Micro-nanofluidic systems for extracellular vesicles

Abstract

The present disclosure relates to a microfluidic system for collecting, extracting, harvesting, isolating, and / or analyzing nucleic acids from extracellular vesicles (EVs). The present disclosure also relates to a microfluidic system that is further coupled to an isolation chamber, a post-processing device, or a combination thereof. The microfluidic system disclosed herein is used for detection of liquid biopsy biomarkers for cancer diagnostics, infectious disease diagnostics, drug discovery, and biomarker analysis.

Description

[0001] Docket No. 103362-085WO1 MICRO-NANOFLUIDIC SYSTEMS FOR EXTRACELLULAR VESICLES STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with Government Support under Grant No. W81XWH-22-1-0531 awarded by the United States Department of Defense. The Government has certain right in the invention.

[0003] RELATED APPLICATION

[0004] This PCT application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63 / 778,762, filed March 27, 2025, entitled “MICRO-NANOFLUIDIC SYSTEM FOR EXTRACELLULAR VESICLES,” which is incorporated by reference herein in its entirety.

[0005] FIELD

[0006] The present disclosure relates to microfluidic systems for collecting, extracting, harvesting, isolating, and / or analyzing nucleic acids from extracellular vesicles (EVs).

[0007] BACKGROUND

[0008] Microfluidics is an emerging field based on the combined principles of physics, chemistry, biology, fluid dynamics, microelectronics, and material science. Various materials are processed into devices made of miniaturized chips containing micro- or nano-sized channels and chambers employed for molecular separation, drug encapsulation, drug delivery, drug targeting, cellular analytics, and cell culture. Such devices are useful instruments for carrying out operations including chemical reactions, molecular separations, and detection of various compounds. However, many devices are limited to being designed to perform one function, such as separation, analytics, or detection. Therefore, what is needed in the field is a device that integrates more than one function onto a single chip.

[0009] The devices and methods of the present disclosure address these needs and other needs.

[0010] SUMMARY

[0011] The present disclosure provides an integrated micro-nanofluidic system for collecting, extracting, harvesting, isolating, and / or analyzing cargo from EVs, including but not limited to nucleic acids (such as for example DNA and / or RNA). The present disclosure provides methodsDocket No. 103362-085WO1 using the integrated micro-nanofluidic system to detect a nucleic acid biomarker and / or treat a subject having a disease or disorder.

[0012] In one aspect, disclosed herein is a micro-nanofluidic system for collecting, extracting, and isolating an extracellular vesicle (EV) cargo from an EV sample, wherein the system comprises four or more fluidly connected components comprising a concentrator, a first microchannel, a second microchannel, and a capture channel, and wherein the concentrator, the first microchannel, the second microchannel, and the capture channel are fluidically connected to allow transport of the EV sample from one component to a second component.

[0013] In some embodiments, the concentrator comprises a first electric field for separating the EV sample into at least two groups. In some embodiments, the concentrator separates EVs based on size of EVs. In some embodiments, a first group of EVs is separated into a storage unit. In some embodiments, a second group of EVs is directed into the first microchannel. In some embodiments, the second group of EVs are further enriched by immunoaffmity capture comprising a surface-immobilized antibody including, but not limited to an anti-CD63 antibody, an anti-CD81 antibody, or a combination thereof.

[0014] In some embodiments, the first microchannel comprises a second electric field for transporting the second group of EVs into the center of the first microchannel. In some embodiments, the second microchannel comprises a lysis buffer and a proteinase K for lysing open the second group of EVs. In some embodiments, the capture channel comprises one or more micropillars for selectively capturing the EV cargo from the second group of EVs. In some embodiments, the EV cargo comprises a nucleic acid, peptides, or other cellular components.

[0015] In some embodiments, the system is further connected to a EV-cargo isolation chamber. In some embodiments, the EV-cargo isolation chamber comprises a EV-DNA isolation chamber. In some embodiments, the system is further connected to a post-processing device (such as for example a thermal cycler, a PCR device, a biosensor, a microarray, a sequencer, a liquid chromatographer, a mass spectrometer, or any combinations thereof). In some embodiments, the EV cargo is collected in a collection container prior to entering the post-processing device. In some embodiments, the EV cargo is immediately directed into the post-processing device.

