwise configured. The baffle geometry can be a triangle, wedge, crescent, etc. They can assume regimented or staggered patterns, including herringbone patterns. In an embodiment, the baffles may be cubes or triangular prisms. The baffles can have a height ranging from about 15 pm to about 3 mm, about 20 pm to about 2 mm, about 25 pm to about 2 mm, about 30 pm to about 2 mm, about 35 pm to about 1 mm, about 40 pm to about 1 mm, or about 45 pm to about 1 mm. The baffles may be spaced from about 25 pm to about 7 mm, about 50 pm to about 6 mm, about 100 pm to about 5 mm, about 100 pm to about 4 mm, about 100 pm to about 3 mm, about 100 pm to about 2 mm, about 100 pm to about 1 mm, about 125 pm to about 5 mm, or about 150 pm to about 5 mm apart. The size, number, and spacing of the baffles may vary and be selected to provide the sample flow dispersion, route, and rate desired for a particular use or particle to be isolated. In some embodiments, each or particular baffles may have gaps formed at both the top and/or the bottom, at one or both sides, all the way around them. In addition, the baffles may be arranged in an array with a regular pattern or an irregular arrangement. And some of the baffles may be larger than other ones.

In another aspect, the exosomal proteins comprise exosomal surface proteins. Exosomal surface proteins may comprise proteins, such as but not limited to, CD63, CD81 , EGFR, VCP, POTEE, FASN, HSPA13, ENO2, DDR1 , CEA, DPEP1 , GPC1 , TGF- 1, ENO1 , other known exosome surface markers, or a combination thereof.

In another aspect, the system isolates exosomes ranging in size from about 20 nm to greater than 500 nm, about 20 nm to greater than 400 nm, about 20 nm to greater than 300 nm, about 20 nm to greater than 200 nm, about 20 nm to greater than 100 nm, about 20 nm to greater than 50 nm, about 30 nm to greater than 500 nm, about 40 nm to greater than 500 nm, about 50 nm to greater than 500 nm, about 60 nm to greater than 500 nm, about 70 nm to greater than 500 nm, about 80 nm to greater than 500 nm, about 90 nm to greater than 500 nm, about 100 nm to greater than 500 nm, about 200 nm to greater than 500 nm, about 300 nm to greater than 500 nm, or about 400 nm to greater than 500 nm. In a particular aspect, the system isolates exosomes ranging in size from about 30 nm to greater than 200 nm.

In another aspect, each filter pore of the plurality of filter pores has a diameter of about 200 nm to about 5 pm, about 300 nm to about 5 pm, about 400 nm to about 5 pm, about 500 nm to about 5 pm, about 600 nm to about 5 pm, about 700 nm to about 5 pm, about 800 nm to about 5 pm, about 900 nm to about 5 pm, about 1 pm to about 5 pm, about 2 pm to about 5 pm, about 3 pm to about 5 pm, about 4 pm to about 5 pm, about 200 nm to about 4 pm, about 200 nm to about 3 pm, about 200 nm to about 2 pm, about 200 nm to about 1 pm, about 200 nm to about 900 nm, about 200 nm to about 800 nm, about 200 nm to about 700 nm, about 200 nm to about 600 nm, about 200 nm to about 500 nm, about 200 nm to about 400 nm, or about 200 nm to about 300 nm. In another aspect, the first diameter of the first nanopore opening is between about 10 nm and about 200 nm, about 20 nm and about 200 nm, about 30 nm and about 200 nm, about 40 nm and about 200 nm, about 50 nm and about 200 nm, about 60 nm and about 200 nm, about 70 nm and about 200 nm, about 80 nm and about 200 nm, about 90 nm and about 200 nm, about 100 nm and about 200 nm, about 110 nm and about 200 nm, about 120 nm and about 200 nm, about 130 nm and about 200 nm, about 140 nm and about 200 nm, about 150 nm and about 200 nm, about 160 nm and about 200 nm, about 170 nm and about 200 nm, about 180 nm and about 200 nm, about 190 nm and about 200 nm, about 10 nm and about 190 nm, about 10 nm and about 180 nm, about 10 nm and about 170 nm, about 10 nm and about 160 nm, about 10 nm and about 150 nm, about 10 nm and about 140 nm, about 10 nm and about 130 nm, about 10 nm and about 120 nm, about 10 nm and about 110 nm, about 10 nm and about 100 nm, about 10 nm and about 90 nm, about 10 nm and about 80 nm, about 10 nm and about 70 nm, about 10 nm and about 60 nm, about 10 nm and about 50 nm, about 10 nm and about 40 nm, about 10 nm and about 30 nm, or about 10 nm and about 20 nm. In another aspect, the second diameter of the second nanopore opening is less than about 2 pm, less than about 1 .5 pm, less than about 1 pm, or less than about 0.5 pm.

In another aspect, the asymmetric nanopore membrane is formed from one or more materials comprising one or more of a polyethylene terephthalate (PET), a polycarbonate (PC), a polypropylene (PP), a polyimides (PI), or a polyethersulphone (PES).

In another aspect, the magnetic or non-magnetic Janus particles are about 200 nm to about 5 pm, about 300 nm to about 5 pm, about 400 nm to about 5 pm, about 500 nm to about 5 pm, about 600 nm to about 5 pm, about 700 nm to about 5 pm, about 800 nm to about 5 pm, about 900 nm to about 5 pm, about 1 pm to about 5 pm, about 2 pm to about 5 pm, about 3 pm to about 5 pm, about 4 pm to about 5 pm, about 200 nm to about 4 pm, about 200 nm to about 3 pm, about 200 nm to about 2 pm, about 200 nm to about 1 pm, about 200 nm to about 900 nm, about 200 nm to about 800 nm, about 200 nm to about 700 nm, about 200 nm to about 600 nm, about 200 nm to about 500 nm, about 200 nm to about 400 nm, or about 200 nm to about 300 nm in diameter size. In a particular aspect, the magnetic or non-magnetic Janus particles are about 500 nm to about 1 pm in diameter size.

In another aspect, the one or more capture antibodies are conjugated to a gold-coated portion of the magnetic or non-magnetic Janus particles. The capture antibody may bind to surface markers on exosomes. In particular, the capture antibody may bind to CD63, CD81 , EGFR, VCP, POTEE, FASN, HSPA13, ENO2, DDR1 , CEA, DPEP1 , GPC1 , TGF- 1 , ENO1 , other known exosome surface markers, or a combination thereof.

In another aspect, the source for inducing fluid crossflow generates a pressure less than about 2 atm, less than about 1.5 atm, less than about 1 atm, or less than about 0.5 atm. In another aspect, the source for inducing fluid crossflow comprises a syringe pump, an electroosmotic pump, a micropump, a centrifuge, or a combination thereof. In another aspect, the image recording device comprises a smartphone. In another aspect, the system achieves an isolated exosome yield of at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% from the sample.

