WO2017062901A2 - Methods to isolate extracellular vesicles in biofluids for biomarker discovery and disease diagnosis - Google Patents

Abstract

EVs are connected to onset and progression of numerous diseases and can be used for their diagnosis. Methods and platform technology to isolate EVs and detect intravesicle phosphoproteins from biofluids are discussed. Secreted extracellular vesicles in biofluids are effectively isolated and phosphoproteins are extracted for detection and identification as biomarkers for disease diagnosis using antibodies, mass spectrometry, or other like methods.

Description

METHODS TO ISOLATE EXTRACELLULAR VESICLES IN BIOFLUIDS FOR BIOMARKER DISCOVERY AND DISEASE DIAGNOSIS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a PCT international patent application which claims the benefit of U.S. provisional patent application serial number 62/238,693, filed October 7, 2016, the disclosure of which is expressly incorporated by reference.

TECHNICAL FIELD

[0002] This disclosure is directed to materials and methods to isolate extracellular vesicles (EVs) in biological fluids.

BACKGROUND

[0003] Disease biomarker discovery and validation is a highly promising task. This stands particularly true for proteomics platforms, and even more so for biomarkers of post-translational modifications. Discovery-based approaches, e.g. proteomics to identify important disease biomarkers from biofluids, have met enormous challenges. Clinically useful protein biomarkers can provide earlier and better diagnosis for more effective treatments, improved prognosis for patient monitoring, and preventative screening that can identify patients at highest risk and subsequently offer intervention.

"Liquid biopsies" - analysis of biofluids such as plasma, serum, urine, tears, saliva - have gained much attention as a potentially useful source of diagnostic biomarkers.

[0004] For example, blood plasma or blood serum is a particularly attractive sample source for the detection of disease biomarker candidates due to its easy accessibility and presence of measurable tissue- derived proteins that hold potential to uncover physiological and pathological changes during diseases. To overcome problems of plasma complexity, some of the recent developments in biofluid analysis include isolation, detection and profiling of cell-secreted extracellular vesicles (EVs).

[0005] EVs are membrane-enveloped vesicles produced in an evolutionally conserved fashion by a variety of cells found in different organisms and species. The major role of microvesicles is their ability to transfer information from the original cell to other cells using different classes of molecules. In humans, EVs can be found in all bodily fluids and sometimes in intercellular regions. Their size ranges between 20 run and 1000 nm.

[0006] The term EV typically includes smaller size exosomes derived from multivesicular endosome-based secretions, and microvesicles derived from plasma membrane. EVs are an effective and ubiquitous method for intercellular communication and removal of excess materials. As EVs are shed by cells of all types into virtually every type of biological fluid, EVs embody a good representation of their parent cells. Analysis of the EV cargo has a great potential for biomarker discovery and disease diagnosis.

[0007] Recently, the interest in studying EVs increased because they are thought to play a role in metastasis and angiogenesis. They can also serve as drug targets and carriers in addition to diagnostic tools for cancer and other diseases. Collecting fluids containing EVs is non-invasive. Currently used methods of isolating EVs include ultracentrifugation, flow cytometry, and antibody-based approaches.

[0008] Multiple publications have analyzed exosomes and microvesicles for DNA, RNA and protein content, finding molecular effectors fully active even after delivery to the target cells.

Interestingly, researchers have also found many differentiating characteristics of the cancer cell-derived cargo, including gene mutations and active miRNA molecules, which possess metastatic properties. Particularly promising are the findings that these EV-based disease markers can be identified well before the onset of symptoms or physiological detection of a tumor, making them a promising candidate for early-stage cancer and other disease detection.

[0009] Designing a non-invasive, early cancer detection method is of great importance. An effective testing method should save time, reduce the cost and the risks involved in the currently -applied cancer diagnosis and prognosis approaches. It should also be applied to other forms of disease by exploiting the different EV populations in a patient's blood or other fluids.

[0010] References discuss analysis of exosomes and microvesicles for intravesicle components, such as DNA, RNA and proteins. As phosphorylation is a major player in cancer and other disease progression, EV phosphoproteins are active targets as indicators of cellular states and for in vitro disease diagnosis.

[0011] US Patent No. 9,081,012 to Park, et al. details a porous polymer monolith filter method and apparatus to isolate extracellular vesicles from body fluid.