[0016] In some embodiments, the EV is extracted from a biological sample selected from tissue, cells, sweat, blood, urine, tears, nasal mucus, saliva, and / or related fluids. In some embodiments, the system is used for detection of liquid biopsy biomarkers for cancer diagnostics, infectious disease diagnostics, drug discovery, biomarker analysis, or a combination thereof.Docket No. 103362-085WO1 In some aspects, disclosed herein is a method of detecting a nucleic acid biomarker, the method comprising obtaining a biological sample; inserting the biological sample into the micro-nanofluidic system of any preceding aspect, wherein said system extracts at least one EV sample from other contents in the biological sample, and wherein said system further separates and isolates an EV cargo from the EV sample; and detecting a biomarker within the EV cargo, wherein the biomarker is a signature of a disease or disorder.

[0017] In some aspects, disclosed herein is a method of treating a subject in need thereof, the method comprising obtaining a biological sample from the subject; inserting the biological sample into a micro-nanofluidic system of any preceding aspect, wherein said system extracts at least one EV sample from other contents in the sample, and wherein said system further separates and isolates an EV cargo from the EV sample; detecting a biomarker (such as, for example detecting one or more biomarkers) within the EV cargo, wherein the biomarker is a signature of a disease or disorder; and administering to the subject a therapy when the biomarker is detected or the biomarker levels are significantly increased compared to a subject having a healthier condition.

[0018] In some embodiments, the biomarker includes, but is not limited to MDM2, CDK- miR-16-5p, miR-20a-5p, miR-25-3p, miR-92a-3p, miR-93-5p, or a combination thereof.

[0019] In some embodiments, the disease or disorder comprises a cancer including, but not limited to liposarcoma (LPS) cancer, prostate cancer, and bladder cancer. In some embodiments, the biological sample comprises tissue, cells, cell conditioned media, sweat, blood, urine, or saliva.

[0020] In some embodiments, the healthier condition comprises a healthy state or a state of health after receiving therapy. In some embodiments, the cancer therapy comprises an immunotherapy, chemotherapy, radiation therapy, surgery, or any combinations thereof. In some embodiments, the system is used for detection of liquid biopsy biomarkers for cancer diagnostics, infectious disease diagnostics, drug discovery, and biomarker analysis.

[0021] BRIEF DESCRIPTION OF FIGURES

[0022] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0023] Figures 1A and IB show the initial setup of the micro-nanofluidic device. Figure 1A shows the schematic of the operating conditions of the microfluidic device with the integrated nanocapillary array membrane (NCAM). Figure IB shows the fabricated device with injectionDocket No. 103362-085WO1 and separation channel highlighted using food color Dye. Magenta (Injection channel), and Green (Separation Channel).

[0024] Figures 2A, 2B, 2C, and 2D show an enhanced iteration of the micro-nanofluidic device by integrating an electric field across a membrane to improve EV isolation. Figure 2A shows the schematic of the microfluidic device with the integrated nanocapillary array membrane (NCAM), featuring gold electrodes for applying the electric field across the injection and separation channels. Figure 2B shows that at 100 V, overheating and media browning were observed, indicating compromised sample integrity. Figures 2C and 2D show the EV capture efficiency (measured as total DNA yield) and purity ratio at various applied voltages. Capture efficiency increased with voltage, peaking at 50 V before declining at 70 V due to potential EV damage.

[0025] Figures 3A, 3B, and 3C show the optimization of the multi-layer microfluidic device. Figure 3A shows the schematic of the operating conditions of the multi-channel microfluidic device with the integrated nanocapillary array membrane (NCAM). Figure 3B shows the CAD model schematic of the vertically integrated microchannels. Figure 3C shows the optical micrograph of the device showing inlet-outlet channel interface. Scale= 100 pm.

[0026] Figure 4 shows the total DNA quantification from LCCM-derived EVs isolated using our microfluidic device. Multiple concentrations and temperatures of glutaraldehyde (GA) were studied to optimize the performance of the device and maximize the isolated EV yield.

[0027] Figures 5 A, 5B, and 5C show the schematic of device with biofluid infusing in injection channel (top). Small EVs (sEVs; <200nm) and other smaller particles (like proteins) gets filtered through NCAM and enters the separation channels (bottom) where only sEVs gets captured by anti-antibodies mobilized on the wall of separation channels. Figure 5C shows the DiO-dye tagged EVs aggregates accumulated on top of NCAM post experiment.