Another embodiment described herein is a method for isolating exosomes and detecting colocalized exosomal proteins from a sample, the method comprising: introducing a sample comprising exosomes into a device for isolating exosomes, the device comprising: a first chamber, wherein the sample comprising exosomes is positioned within the first chamber; a second chamber; a third chamber; a filter positioned between the first chamber and the second chamber, the filter comprising a first filter surface facing and at least partially defining the first chamber, a second filter surface facing and at least partially defining the second chamber, and a plurality of filter pores extending between the first and second filter surfaces; an asymmetric nanopore membrane positioned between the second chamber and the third chamber, the asymmetric nanopore membrane comprising a first membrane surface facing and at least partially defining the second chamber, a second membrane surface facing and at least partially defining the third chamber, and a plurality of asymmetrically-shaped nanopores extending between the first and second membrane surfaces, wherein each nanopore includes a first nanopore opening at the first membrane surface having a first diameter, and a second nanopore opening at the second membrane surface having a second diameter that is greater than the first diameter; and a source for inducing fluid crossflow through the asymmetric nanopore membrane from the second chamber to the third chamber by pressure driven flow, electroosmotic flow, centrifugal force, or a combination thereof; inducing fluid crossflow through the asymmetric nanopore membrane from the second chamber to the third chamber, whereupon the exosomes are isolated in the third chamber; mixing the isolated exosomes with magnetic or non-magnetic Janus particles conjugated to one or more capture antibodies having specific binding affinity to one or more exosomal proteins to generate exosome-bound magnetic or non-magnetic Janus particles; optionally, isolating the exosome-bound magnetic or non-magnetic Janus particles using a magnetic pulldown, and removing unbound exosomes; mixing the exosome-bound magnetic or non-magnetic Janus particles with one or more fluorophore-conjugated detector antibodies having specific binding affinity to one or more exosomal proteins to generate magnetic or nonmagnetic Janus particles bound to exosomes and fluorophore-conjugated detector antibodies; optionally, isolating the magnetic or non-magnetic Janus particles bound to exosomes and fluorophore-conjugated detector antibodies using a magnetic pulldown, and removing unbound fluorophore-conjugated detector antibodies; detecting and measuring changes in the rotational frequency of the magnetic or non-magnetic Janus particles bound to exosomes and fluorophore- conjugated detector antibodies using an image recording device, thereby detecting colocalized exosomal proteins; and tracking individual magnetic or non-magnetic Janus particle with an image analysis software to isolate the magnetic or non-magnetic Janus particle rotation from translation.

In one aspect, the fluorophore-conjugated detector antibodies may be conjugated to any fluorophore known in the art such as, but not limited to, StarBright UltraViolet 400, DyLight 405, StarBright Violet 440, StarBright UltraViolet 445, Pacific Blue, StarBright Violet 475, StarBright UltraViolet 510, StarBright Violet 515, DyLight 488, Alexa Fluor 488, FITC, Amethyst Orange, StarBright UltraViolet 575, StarBright Violet 570 , DyLight 550, PE, hFab Rhodamine*, StarBright Violet 610, StarBright Ultra Violet 605, Texas Red, APC, Alexa Fluor 647, StarBright Violet 670, Cy5, DyLight 650, PerCP, StarBright UltraViolet 665, StarBright Blue 700, DyLight 680, StarBright Violet 710, Alexa Fluor 700, StarBright UltraViolet 740, StarBright Violet 760, DyLight 755, StarBright Violet 790, StarBright UltraViolet 795, and DyLight 800.

In one aspect, the sample comprises whole blood, plasma, serum, urine, saliva, breast milk, or cell media. In another aspect, inducing fluid crossflow through the asymmetric nanopore membrane from the second chamber to the third chamber comprises a flow rate between about 0.01 mL/hour to about 1000 mL/hour, about 0.1 mL/hourto about 1000 mL/hour, about 1 mL/hour to about 1000 mL/hour, about 10 mL/hour to about 1000 mL/hour, about 50 mL/hour to about 1000 mL/hour, about 100 mL/hour to about 1000 mL/hour, about 200 mL/hour to about 1000 mL/hour, about 300 mL/hour to about 1000 mL/hour, about 400 mL/hour to about 1000 mL/hour, about 500 mL/hour to about 1000 mL/hour, about 600 mL/hour to about 1000 mL/hour, about 700 mL/hour to about 1000 mL/hour, about 800 mL/hour to about 1000 mL/hour, about 900 mL/hour to about 1000 mL/hour, about 0.01 mL/hour to about 900 mL/hour, about 0.01 mL/hour to about 800 mL/hour, about 0.01 mL/hour to about 700 mL/hour, about 0.01 mL/hour to about 600 mL/hour, about 0.01 mL/hour to about 500 mL/hour, about 0.01 mL/hour to about 400 mL/hour, about 0.01 mL/hour to about 300 mL/hour, about 0.01 mL/hour to about 200 mL/hour, about 0.01 mL/hour to about 100 mL/hour, about 0.01 mL/hour to about 50 mL/hour, about 0.01 mL/hour to about 10 mL/hour, about 0.01 mL/hour to about 1 mL/hour, or about 0.01 mL/hour to about 0.1 mL/hour.

In another aspect, the method achieves an isolated exosome yield of at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% from the sample.

In another aspect, the method is performed in at most 65 minutes, at most 60 minutes, at most 55 minutes, at most 50 minutes, at most 45 minutes, or at most 40 minutes.

In another aspect, the method has an about 300-, about 200-, about 100-, about 90-, or about 80-exosome particle detection sensitivity and a 4-log dynamic range.

It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof. Various embodiments and aspects of the inventions described herein are summarized by the following clauses:

Clause 1. A system for isolating exosomes and detecting colocalized exosomal proteins from a sample, the system comprising: a device for isolating exosomes from a sample, the device comprising: a first chamber; a second chamber; a third chamber; a sample comprising exosomes positioned within the first chamber; a filter positioned between the first chamber and the second chamber, the filter comprising a first filter surface facing and at least partially defining the first chamber, a second filter surface facing and at least partially defining the second chamber, and a plurality of filter pores extending between the first and second filter surfaces; an asymmetric nanopore membrane positioned between the second chamber and the third chamber, the asymmetric nanopore membrane comprising a first membrane surface facing and at least partially defining the second chamber, a second membrane surface facing and at least partially defining the third chamber, and a plurality of asymmetrically-shaped nanopores extending between the first and second membrane surfaces, wherein each nanopore includes a first nanopore opening at the first membrane surface having a first diameter, and a second nanopore opening at the second membrane surface having a second diameter that is greater than the first diameter; and a source for inducing fluid crossflow through the asymmetric nanopore membrane from the second chamber to the third chamber by pressure driven flow, electroosmotic flow, centrifugal force, or a combination thereof; magnetic or non-magnetic Janus particles conjugated to one or more capture antibodies having specific binding affinity to one or more exosomal proteins, wherein the Janus particles are coated with a scattering material; one or more fluorophore-conjugated detector antibodies having specific binding affinity to one or more exosomal proteins; optionally, a magnet; and an image recording device to detect and measure changes in the rotational frequency of the magnetic or non-magnetic Janus particles.