[0012] PCT Published Patent Application WO/2015/139019 to He et al. discloses lysis and in situ analysis of exosomes using a microfluidic microfluidic device.

[0013] U.S. Patent No. 8,501,486 to Tao details a polymer-based metal ion or metal oxide capturing (Poly MAC) reagent suitable for the capture of phosphopcptides from mixtures. U. S. Published Patent Application 2013/0095502 to Tao et al. describes a reagent for the detection of phosphorylated molecules (pIMAGO).

SUMMARY OF INVENTION

[0014] Methods to identify and detect phosphoproteins in biological fluids.

[0015] The first method includes isolation of EVs, exosomes or microvesicles. Intravesicle components like RNA, DNA, proteins are then extracted from the EVs, exosomes or microvesicles. The extract will likely include a variety of modified proteins. Finally, affinity -based detection with specific phospho-antibodies, aptamers or chemical detection such as small molecules/nanopolymers (e.g.

pi MA GO, etc.) is used to detect the phosphoproteins in the extract.

[0016] The biological matrices include plasma, serum, cerebrospinal fluid, saliva, interstitial fluid, urine, rectal discharge and tears. BRIEF DESCRIPTION OF DRAWINGS

[0017] The above-mentioned and other features of this disclosure, and the manner of attaining them, will become more apparent and the disclosure itself will be better understood by reference to the following description of embodiments of the disclosure taken in conjunction with the accompanying drawings, wherein:

[0018] Figures 1 and 2 are schematics of a microfluidic device including a microchannel used to capture the EVs.

[0019] Figure 1A illustrates a cross sectional top view of a polymer previously defined with channel inlet, channel outlet and larger microchannels.

[0020] Figure IB illustrates a top view of material deposited on a flat polymer (glass layer not shown for clarity).

[0021] Figure 1C illustrates a top view of the polymer of Figure 1 A conformed to the material of Figure IB.

[0022] Figure ID illustrates a top view of a microfluidic device according to an embodiment of the present disclosure including glass layer and proposed dimensions for this embodiment.

[0023] Figure IE illustrates a cross sectional side view of the microfluidic device of Figure ID according to an embodiment of the present disclosure including glass layer and proposed dimensions for this embodiment.

[0024] Figure IF illustrates a cross sectional side view of the microfluidic device of Figure ID according to an embodiment of the present disclosure including glass layer and illustrating syringe and power supply.

[0025] Figure 2A illustrates a cross sectional side view of a microfluidic channel according to Figures 1 A - 1C.

[0026] Figure 2B illustrates the microfluidic channel of Figure 2A with large and small microspheres packed in the larger microchannel.

[0027] Figure 2C illustrates the microfluidic channel of Figure 2B with trapped extracellular vesicles.

[0028] Figure 2D illustrates the microfluidic channel of Figure 2C after release of intravesicle components from trapped EVs by applying pulse high voltage.

[0029] Figure 3A illustrates a top view of a microfluidic device according to an embodiment of the present disclosure. [0030] Figure 3B illustrates a cross sectional side view of the microfluidic device of Figure 3 A according to an embodiment of the present disclosure.

[0031] Figure 3C illustrates an end view of the microfluidic device of Figure 3A according to an embodiment of the present disclosure.

[0032] Figure 4A shows a chart of CD31 -labeled EV accumulation in a microfluidic device according to an embodiment of the present disclosure.

[0033] Figure 4B shows CD31 -labeled extracellular vesicles zero minutes after injection in the microfluidic device of Figure 4A following column packing. Pictures are taken on EVOS FL microscope with Texas Red filter and quantified using ImageJ software.

[0034] Figure 4C shows CD31 -labeled extracellular vesicles three minutes after injection in the microfluidic device of Figure 4A following column packing. Pictures are taken on EVOS FL microscope with Texas Red filter and quantified using ImageJ software.

[0035] Figure 4D shows CD31 -labeled extracellular vesicles six minutes after injection in the microfluidic device of Figure 4A following column packing. Pictures are taken on EVOS FL microscope with Texas Red filter and quantified using ImageJ software.

[0036] Figure 4E shows CD31 -labeled extracellular vesicles nine minutes after injection in the microfluidic device of Figure 4A following column packing. Pictures are taken on EVOS FL microscope with Texas Red filter and quantified using ImageJ software.