Clause 2. The system of clause 1 , wherein the sample comprises whole blood, plasma, serum, urine, saliva, breast milk, or cell media.

Clause 3. The system of clause 1 or 2, wherein the first chamber comprises one or more baffles on a wall opposite of the first filter surface.

Clause 4. The system any one of clauses 1-3, wherein the exosomal proteins comprise exosomal surface proteins.

Clause 5. The system any one of clauses 1-4, wherein the system isolates exosomes ranging in size from about 30 nm to >200 nm.

Clause 6. The system any one of clauses 1-5, wherein each filter pore of the plurality of filter pores has a diameter of about 200 nm to about 5 pm.

Clause 7. The system any one of clauses 1-6, wherein the first diameter of the first nanopore opening is between about 10 nm and about 200 nm.

Clause 8. The system any one of clauses 1-7, wherein the second diameter of the second nanopore opening is less than about 2 pm.

Clause 9. The system any one of clauses 1-8, wherein the asymmetric nanopore membrane is formed from one or more materials comprising one or more of a polyethylene terephthalate (PET), a polycarbonate (PC), a polypropylene (PP), a polyimides (PI), or a polyethersulphone (PES).

Clause 10. The system any one of clauses 1-9, wherein the magnetic or non-magnetic Janus particles are about 500 nm to about 1 pm in diameter size.

Clause 11. The system any one of clauses 1-10, wherein the one or more capture antibodies are conjugated to a gold-coated portion of the magnetic or non-magnetic Janus particles.

Clause 12. The system any one of clauses 1-11 , wherein the source for inducing fluid crossflow generates a pressure less than about 1 atm.

Clause 13. The system any one of clauses 1-12, wherein the source for inducing fluid crossflow comprises a syringe pump, an electroosmotic pump, a micropump, a centrifuge, or a combination thereof.

Clause 14. The system any one of clauses 1-13, wherein the image recording device comprises a smartphone.

Clause 15. The system any one of clauses 1-14, wherein the system achieves an isolated exosome yield of at least > 85% from the sample. Clause 16. A method for isolating exosomes and detecting colocalized exosomal proteins from a sample, the method comprising: introducing a sample comprising exosomes into a device for isolating exosomes, the device comprising: a first chamber, wherein the sample comprising exosomes is positioned within the first chamber; a second chamber; a third chamber; a filter positioned between the first chamber and the second chamber, the filter comprising a first filter surface facing and at least partially defining the first chamber, a second filter surface facing and at least partially defining the second chamber, and a plurality of filter pores extending between the first and second filter surfaces; an asymmetric nanopore membrane positioned between the second chamber and the third chamber, the asymmetric nanopore membrane comprising a first membrane surface facing and at least partially defining the second chamber, a second membrane surface facing and at least partially defining the third chamber, and a plurality of asymmetrically-shaped nanopores extending between the first and second membrane surfaces, wherein each nanopore includes a first nanopore opening at the first membrane surface having a first diameter, and a second nanopore opening at the second membrane surface having a second diameter that is greater than the first diameter; and a source for inducing fluid crossflow through the asymmetric nanopore membrane from the second chamber to the third chamber by pressure driven flow, electroosmotic flow, centrifugal force, or a combination thereof; inducing fluid crossflow through the asymmetric nanopore membrane from the second chamber to the third chamber, whereupon the exosomes are isolated in the third chamber; mixing the isolated exosomes with magnetic or non-magnetic Janus particles conjugated to one or more capture antibodies having specific binding affinity to one or more exosomal proteins to generate exosome-bound magnetic or non-magnetic Janus particles; optionally, isolating the exosome-bound magnetic or non-magnetic Janus particles using a magnetic pulldown, and removing unbound exosomes; mixing the exosome-bound magnetic or non-magnetic Janus particles with one or more fluorophore-conjugated detector antibodies having specific binding affinity to one or more exosomal proteins to generate magnetic or non-magnetic Janus particles bound to exosomes and fluorophore-conjugated detector antibodies; optionally, isolating the magnetic or non-magnetic Janus particles bound to exosomes and fluorophore-conjugated detector antibodies using a magnetic pulldown, and removing unbound fluorophore-conjugated detector antibodies; detecting and measuring changes in the rotational frequency of the magnetic or nonmagnetic Janus particles bound to exosomes and fluorophore-conjugated detector antibodies using an image recording device, thereby detecting colocalized exosomal proteins; and tracking individual magnetic or non-magnetic Janus particle with an image analysis software to isolate the magnetic or non-magnetic Janus particle rotation from translation.

Clause 17. The method of clause 16, wherein the sample comprises whole blood, plasma, serum, urine, saliva, breast milk, or cell media.

Clause 18. The method of clause 16 or 17, wherein inducing fluid crossflow through the asymmetric nanopore membrane from the second chamber to the third chamber comprises a flow rate between about 0.01 mL/hour to about 1000 mL/hour.

Clause 19. The method any one of clauses 16-18, wherein the method achieves an isolated exosome yield of at least > 85% from the sample.

Clause 20. The method any one of clauses 16-19, wherein the method is performed in about < 60 minutes.

Clause 21. The method any one of clauses 16-20, wherein the method has an about 100- exosome particle detection sensitivity and a 4-log dynamic range.

EXAMPLES

Example 1

Assay Platform

Two innovative technologies currently being developed will be integrated, the Asymmetric Nanopore Membrane (ANM) nanofiltration technique and the rotational diffusivity Immuno-Janus Particle (IJP) technique using magnetic or non-magnetic Janus particles (MJP), for exosome isolation from plasma samples and profiling of their colocalized surface proteins in 60 minutes with 100 exosome particles detection sensitivity and 4 log dynamic range. The first AMN technology provides high-throughput isolation and purification of exosomes in plasma or cell media (FIG. 1) while the second UP technology allow profiling of colocalized exosome proteins with an ultrasensitive MJP with conjugated antibodies (FIG. 1). For the proof-of-concept study, exosomes will be captured with MJPs attached to anti-CD26 antibodies and profile five colocalized exosomal surface proteins (CD59, CD13, CD107a, EpCAM, EGFR) in PC-3 cell media samples. After the selective capture of exosomes, MJPs will be pulled-down with a magnet to remove the unbound vesicles (FIG. 1). They will then be exposed to MJPs functionalized with five anti-antibodies of surface proteins, each labelled with Qdots of different colors for sensitive detection and profiling of exosomal surface proteins followed by another MPJ pull-down wash to remove free-floating unbound Qdots (FIG. 1). The total assay time (purification and profiling) for a 100 pL plasma sample will be ~1 hour. PC-3 cell lines will be used for the initial ANM and MJP optimization study. Because of the Janus design with highly intense blinking at ~ 1 Hz, the MJP rotation frequency and colocalized protein fluorescent signals can be detected with low-cost imaging equipment, even a smart-phone camera. The optimized platform will be validated against a cohort of 25 prostate cancer patient plasma samples and benchmark the platform by analyzing the MJPs with conventional flow cytometry.