[0037] Figure SA1 shows CD31 -labeled extracellular vesicles after injection in a microfluidic device following column packing according to an embodiment of the present disclosure. Pictures are taken on a Nikon i90 microscope using a Texas Red filter before the application of 1 pulse of high- voltage electricity.

[0038] Figure 5 A2 shows CD31 -labeled extracellular vesicles after injection in the microfluidic device of Figure 5A1. Pictures are taken on a Nikon i90 microscope using a Texas Red filter after the application of 1 pulse of high-voltage electricity.

[0039] Figure SB1 shows CD31 -labeled extracellular vesicles after injection in a microfluidic device following column packing according to an embodiment of the present disclosure. Pictures are taken on a Nikon i90 microscope using a Texas Red filter before the application of 1 pulse of high- voltage electricity.

[0040] Figure SB2 shows CD31 -labeled extracellular vesicles after injection in the microfluidic device of Figure 5B1. Pictures are taken on a Nikon i90 microscope using a Texas Red filter after the application of 1 pulse of high-voltage electricity. [0041] Figure SCI shows CD31 -labeled extracellular vesicles after injection in a microfluidic device following column packing according to an embodiment of the present disclosure. Pictures are taken on a EVOS FL microscope using a Texas Red filter before the application of 1 pulse of high- voltage electricity.

[0042] Figure 5C2 shows CD31 -labeled extracellular vesicles after injection in the microfluidic device of Figure 5C1. Pictures are taken on a EVOS FL microscope using a Texas Red filter after the application of 1 pulse of high-voltage electricity.

[0043] Figure 6A illustrates a side view of a capillary device according to an embodiment of the present disclosure.

[0044] Figure 6B illustrates a top view of the capillary device of Figure 6A according to an embodiment of the present disclosure.

[0045] Figure 7 illustrates a schematic analysis procedure to isolate and identify EV

phosphoproteome.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0046] The embodiments disclosed below are not intended to be exhaustive or limit the disclosure to the precise forms disclosed in the following detailed description. Rather, the embodiments are chosen and described so that others skilled in the art may utilize their teachings.

[0047] While this disclosure has been described as having an exemplary design, the present disclosure may be further modified within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses, or adaptations of the disclosure using its general principles. Further, mis application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this disclosure pertains.

[0048] It has been previously demonstrated mat bacterial cells with diameters at ~ 1 μπι could be physically trapped by packing microspheres inside a microfluidic device. EV diameters range from 20 nm to 1 μιτι. Therefore a microfluidic device including microfluidic channels can be designed and constructed with the pores of similar sizes based on the spaces between the microspheres accumulated in the microchannel.

[0049] This disclosure is an integrated platform technology to effectively isolate, enrich and detect EVs, exosomes, and intravesicle components such as DNA, RNA, proteins and specifically

phosphoproteins in high- or low-throughput manner from biological matrices. High throughput is multiple simultaneous analysis (-100 samples in one hour) versus low throughput being described as analysis of one sample at a time. The biological fluids may include plasma, serum, urine and cerebrospinal fluid. The underlying principle of the method includes an efficient and convenient way to isolate exosomes, EVs, microvesicles, or a combination thereof and in situ extraction of the isolated intravesicle components. An additional principle of the method includes detection of the intravesicle components such as phosphorylated target proteins by antibodies, aptamers, small molecules, or enrichment of the comprehensive phosphoproteome followed by mass spectrometry.

[0050] A microfluidic device including microfluidic channels may be fabricated according to previous protocols of soft lithography utilizing polydimethylsiloxane (PDMS).

[0051] Figures 1 - 4 illustrate a microfluidic device forming a microchannel according to embodiments of the present disclosure. The sizes disclosed in Figure 1 are exemplary. Briefly, the patterns of microchannels may be designed using drawing software, such as Adobe Illustrator, and printed on a transparency film with a resolution of 5080 dpi. The transparency is then used as the photomask in photolithography for the fabrication of a master on a silicon wafer. Smaller microscale objects having a height of about 3 - 4 um are deposited on a flat surface in order to create an imprinting surface (Figure IB). It is envisioned that smaller microscale objects may have any height within the range of about 1 um to about 5 um. The structure of reduced-depth and normal microchannels may be fabricated separately with photoresists of SU-82002 and AZ 2025 (Clariant, Somerville, NJ, USA), respectively, on a silicon wafer to create two depths (2 and 40 um, respectively).

[0052] A polymer, such as PDMS (DOW Coming sylgard-184), (Figure 1A) is placed on top of the imprinting surface and allowed to conform to the imprinting surface. The polymer mixture of the monomer and the curing agent with the mass ratio of 10: 1 may be poured on the master and then heated at the temperature of 80 °C for 1.5 h. The conformed polymer already defines relatively large microscale channels having a height of about 60 um. It is envisioned that relatively large microscale channels may have any height within the range of about 10 um to about 100 um. After the polymer sets, the conformed polymer defines larger microchannels 2 and defines smaller microchannels 3 in the shape of the imprinted surface (Figure 1C). The conformed polymer is removed from the imprinting surface. Larger microchannels 2 of bigger depth have a height of approximately 60 um and smaller microchannels 3 of smaller depth have a height of -3-4 um. The peeled PDMS stamp (approximately 5 mm thick) with structures of the microchannels at the surface is then punched to generate two access holes, one will serve as channel inlet 1 and the other as channel outlet 5 (Figure 2A).

[0053] The peeled-off PDMS layer may then be treated with oxygen plasma and combined with a clean glass slide 11 (Figures 3A - 3C) to form a PDMS micromicrofluidic device with designed structure. Glass slide 11 has been pre-cleaned in (H20: NH40H (27%):H202 (30%) = 5:1:1, volumetric ratio) solution with stirring at around 75 °C for an hour and then rinsed with Dl water and dried. The PDMS microfluidic device and the glass slide are oxidized using an Oxygen Plasma Cleaner and then immediately put into contact to form the PDMS/glass microfluidic device according to embodiments of the present disclosure. [0054] Each microfluidic device may contain 16 channels where each channel includes inlet 1 and outlet 5. Materials are inserted into channel inlet 1 using a tube attached to a glass syringe (lmL BD glass syringe Luer-Lock tip) operated by a pump (Harvard Apparatus PHD2000) that has refill and infuse options. After protocol optimization, we establish loading quantities of beads and sample and how to load mem into the microfluidic device. A first syringe is filled sequentially with 400 uL of PBS, 40-60 uL of air plug, 2-5 uL of 3.98 um beads (wherein the 3.98 um beads are within the range of 3 - 5 um), 2-10 uL of air plug, 1-2 uL of 3.17 um beads (within the 3.17 um beads are within the range of 2-4 um), 2-10 uL of air plug, and 4 uL of 6.46 um beads (wherein the 6.46 um beads are within the range of 5-7 um). It is envisioned that the beads could be in above ranges and/or any combination thereof. Beads are purchased from Bangs Laboratories and diluted 10 times in PBS. It is envisioned mat the beads could be in the range of approximately 2 to approximate!}' 8 um, approximately 2 to approximately 4 um, approximately 4 to approximately 6 um, approximately 6 to approximately 8 um, or any combination thereof. A platinum wire is embedded in second syringe to permit the passage of the electric field after sample application. A second syringe is filled respectively with 400 uL of PBS and 20 uL of 1/10 diluted plasma sample.

[0055] First, first syringe is connected to channel inlet 1 and the pump is turned on at a flow rate of 5 uL/min. Mechanical force is applied to the microfluidic device to help the beads pack more uniformly. It is also envisioned mat an air plug will stabilize the packed beads and prevent them from flowing backwards out of the channel even if inverted. After having loaded all the beads, second syringe is applied to the inlet. Sample is injected at a flow rate of 5 uL/min. It is envisioned that the flow rate could be in the range of approximately 2 to approximately 20 uL/min.

EV ISOLATION

[0056] In general, the microfluidic device and its mode of action would include the following components and process steps: introduction of microspheres 6 in larger microchannel 2 of microfluidic device such that reduced size microchannel 3 leads to capture of microspheres 6. Packed microspheres 6 are tuned in size to capture EVs.