The integrated platform will be the first technology that can isolate exosomes from plasma and profile exosomal biomarkers with sufficient specificity and sensitivity required for the reliable detection of cancer markers. To ensure analytical accuracy, it is necessary to remove all free- floating proteins that can interfere with downstream immunocapture or assay steps. To address this issue, a high-throughput nanofiltration device, ANM will be integrated for high yield and pure isolation of exosomes from plasma. To achieve specificity, exosomal colocalized protein profiling with the MJP will be conducted. Importantly, the assay time for the integrated platform is 1 hour. As the two technologies involve only small equipment like pumps and imaging camera, they can be easily integrated and automated. Easy production of the proposed device into a portable commercialized product after complete. Other than eventual diagnostic applications, this integrated platform will also significantly elevate the research community, as exosome biomarker discovery and therapeutic exosome development are active areas of research. Both areas will benefit from the rapid purification and characterization offered by the integrated platform.

ANM-based Filtration Technology for Isolation of Exosomes A novel technology for exosome isolation from cell media and plasma samples is described. A size-based patent-pending Asymmetric Nanopore Membrane (ANM) filtration technology is being developed for high-throughput (<30 minutes) and high-yield (>85%) isolation and purification of exosomes (30 to 200 nm) from other nanocarriers. ANMs (as opposed to conventional cylindrical or irregularly shaped pores in ultrafiltration membranes) offer important advantages for exosome isolation applications (FIGS. 2A-B). The asymmetrical pore shape allows a dramatic -500% reduction in the applied pressure/force to drive the sample through the filter membrane at the same throughput, compared to an analogous cylindrical pore membrane. This significant reduction in applied pressure prevents exosome deformation, lysing and fusion and thus significant increases the yield while preserving other advantages of size-based ultrafiltration. Moreover, the chance of clogging and vesicle trapping is significantly reduced due to a dramatic enhancement in the rate of transport through the membrane, relative to an analogous cylindrical pore membrane. This new pore geometry design allows high yield and high throughput and permits trapping designs shown in FIG. 2B. The trapping design allows for the concentration of exosomes of a specific size range from the larger and smaller debris, molecules, and EVs. The concentration factor can be as large as 100. Importantly, it allows for the flushing of trapped exosomes with a rinsing buffer to remove all contaminants, including the abundant free-floating proteins, nucleic acids, ribonucleoprotein, and lipoproteins, resulting in the isolation of exosomes with very high purity.

For accurate downstream analysis of exosomes, it is desirable to isolate exosomes and remove free-floating interfering biomarkers before analysis. It would also be beneficial to significantly concentrate the volume of the purified exosomes to enhance the sensitivity of the downstream assay. The challenge is then to isolate all exosomes with a high-yield and low-cost device from plasma samples, as the standard ultracentrifugation (UC) equipment is too costly and personnel-intensive and not suitable for the personalized device. Tha ANM from the NIH UG3UH3CA241684 is a low-cost ultrafiltration membrane for isolating exosomes by size. The ANM is prepared by low-cost etching techniques, based on the irradiation of material with chemical and plasma etching. The pore size was fine-tuned by adjusting the etching time, and highly uniform 30 nm (tip) with less than 3% variation and 500 nm (base) conical nanopores were fabricated for selective isolation of EV particles (FIGS. 3A-B). 99% of the protein can be removed by washing with different amounts of the buffer (FIG. 4B). The ANM-isolated EV size distribution has been characterized with Nanoparticle Tracking Analysis (NTA) (FIG. 4D). Preliminary results showed about 30% higher yield and significantly less aggregation with ANM compared to an analogous cylindrical pore (symmetric) membrane (FIG. 4G). The performance of ANM with qEV (a size exclusion chromatography from Izon), ultracentrifugation (UC), and precipitation isolation techniques was also benchmarked. The preliminary study shows the isolation yield was two times better than qEV and 10-fold better than UC (FIG. 4F). Correspondingly, Western blot results of ANM-isolated sEV sample show a strong band for tetraspanin CD63 and TSG101 (FIG. 4C). Purity with respect to free-floating protein, as determined by the BCA test, was also significantly better (>3x) for ANM than qEV (FIG. 4E). The biggest advantage of ANM EV purification is, however, the high throughput provided by the low-resistance conic pores (FIG. 4A). By recycling through the chip, exosomes can be steadily purified from protein with additional wash (recycle) without EV loss.

These studies also indicate that the ANM isolated sEV preserves the same power-law and asymmetric size distribution as the original plasma or cell culture media samples, with mean/mode size that does not change by more than 10% from the original sEV population (see FIG. 5 for one particular plasma sample). (Note: all runs are conducted after pre-filtering with a 200 nm filter to remove microvesicles). Extensive testing was done with different conditioned cell media and commercial plasma samples. The average sEV isolation yield is found to be 85%, which varies less than 5% for different ANM isolation runs of the same sample and less than 10% for different cell media or diluted plasma samples. The sEV isolation yield by ANM is hence estimated to be 85% with 10% error for cell media and 30* diluted plasma at 10 mL/hour throughput.

Janus Particle Based Assay Platform to Detect Exosomes and Profile Exosomal Surface Proteins Unlike other optical exosome technologies, the MJP technology described herein images the rotation of the much larger MJPs with low-cost cameras, rather than the smaller exosomes, to overcome the resolution limitation. Micron-sized Janus particles undergo rotational diffusion because of thermal noise, with a rotational frequency of about 1 Hz that can be easily detected with low-cost imaging systems. Studies have shown that an immuno-Janus particle (UP) can have a significantly lower rotation frequency when attached to nanobeads with an autocorrelation video analysis of the blinking Janus beads. Unlike translational diffusivity with first-order scaling, the Einstein-Stokes relationship for rotational diffusivity stipulates the rotation diffusivity (frequency) scales as the third order of the particle size. Hence, coupling with a 100-nm bead on a micron-size UP can reduce its frequency by as much as 33%. By using parallelized singleparticle tracking (~5 minutes), it is possible to deconvolve the translational and rotational diffusivity to achieve a much higher resolution and approach this theoretical limit. With antibodies for CDF63, a common EV tetraspanin, functionalized onto 1-micron JPs, CD63-EV was able to be assayed directly with a dynamic range of 10 fM to 10 pM in 2 L of diluted plasma between coverslips (FIG. 4), corresponding to 103 to 106 EVs (3 log dynamic range). However, the sensitivity should be a function of the Janus particle concentration, which is at 10 fM for FIG. 4F. By tuning the Janus particle concentration, it is expected to be able to reduce the detection limit 10* to 100 EVs, which will be an ideal cancer screening platform with 4 log dynamic range. Although the significant upregulation of exosomal surface proteins for samples from early-stage patients may not be seen, the identification of cancer by only detecting cancer-derived exosomes is anticipated. The incubation and image analysis time for each IJP assay is less than 15 minutes due to the bulk docking of the particles. It hence represents a rapid exosome quantification technology based on its membrane proteins. For profiling of multiple colocalized surface proteins, anti-antibody-Qdots will be used specific to the different surface proteins of interest and the protein amount will be quantified by monitoring the intensity of different fluorescence signals coming from exosome-MPJ conjugate (see FIG. 1). The 1 Hz blinking due to Browning rotation of the stationary one-micron IJP allows us to discern the intensity of fluorescent signals, with much higher resolution than direct detection from the exosomes in fast-moving flow cytometry.