[0057] In one embodiment, microspheres 6 will flow into the normal microchannel and would be blocked by smaller microchannel 3. In another embodiment, larger microspheres 6 can be initially used to block smaller microchannels 3 and allow for use of microspheres 6 smaller than the height of smaller microchannels 3. In yet another embodiment, combinations of microspheres 6 of different diameters may be used to tune the capture EVs. The pore sizes can be modified using microspheres 6 with different diameters. The spacing between microspheres 6 may be carefully adjusted to trap EVs while removing the extravesicle components, such as proteins and small molecules in the biological fluid.

[0058] A microfluidic device according to an embodiment of the present disclosure may be used to capture and isolate EVs from biological fluid. FIG. 2 summarizes the process. As illustrated in Figure 2B, when microspheres 6, having a diameter of at least 3 um, are placed in channel inlet 1 of larger microchannel 2 (Figure 2A), microspheres 6 are effectively blocked by smaller microchannels 3 while fluid and extravesicle components, such as proteins in fluid, are small enough to pass through smaller microchannels 3 and flow out of channel outlet 5.

[0059] As previously described, microspheres 6 are packed into larger microchannel 2. A biological sample including fluid, free proteins in fluid and EVs 7 including proteins, is passed through packed microspheres 6. The combination of smaller microchannel 3 and microspheres 6 do not allow EVs 7 to pass based on EVs 7 size range from 20 nm to 1 um (Figure 2C). EVs 7 in the biological fluid are trapped by microspheres 6 as the rest of the sample flows out of channel outlet 5.

[0060] Blood Plasma EV isolation Example

[0061] We use blood plasma as one example to describe the procedure.

[0062] 1 mL aliquots of a plasma blood sample are thawed and centrifuged at 20,000 g for 30 minutes. Supernatant is collected to a new tube and is suspended as a pellet in 300 uL of phosphate buffered saline (PBS). EVs are labeled with microvesicle marker endothelial anti-human CD31 (available from Biolegend at www.biolegend.com/pe-anti-human-cd31-antibody-882.html) in dark for 20 mins. Labeled EVs sample is centrifuged at 20000g for 30 mins. Supernatant is discarded and plasma is added to collected pellet. Sample is diluted 10 times with PBS.

[0063] Following the collection of the relevant biofluid (e.g. removal of blood cells for plasma use), EVs are isolated by a microfluidic device according to an embodiment of the present disclosure. As second membrane or microsphere accumulation with larger diameter holes can be used in front of the device to collect larger blood cells when using blood directly, allowing for EVs to pass before being captured and concentrated as described above.

[0064] After packing the column with microspheres, we apply the labeled EVs sample to the microfluidic device including the microchannel having microspheres. Pictures are taken every 20 seconds for 10 mins. Figs. 4A - 4E show gradual increase of red fluorescent signal. Red fluorescence signal reaches a plateau after approximately 8 mins. The plateau indicates a gradual accumulation of extracellular vesicles in the beads. These results show that 3.98 um beads can isolate extracellular vesicles in the microfluidic device and separate them from other blood plasma components.

[0065] Alternatively to a microfluidic device including trapping microspheres, EVs may be isolated by centrifugation (ultracentrifugation for exosomes), precipitation of vesicles, or antibody enrichment of EVs based on the EVs surface proteins. If necessary, differential centrifugation can be carried out to separate microvesicles from exosomes (lower speed for microvesicle collection and higher

ultracentrifugation speed for exosome collection). Otherwise, high speed can be used to collect both types of EVs. Either of the methods listed is designed to separate the EVs from small molecules, proteins and other components present in a biofluid (this step is particularly vital for plasma/serum analysis). EXTRACTION OF INTRA VESICLE COMPONENTS FROM EV

[0066] Following collection of the EVs in the microchannels on a microfluidic device, proteins/phosphoproteins and other materials inside the concentrated EVs are extracted by applying pulsed electric field or other approaches on the microchannel or outside. (Figure 2D)

[0067] Following a continuous PBS flow of 5 mins to wash away all untrapped molecules and particles, a high-voltage power supply is connected to the microfluidic device. A high voltage power supply is operated by computer software (Lab VIEW 2015) to generate a 1100V pulse for 1 second every 9 seconds. This power supply is also connected to the inlet through the platinum wire in syringe 2 and to the outlet by another platinum wire. Once the power supply gets a signal, the current may go through the channel. (Fig. 3) A current of 1100V may go through the channel inducing tysing or at least electroporation of the captured EVs.