The ANM exosome purification module and rotational diffusivity MJP assay module will be utilized for the development of a diagnostic tool for clinical research. The proposed platform will be developed with a PC-3 model cell line. The exosomes will first be isolated and enriched from PC-3 cell media for downstream exosome detection and then profiling of multiple surface protein biomarkers will be conducted. Enriched exosomes will be immunocaptured with MJP attached to CD26 and then anti-antibody attached to 5 different colors of 5 Qdots will be used to profile 5 different exosomal surface proteins in captured exosomes. Upon successful optimization, prostate cancer plasma samples will be used to validate the platform. The performance of the platform will be benchmarked and compared with conventional flow cytometry used for cell analysis.

Detection of Exosome and Single Exosomal Surface Protein

Optimize the ANM isolation protocol for the enrichment and purification of exosomes in PC-3 cell media and healthy human plasma samples. The protocol of exosome isolation by ANM will be optimized for PC-3 cell line media and healthy human plasma samples. The module optimization with plasma samples will be done, where the assay platform will be validated with clinical samples.

An optimized protocol will be used to fabricate the 30 nm ANM membrane. Briefly, commercial track-etch membranes are used and then processed to develop a conical shape pore of 30 nm (tip) and 500 nm (base) (see FIG. 2) by performing a plasma etch at an optimized time and oxygen flow rate (see FIG. 3). A scanning electron microscope (SEM) will be performed to check the pore size and its uniformity. Upon successful ANM module fabrication, the exosome isolation protocol will first be optimized and refined for PC-3 cell media samples. 10 mL of supernatant of cell culture media samples will be pumped through a 0.2-micron syringe filter to remove the larger EVs and cell debris followed by passing the resultant solution through an ANM nanofiltration module to isolate and enrich the exosomes with a high yield (>90%) and good purity. TEM and NanosightTM will be used to check the isolation yield and exosome size. The isolation purity will be determined by nanodrop and standard Bradford Protein Assay (BioRad) or BCA protein assay (Fisher) by analyzing before and flow-through solutions. Western Blot will be performed on the ANM- isolated fraction to determine the quality of the isolated particles by checking the EV proteins like Actin, ALIX, and TSG101. All experiments will be performed in triplicate.

Upon successful optimization with the cell media sample, the module will be optimized for plasma samples. A low-viscus 30* diluted plasma sample will be pumped sequentially through 0.2-micron syringe filter and the ANM module and refined the protocol to achieve a high yield (>85%) and good purity. The plasma is more heterogeneous and complex than cell media. Hence, to estimate the purity of the ANM-isolated EVs, the pressure drop across the ANM will be monitored, as the viscosity of the sample decreases with protein removal. If needed, a control strategy will be implemented to recycle the exosomes until the desired purity is achieved. In addition to the analytical characterization tools used for cell media samples, the isolation purity will also be tested against lipoproteins such as HDLs using APOA1 ELISA and western blot. A goal will be purity that is sufficient and does not interfere with the downstream exosome immunocapturing step.

Optimize the Magnetic Janus Particle Size and Number for Sensitive Exosome Detection

Different sizes of MJPs will be used and optimized for the right size, and MJP number will also be optimized to achieve high exosome detection sensitivity. Ideally, the exosome detection sensitivity will be the highest with the comparable size MJPs to amplify the change in the particle rotational diffusivity. However, since microscopy resolution of the blinking MJP is around 500 nm for low-cost cameras, the MJP size shall be optimized from 500 nm to 1 micron. As the rotational diffusivity is greatly affected by the particle diameter, the size reduction will increase the sensitivity, thus leading to a better limit of detection (LOD). Different sizes of MJPs will first be synthesized using the optimized protocol developed for Janus particles. A gold side of the MPJ will be used to functionalize CD26 anti-antibodies using a commercial gold-antibody conjugation kit (FIG. 7A). Fixed concentrations of MJPs with exosomes will show a different rotational diffusivity distribution than that of unoccupied MJPs provided rotational and translational components are de- convolved. The difference in the distribution of the rotational diffusivity is directly related to the concentration of MJPs-EV complexes as well as the bulk EV concentration. As the MJP can capture multiple exosomes, it would be desirable if the blinking frequency is also sensitive to the average number of exosomes per MJP to increase the sensitivity (FIG. 7B). A preliminary experiment shows that a detectable 5% drop in the average rotation frequency for 10* increase in the exosome concentration (attached number of exosome per MJP) (FIG. 6G) or about 50% drop with 100* more exosomes. With the micron-sized MJP and 100 nm exosomes, it is estimated from the Einstein-Stokes rotational diffusivity that the rotation frequency will decrease by 40% if the MJP is saturated with 400 exosomes. The preliminary results suggest that partially covered MJPs can be detected, down to about 40 exosomes per MJP. This sensitivity can improve with smaller submicron MJPs. In theory, 40 100-nm exosomes can reduce the rotation frequency of a 500 nm MJP by 60% at a higher frequency of 10 Hz. With the MJP size down to about 300 nm, the particle Peclet number is below unity and the MJP does not settle. However, there is an advantage to larger particles that do settle at the 1 -micron range. A continuous sedimentation flux of particles across the focal plane allows interrogation of all the MJPs, without sophisticated microfluidics or alternation of the focal plane. Hence, an optimum MJP size of about 500 nm (~3% Af with one exosome and 10 Hz) is expected that will provide the highest exosome detection sensitivity without compromising the single particle tracking resolution.

The platform can be calibrated and benchmarked against Nanoparticle-tracking analysis, flow cytometry-based methods, and Surface Plasmon Resonance (SPR), with proper accounting of their resolution limitation because of their direct imaging of the exosomes, whereas the current technology images the much larger MJPs. The high shear due to rotating MJPs also means that no additional washing step will be necessary to prevent any non- specific adsorption of antibodies. For clinical samples, the MJPs can capture multiple exosomes depending on the cancer-derived exosome concentration in the samples. As the rotational time of MJP-exosome conjugate is dependent on the number of captured exosomes, the development of a correlation plot between average captured exosome number and rotational time is anticipated that can estimate the total captured exosomes by Poisson statistics. For this study, a detection sensitivity of 1 exosome per MJP will be targeted with the smallest detectable MJP, the digital limit. The tradeoff between sensitivity and colocalization number may require two different MJP suspensions of different size. The exosome stock solution will be prepared for this study by calculating the exosome concentration with NanoSight™. Optimize the Assay Parameters for Exosome Detection and Profiling of colocalized Surface Proteins on Exosomes Enriched from the Cell Media Samples

Upon successful optimization of MJP size and concentration, individual surface proteins will be profiled with specific antibodies tagged with different Qdots. Five Qdots with nonoverlapping spectral wavelengths (for example, Qdots from Thermo Fisher that sells five distinct color Qdots excitable with a long-wavelength UV light source) will be selected. Qdot-conjugated antibodies that bind to the MJP-exosome conjugate will blink at the same rotational frequency as that of the MJP-exosome conjugate. Hence, tracking the rotational frequency of MJP-exosome- Qdot will allow to differentiate it from unbound Qdots, even though most of the interfering free- floating Qdots have been removed by a magnetic pulldown step. Current state-of-art laser excitation/emission system can detect several dozen fluorophores at the same time by employing different lasers and the platform can be extended to do the same. Moreover, the five different Qdots on two different size MJPs can be used as their rotational frequencies will be different. This should theoretically be able to detect 10 surface protein biomarkers. Nevertheless, 5 colocalized markers on each exosome will be focused, as it will already transform exosome diagnostics. This platform will be optimized with exosomes from the PC-3 cell line. All tests will be done in triplicate.

A calibration plot will be developed for each of the five protein biomarkers with SPR. A different concentration of exosomes will be taken and a similar sandwich with reporter antibody conjugated to gold instead of Qdots will be used. This will be done for each protein biomarker. The plot will be used to determine the exosomal surface protein concentration in MJP-exosome- Qdot by calculating the average fluorescence intensity coming from MJP-exosome. The assay performance will be benchmarked for each protein biomarker by flowing the MJP-exosome-Qdot conjugate in conventional flow cytometry. The use of MJP will make the conjugate sufficiently bigger and will fall within the size range of conventional flow cytometry, which is about 270-600 nm. After completing the assay for one surface protein, the same optimized assay protocol (such as optics parameter) will be used for other protein targets and the parameters such as exposure time, gain, etc. will be determined that will work for all five Qdots.

The LOD is a key to enhance the performance of the proposed JP diagnostic platform. Although the background noise due to the environmental temperature and liquid viscosity variations can be well controlled below 1% during ANM isolation, other factors, such as light intensity variation and camera noise, may impede the measurement accuracy. To minimize the effects of these variations on accurate quantification, reference fluorescence JPs will be introduced. The reference JPs will not capture any exosome or protein because of absence of functional ligands on the surfaces. Both the reference and probe JPs will experience the same conditions in the sample. By simply dividing the rotational diffusivity of probe JPs by that of reference JPs, the ratio should be only proportional to the particle diameter cubed which correlates with the exosome number on the MJP.

The profiles of the colocalized proteins would be more accurate if each MJP captures only one exosome in the Poisson limit. Hence, 5 MJP concentrations will be prepared, with sequential 10x dilution, and the profiling statistics will be recorded at different concentrations to ensure accurate profiles. This will be done in 5 different observation wells with aliquoted samples. The imaging results will be benchmarked with confocal imaging, which should be able to decipher fluorescent signals from two different exosomes on the same MJP.

According to previous studies, 10 Hz is sufficient to resolve the blinking frequency of exosomes. Most modern smartphones are capable of resolving the signal and recording a quality video for analysis (see FIG. 8). An Android smartphone App coded by Java can be programmed to enable the recording and frequency computation. The rotational diffusion is associated with low blinking frequency, so the requirements for the smartphone camera resolution and framerate are not stringent.

If the exosome isolation purity is an issue due to the presence of excess free proteins in the complex plasma sample, a baffle on the top surface of the ANM case will be designed (see FIG. 9) to remove the filter cakes more efficiently. If the non-specific adsorption is an issue, the surfaces of MJPs will be blocked with BSA to prevent non-specific binding. If the antibodies do not have good affinity and cannot capture the exosomes efficiently, antibodies from different suppliers will be tested and the optimal antibodies will be selected by determining their k(on) and k(off) from SPR. If profiling of colocalized proteins does not have sufficient resolution, the rotation of the MJP shall be enhanced by forcing with a rotating magnetic field supplied by a magnetic stirrer. The forcing field will prevent translational Brownian motion of the MJP. Importantly, since the angle between the magnetic dipole of the MJP and magnetic field is a nonlinear function of the dipole orientation, the periodic forcing results in a nonlinear entrainment of the MJP rotation at a lower frequency than the forcing frequency. This lower rotation frequency about a fixed axis of MJP localized at a particular location allows easier scrutiny of the intensity of all 5 Qdots at each MJP.

Detection of Exosomes and Five Exosomal Surface Proteins

All five proteins will be profiled together on the exosome surface. The protocol and parameters optimized as described herein will be used and the five exosomal surface proteins will be profiled. The assay parameters will first be optimized to detect two exosomal surface biomarkers by incubating the exosomes from the PC-3 cell line. The protein number will then be gradually increased to detect three, four, and five exosomal protein biomarkers. Similar to single protein biomarkers, all parameters will be re-optimized to map the MJP-exosome conjugates and determine the average fluorescence intensity coming from all five surface protein biomarkers. The developed SPR calibration plot will be used to determine the surface protein concentration. The assay platform will then be tested with exosomes collected from healthy plasma. Although no issues with plasma samples are anticipated as ANM isolates will be used, any troubleshooting will be done with the validation study. The profiling of five protein biomarkers will be benchmarked for PC-3 and plasma exosomes using MJP-exosome-Qdot conjugates and conventional flow cytometry.

If the MJP particle size is too small to resolve the fluorescence signals of five surface proteins, a bigger sized MJP may be used, and the MJP concentration may also be increased to minimize multiple exosome docking. If necessary, the smartphone camera will be replaced with a confocal imaging system, but this will reduce the functionality of the platform.

Validate the Performance of the Platform with Prostate Cancer Plasma Samples

Finally, a small-cohort blind test with commercial prostate cancer plasma samples will be conducted to validate the optimized assay platform. Twenty-five de-identified healthy and 25 deidentified prostate cancer plasma samples will be tested. Similar blind tests for HDL with PON1 surface protein have been conducted. The performance of the platform will be benchmarked with conventional flow cytometry. After the magnetic pulldown step of MJP-exosome-Qdot conjugates, the sample will be divided into two parts. One part will be used to test the platform while the other part will be used for flow cytometry. The samples will be acquired from commercial clinical sample suppliers like Precision for Medicine or BioIVT.