[0068] While a 1100 V pulse is utilized, it is envisioned that an approximately 500 V to approximately 1.5 KV pulse would accomplish a similar result. While 1 pulse is utilized, it is envisioned that multiple pulses, such as no more than thirty pulses, no more than twenty pulses, or no more than ten pulses, may accomplish a similar result even at a lower voltage per pulse. While a pulse lasting one second is utilized, it is envisioned that pulses of different lengths of time, such as no more than three second, no more than two seconds, or no more than ten seconds, may accomplish a similar result even at a lower voltage per pulse or with fewer pulse cycles. Finally, while a ten second pulse cycle is utilized, it is envisioned that pulses cycles of shorter, longer, or varying length, such as approximately five second pulse cycles, approximately fifteen second pulse cycles, or combinations thereof, may accomplish a similar result of either lysing or electroporation of EVs.

[0069] As illustrated in Figures 4 and 5 as one of many extraction techniques, pulsed high voltage is applied across the microfluidic device, such as large microchannels 2 and 4, by electrodes 8 to lyse EVs or at least electroporate or release intravesicle components, such as proteins, out of EVs. Proteins are extracted from the EVs, exosomes or microvesicles by applying pulsed electric field or other approaches in the microchannel or outside of the microchannel.

[0070] Fluorescence was detected using Texas Red filter on EVOS FL microscope or Nikon i90 microscope. Fluorescence quantification was analyzed on ImageJ software and compared to time 0 mins.

[0071] Once the EVs are lysed or electroporated, the fluorescent signal disappears and the vesicles release their content into the channel (Figs. 4 and 5). The content of those particles can go through the beads reaching channel outlet 5 where it can be collected for further analysis.

[0072] Other approaches include protein extraction with urea, thiourea, NP-40, Tween20, Triton-X, SDS or any other detergents capable of disrupting the lipid membrane of EVs 7. An advantage of using a pulsed electric field is that no additional reagent is needed and thus no additional procedure is required to remove the reagents. ALTERNATIVE CAPILLARY EMBODIMENT

[0073] An alternative embodiment is illustrated as Figures 6A and 6B. Similar components of a microfluidic channel device are given die same reference character. Capillary 20 may be fabricated by joining a plastic or polymer tube 13 as a holder for a capillary IS containing a microchannel 16 having a channel diameter of 75 um to about 1000 um. Figures 6A and 6B illustrate the construction showing the resulting capillary containing space for an array of beads 18 or microspheres 18 appropriately sized and packed within microchannel 16. The packed bead array 18 simulates a variably restrictive microporous flow-through aperture with the restrictive porosity determinable by the chosen diameter of the beads 18 in the array with a frit 17 or mesh filter 17 at the posterior end of channel outlet 14 securing beads 18 while allowing fluid flow. As in other embodiments, the spacing between the beads, number type, shape and different sizes of beads 18 may be carefully adjusted to trap the EVs while releasing the proteins and small molecules in the flow-through biological fluid.

DETECTION OF PHOSPHOPROTEINS FROM EV

[0074] A short high voltage pulse may be generated to ryse the EVs and extract intravesicle components including phosphoproteins for subsequent collection and analysis with MS, ELISA, microarrays or other methods such as Poly MAC, pIMAGO or International Publication Number WO 2016/144843 Al, titled Chemically functionalized array to analyze protein modifications, the subject matter of each are expressly incorporated by reference.

[0075 J Released proteins 9 flow through smaller microchannels 3 where released proteins 9 are collected in channel outlet 5 (Figure 3) or eluted out of channel outlet 14 (Figure 6) for further analysis. As alternatives to smaller microchannel 3, filter-like membrane, centrifugation, precipitation, or antibody enrichment can be used to isolate EVs 7. Exosomes can also be isolated by ultracentrifugation.

Differential centrifugation can be used to separate microvesicles from exosomes.

[0076] The extracted intravesicle components, such as phosphoproteins, as well as other post translational modifications (PTMs) such as glycoproteins, are directly analyzed, or affinity purified with specific phospho-antibodies, aptamers or small molecules/nanopolymers (e.g. pIMAGO, etc.), for both biomarker discovery and diagnostics purposes. For a more convenient procedure, the detection may be carried out directly on the microfluidic device (in situ) following EV capture protein extraction.