Example 2

Methods

Janus Particle Fabrication

The Janus Particles (“JPs” or “the particles”) were produced in-house using 1 pm fluorescent polystyrene beads from Thermo Fisher Scientific. The stock solution was diluted to 0.1 % solids in isopropyl alcohol. 1 ml_ of the suspension was dispersed onto a plasma-treated microscope slide and allowed to dry entirely. Once dry, the slide was inserted into an AIRCO Temescal FC 1800 electron beam vacuum deposition/thin-film coater system and coated with 30 nm of gold at a rate of 0.5 A/s. The slide was then sonicated in a sonicator for 15 minutes. The released particles were collected in a 1% (v/v) Tween20/DI H₂O solution and filtered three times using a 5 pm disk filter to remove any aggregates and other impurities. The particle suspension was concentrated to 1 x 10⁸ particles/mL and subsequently stored in a 4°C fridge.

Janus Particle Functionalization

The gold hemisphere of the JPs was functionalized with select antibodies using the Abeam Gold Conjugation Kit. Antibodies underwent a buffer exchange using a 10 kDa centrifugal filter and DI H₂O. Antibodies were diluted to 0.1 mg/mL using the gold antibody diluent. 6 pL of the mixture was combined with 21 pL of the gold conjugation buffer in a 0.2 ml_ PCR tube. 22.5 pL of this solution was then combined with 50 pL of JPs and mixed for 15 minutes at 1000 rpm on a shaker. Gold conjugation quencher was added (2.5 pL) to the solution, which reacted for 15 minutes at room temperature or overnight at 4 °C. The particles were centrifuged at 6000g for 5 minutes and washed with 1:400 Tween20:DI H₂O once, and with 0.1 x PBS twice, before being reconstituted in 50 pL of 0.1 x PBS. This solution was combined with exosomes in a 2:1 ratio and allowed to incubate for an hour.

Fluorescent Imaging of Janus Particles

A 2 pL drop of the JP solution was pipetted onto a standard glass microscope slide. A cover slip was placed over the JP solution with a spacer of 440 pm. The slide was then placed on an Olympus IX-71 inverted fluorescent microscope above a 10x objective. The fluorescence was produced from a mercury bulb. The objective plane was set in the middle of the slide and coverslip to avoid boundary effects from either. Videos ranging from 60 sec to 180 sec were captured using a suitable camera and a fly camera at a frame rate of 10 Hz. All trials were conducted with a minimum of three technical replicates.

The Platform

FIG. 15A shows a schematic of the I JPs, which is essentially a micron-sized sphere with one half being fluorescent while the other half is coated in gold. When the gold side is up, there is weak fluorescence compared to the fluorescent side, creating a blinking effect. The gold side also has antibodies functionalized to it for exosomes to bind. Transmission electron microscopy (TEM) images of these particles show this (FIG. 15B). The experiment has a simple to follow workflow where the particles are mixed with the sample and the resulting signals are read. Most biological samples have a viscosity within the same order of magnitude as PBS, therefore the biological samples were diluted a hundred times to remove any viscosity-induced changes. For highly viscous samples, another method may be used as discussed below. Once the sample is incubated for long enough, a net shift in distribution is observed as shown in FIG. 15B with a typical blinking behavior as shown in FIG. 6B.

Tracking the blinking frequency of every JP rather than using a gross two-dimensional autocorrelation function requires a computational strategy which, if left unchecked, can be computationally very demanding and unsuitable in a point-of-care setting. The blinking frequency is the key readout from the assay, therefore, taking high-resolution images of the particles is unnecessary. In addition, having as many JPs in the frame as possible allows for better statistics to be performed, such as determination of the distribution of the periods. Typically for high- resolution images, one can rely on image segmentation and mask-based methods using discontinuity detection or grayscale similarity, often also requiring correction based on its point spread function, diffraction patterns, and is computationally more expensive. A different approach is used for low-resolution images. The image is not merely multiplied with a dilation matrix associated with the feature size of JPs in the low-resolution image whose asymptotic computation complexity is O(n^{2 373}). Instead, once dilated, a disk detection method using accumulation points and Circular Hough Transform is used for the detection of the image with a computational complexity of O(n³ log n). The detected centers are then mapped onto the original image and are tracked frame-by-frame. The meaningful information, such as the intensity of the particle, can then be used to perform wavelet analysis and its frequency of blinking can be determined. The larger aggregates have a much lower blinking frequency and thus can be removed by only considering the range of frequencies in which the JP can lie. The overall algorithm is highlighted in FIG. 10A.

Sensitivity and Specificity of the Platform: Characterizing Directly from Multiple Biofluids

In initial experiments, small extracellular vesicles (sEVs) were isolated from human plasma to examine how the detection signal varied with changes in sEV concentration across a wide range. A detection limit of 1000 sEVs per microliter of sample was achieved and this demonstrated a dynamic range of nearly three orders of magnitude, as illustrated in FIG. 16A. The goal was to develop a detection platform unaffected by non-target substances, such as soluble or highly abundant proteins like albumin. To demonstrate the platform's effectiveness, Asymmetric Nanopore Filtration (ANF) was applied to the sample to separate sEVs from the plasma and the sEV-depleted fraction in the flow-through (FT). Under typical conditions, mixing sEVs and FT in a 1 :1 ratio replicates the original plasma composition. However, by altering the sEV to FT ratio, the presence of soluble proteins does not significantly affect the detection signal. Even with a ten-fold increase in the FT proportion, the detection signal was the same as the isolated exosomes with no FT, while no exosomes were detected in FT (FIG. 12B), this is also confirmed by the NTA data in FIG. 16B. In addition, lysed exosomes or an isotype control do not produce any signal (FIG. 16C). The lysed exosome sample comprises the same proteins that were present prior to lysis of the exosomes because lysing only solubilizes the lipids of the exosomes. This shows that the presence of the proteins alone is not enough to produce a signal. This is because the proteins need to be on a particle >50 nm. This confirms the hypothesis that soluble free-floating proteins will not produce a strong signal. Additionally, it can be shown that exosomes filtered through a 50 nm pore do not produce a strong signal when compared to exosomes filtered through a 100 nm filter or a 220 nm filter.

Interestingly, the calibration curves shown in FIG. 16A for all the cases nicely overlap at the same concentration. This suggests that the calibration curve is universal due to mass transfer effect dominating. Additionally, to show that sEVs can be characterized from plasma, serum, urine, and cell culture media, a test was conducted without performing any isolation or sample treatment other than dilution and 220 nm filtration.

Pilot study with Diseased Patients

One of the major challenges in biomedical field is to have a screening test that can not only tell if you are healthy, but also determine the type of disease. sEVs can potentially allow for a screening test that indicates whether an individual is healthy or not and, if not, the type of disease the individual has. Instead, about 100 subjects belonging to one of the following five categories were analyzed: healthy, colorectal cancer (CRC), glioblastoma (GBM), pancreatic ductal adenocarcinoma (PDAC), and Alzheimer’s disease (AD). Using a panel of markers, the subjects were analyzed to determine which disease the subject had. A simple decision tree is shown in FIG. 17A that allows one to decide which disease each patient had. A summary of the data is shown in FIGS. 17B-E.

For example, as shown in FIGS. 17G-H, aEGFR is able to distinguish between the aEGFR-positive group (CRC/GBM) or aEGFR-negative group (Healthy, PDAC, and AD). Further, when proteins were studied together, the type of disease was able to be determined with high sensitivity (FIGS. 17A-H).

Validation with Orthogonal UC+SPR To make sure that the concentrations are consistent with other orthogonal methods, UC+SPR was used to analyze the samples analyzed by UP. From that analysis, consistent results were seen between the two methods across all the proteins studied as well as biofluids.

Claims

CLAIMS What is claimed:

1. A system for isolating exosomes and detecting colocalized exosomal proteins from a sample, the system comprising: a device for isolating exosomes from a sample, the device comprising: a first chamber; a second chamber; a third chamber; a sample comprising exosomes positioned within the first chamber; a filter positioned between the first chamber and the second chamber, the filter comprising a first filter surface facing and at least partially defining the first chamber, a second filter surface facing and at least partially defining the second chamber, and a plurality of filter pores extending between the first and second filter surfaces; an asymmetric nanopore membrane positioned between the second chamber and the third chamber, the asymmetric nanopore membrane comprising a first membrane surface facing and at least partially defining the second chamber, a second membrane surface facing and at least partially defining the third chamber, and a plurality of asymmetrically-shaped nanopores extending between the first and second membrane surfaces, wherein each nanopore includes a first nanopore opening at the first membrane surface having a first diameter, and a second nanopore opening at the second membrane surface having a second diameter that is greater than the first diameter; and a source for inducing fluid crossflow through the asymmetric nanopore membrane from the second chamber to the third chamber by pressure driven flow, electroosmotic flow, centrifugal force, or a combination thereof; magnetic or non-magnetic Janus particles conjugated to one or more capture antibodies having specific binding affinity to one or more exosomal proteins, wherein the Janus particles are coated with a scattering material; one or more fluorophore-conjugated detector antibodies having specific binding affinity to one or more exosomal proteins; optionally, a magnet; and an image recording device to detect and measure changes in the rotational frequency of the magnetic or non-magnetic Janus particles.

2. The system of claim 1 , wherein the sample comprises whole blood, plasma, serum, urine, saliva, breast milk, or cell media.

3. The system of claim 1 , wherein the first chamber comprises one or more baffles on a wall opposite of the first filter surface.

4. The system of claim 1 , wherein the exosomal proteins comprise exosomal surface proteins.

5. The system of claim 1, wherein the system isolates exosomes ranging in size from about 30 nm to >200 nm.

6. The system of claim 1 , wherein each filter pore of the plurality of filter pores has a diameter of about 200 nm to about 5 pm.

7. The system of claim 1 , wherein the first diameter of the first nanopore opening is between about 10 nm and about 200 nm.

8. The system of claim 1 , wherein the second diameter of the second nanopore opening is less than about 2 pm.

9. The system of claim 1 , wherein the asymmetric nanopore membrane is formed from one or more materials comprising one or more of a polyethylene terephthalate (PET), a polycarbonate (PC), a polypropylene (PP), a polyimides (PI), or a polyethersulphone (PES).

10. The system of claim 1 , wherein the magnetic or non-magnetic Janus particles are about 500 nm to about 1 pm in diameter size.

11. The system of claim 1 , wherein the one or more capture antibodies are conjugated to a gold-coated portion of the magnetic or non-magnetic Janus particles.

12. The system of claim 1 , wherein the source for inducing fluid crossflow generates a pressure less than about 1 atm.

13. The system of claim 1 , wherein the source for inducing fluid crossflow comprises a syringe pump, an electroosmotic pump, a micropump, a centrifuge, or a combination thereof.

14. The system of claim 1 , wherein the image recording device comprises a smartphone.

15. The system of claim 1, wherein the system achieves an isolated exosome yield of at least > 85% from the sample.

16. A method for isolating exosomes and detecting colocalized exosomal proteins from a sample, the method comprising: introducing a sample comprising exosomes into a device for isolating exosomes, the device comprising: a first chamber, wherein the sample comprising exosomes is positioned within the first chamber; a second chamber; a third chamber; a filter positioned between the first chamber and the second chamber, the filter comprising a first filter surface facing and at least partially defining the first chamber, a second filter surface facing and at least partially defining the second chamber, and a plurality of filter pores extending between the first and second filter surfaces; an asymmetric nanopore membrane positioned between the second chamber and the third chamber, the asymmetric nanopore membrane comprising a first membrane surface facing and at least partially defining the second chamber, a second membrane surface facing and at least partially defining the third chamber, and a plurality of asymmetrically-shaped nanopores extending between the first and second membrane surfaces, wherein each nanopore includes a first nanopore opening at the first membrane surface having a first diameter, and a second nanopore opening at the second membrane surface having a second diameter that is greater than the first diameter; and a source for inducing fluid crossflow through the asymmetric nanopore membrane from the second chamber to the third chamber by pressure driven flow, electroosmotic flow, centrifugal force, or a combination thereof; inducing fluid crossflow through the asymmetric nanopore membrane from the second chamber to the third chamber, whereupon the exosomes are isolated in the third chamber; mixing the isolated exosomes with magnetic or non-magnetic Janus particles conjugated to one or more capture antibodies having specific binding affinity to one or more exosomal proteins to generate exosome-bound magnetic or non-magnetic Janus particles; optionally, isolating the exosome-bound magnetic or non-magnetic Janus particles using a magnetic pulldown, and removing unbound exosomes; mixing the exosome-bound magnetic or non-magnetic Janus particles with one or more fluorophore-conjugated detector antibodies having specific binding affinity to one or more exosomal proteins to generate magnetic or non-magnetic Janus particles bound to exosomes and fluorophore-conjugated detector antibodies; optionally, isolating the magnetic or non-magnetic Janus particles bound to exosomes and fluorophore-conjugated detector antibodies using a magnetic pulldown, and removing unbound fluorophore-conjugated detector antibodies; detecting and measuring changes in the rotational frequency of the magnetic or nonmagnetic Janus particles bound to exosomes and fluorophore-conjugated detector antibodies using an image recording device, thereby detecting colocalized exosomal proteins; and tracking individual magnetic or non-magnetic Janus particle with an image analysis software to isolate the magnetic or non-magnetic Janus particle rotation from translation.

17. The method of claim 16, wherein the sample comprises whole blood, plasma, serum, urine, saliva, breast milk, or cell media.

18. The method of claim 16, wherein inducing fluid crossflow through the asymmetric nanopore membrane from the second chamber to the third chamber comprises a flow rate between about 0.01 mL/hour to about 1000 mL/hour.

19. The method of claim 16, wherein the method achieves an isolated exosome yield of at least > 85% from the sample.

20. The method of claim 16, wherein the method is performed in about < 60 minutes.

21. The method of claim 16, wherein the method has an about 100-exosome particle detection sensitivity and a 4-log dynamic range.