Individual phosphoproteins and a panel of proteins and phosphoproteins extracted from EV are measured as the indication of disease status.

[0077] For unbiased discovery studies analysis, the extracted proteins can be digested with a protease or a combination of proteases, and the phosphopeptides enriched using metal-ion based methods (PoryMAC, IMAC, TiC¾, ZrQz, AI2O3, SnO_, etc.), antibodies (pTyr antibody Immunoprecipitation), aptamers, or chromatography -based separation (HILIC, ERLIC, SCX, SAX, etc.). Eluted phosphopeptides can be analyzed and identified by LC-MS or other similar methods (a simplified representation of the approach is shown in Figure 7).

[0078] The described procedure is presented as particularly useful for biomarker discovery studies and/or diagnostic assays using protein phosphorylation as a marker. A preliminary study using lmL of human plasma sample from a breast cancer patient using the described procedure with LC-MS/MS analysis identified >5,000 phosphopeptides from >1,500 unique phosphoproteins with 1% false discovery rate. These data are a significant improvement over any previous studies while using much less starting material, representing a revolutionary method for discovery and detection of important phosphoproteins in biofluids for disease diagnosis.

Claims

1. A device comprising:

a conformed polymer defining smaller microchannels having a height within the range of about 1 um to about 5 μιη and defining larger microchannels having a height within the range of about 10 um to about 100 um,

wherein the conformed polymer defines smaller microchannels by placing the conformed polymer on deposited material and the smaller microchannels are formed by the conforming polymer conforming to the deposited material,

a plurality of microspheres located in one of the larger microchannels, the plurality of microspheres trapped by the smaller microchannels, and

a series of electrodes coupled to the larger microchannels, wherein the electrodes provide for a pulsed current to traverse the microspheres.

2. The device of claim 1 wherein the plurality of microspheres include at least two groups of microspheres having different diameters.

3. A device comprising:

a capillary tube, and a polymer tube,

wherein the polymer tube is a holder for the capillary tube,

wherein the capillary tube defines a microchannel having an effective inner diameter within the range of about 75 um to about 1000 um and,

wherein the capillary tube has an outer diameter essentially the same as the inner diameter of the polymer tube,

wherein the capillary tube is adjoined to the polymer tube,

wherein the capillary tube contains a frit,

wherein a plurality of microspheres are located in the capillary tube, the plurality of microspheres trapped by the frit, and

a series of electrodes coupled to the capillary, wherein the electrodes provide for a pulsed current to traverse the microspheres.

4. A method to isolate extracellular vesicles from biological fluid comprising the steps of:

providing packed microspheres sized to physically trap extracellular vesicles, and

isolating extracellular vesicles, exosomes or microvesicles from biological fluids.

5. The method of claim 4 wherein the biological fluid is selected from the group consisting of plasma, serum, urine, cerebrospinal fluid and tears.

6. The method of claim 4 wherein high throughput analysis is used to analyze multiple samples.

7. A method of claim 4 comprising the steps of:

introducing the biological fluid including free proteins in fluid and EVs including intravesicle proteins among the packed microspheres.

8. A method to isolate and detect intravesicle components from biological fluid comprising the steps of:

isolating the EVs, exosomes or microvesicles from biological fluids,

in situ extraction of intravesicle components from the isolate;

followed by enrichment of the intravesicle components by metal-ion based methods, antibodies, aptamers or chromatography -based separation, followed by detection.

9. The method of claim 9 further comprising the step of applying a pulsed high voltage across microspheres in order to extract proteins out of EVs.

10. The method of claim 10 wherein the pulsed high voltage is a single one second long 1100 V pulse

11. The method of claim 10 wherein the pulsed high voltage is within the range of approximately 500 V to approximately 1.5 KV

12. The method of claim 10 wherein the pulsed high voltage includes multiple pulses.

13. The method of claim 10 further comprising a pulse cycle is selected from the group consisting of a ten second pulse cycle, an approximately five second pulse cycle, an approximately fifteen second pulse cycle, or combinations thereof.

14. The method of claim 18 further comprising collecting proteins released from EVs, wherein the proteins flowed through smaller microchannels.