[0028] Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, and 6H show the demonstration benefit of incorporating an electric field across the NCAM. Figures 6A and 6B show the total isolated EV-DNA, EV capture efficiency. Figure 6C illustrates the filtration of DiO-tagged EVs through NCAM. Regions DD’C’B,’ BCDD,’ and above AA’ denote separation channel, NCAM and injection channel, respectively. Figure 6D shows an increase in the fluorescence intensity in the separation channel (DD’C’B’) with time (frames) due to the inclusion of filtered EVs (via NCAM) in the separation channel. The intensity fluctuates initially as particles cross the NCAM and move through the channel, then saturate over time as the separation channel fills with EVs. Figure 6E shows the sample DNA purity (assessed via 260 / 280 absorbance). Figure 6F showsDocket No. 103362-085WO1 the illustrated TEM images showing electron-dense, cup-shaped EVs isolated via the device. For device, each data point contains pooled fluid from 4 devices, each contributing -180 pL of fluid per test run. Figures 6G and 6H show the EV capture efficiency (measured as total DNA yield) and purity ratio at various applied voltages. Capture efficiency and purity (260 / 280 absorbance ratio between 1.6-2) increased with voltage, peaking at 50 V before declining at 70 V due to potential EV damage.

[0029] Figure 7 shows the proposed design for on-chip DNA / RNA extraction.

[0030] Figure 8 shows the post-EV capture DNA extraction process flow.

[0031] Figure 9 shows the western blot results. Urine derived EVs expressed exosomal markers TSG101 and CD63 and did not express the cell marker calnexin. A smeared band for uromodulin was detected in isolated EVs sample.

[0032] Figures 10A and 10B show the NanoFCM-based characterization of EVs. Phenotypes of isolated (Figure 10 A) healthy donor urine and (Figure 10B) lipo246 cell line conditioned media derived EVs were tested. 500 ms of flow intensity traces for detecting EV particles were recorded in two channels, including size-scattering intensity (Blue; P2) and fluorescent intensity from PE-CD81 (Red; Pl), respectively. The size distribution histograms and scatter plots of the isolated EVs samples are shown.

[0033] Figures 11 A, 11B, 11C, 11D, HE, 1 IF, 11G, and 11H show the particle concentration and size analysis of urinary EVs isolated from healthy donors and LPS patients by Nano flow cytometry (NanoFCM). Figures 11 A, 11B, 11C, and HD show the NanoFCM analysis of urinary EVs isolated by differential centrifugation (10k pellet) showed a significantly higher EV particle concentration in LPS patients compared to healthy controls when normalized to both (Figure 11 A) urine volume and (Figure 1 IB) urinary creatinine (p < 0.005). While mean particle size (Figure 11C) showed no significant difference, (Figure 11D) median particle size was significantly larger in LPS patient samples (p < 0.05). For ultracentrifugation (UC) pellets, Figures HE, 1 IF, 11G, and 11H show that NanoFCM measurements revealed significantly higher EV concentrations in LPS patient samples compared to healthy donors when normalized to (Figure HE) urine volume and (Figure 1 IF) urinary creatinine (p < 0.005). No significant differences were observed in (Figure 11G) mean or (Figure 11H) median particle size between the two groups, indicating that the elevated EV yield in patients primarily reflects increased EV abundance rather than altered size distribution. **p < 0.01. Data reflects 19 LPS patient samples and 6 healthy donor samples.Docket No. 103362-085WO1 Figures 12A, 12B, 12C, 12D, 12E, and 12F show the quantification of EV-associated DNA from urinary EVs isolated centrifugation methods. Figures 12A, 12B, and 12C show that EV-DNA extracted from 10k pellet fractions using the QIAamp DNA Mini Kit (QIAGEN) and quantified with the Qubit dsDNA HS Assay Kit (Invitrogen). Figure 12A shows the mean EV-DNA concentration per mL of urine and Figure 12B shows the EV-DNA normalized to urinary creatinine (U-creatinine) levels were higher in LPS patient samples compared to healthy controls. Figure 12C shows the DNA density per EV, calculated as the total EV-DNA divided by the product of particle concentration and EV volume (4 / 37tr3, where r is the mean EV radius), showed a similar increasing trend in patient samples. Figures 12D, 12E, and 12F show the EV-DNA quantification from UC pellet fractions exhibited comparable patterns, with LPS patients showing higher (Figure 12D) EV-DNA concentration, (Figure 12E) creatinine-normalized EV-DNA, and (Figure 12F) DNA density per EV relative to healthy donors. Although differences were not statistically significant, consistent increases across both pellet types show elevated EV-associated DNA content in LPS patient urine.

[0034] Figures 13A, 13B, 13C, 13D, 13E, 13F, 13G, and 13H show the quantification of MDM2 and CDK4 molecules in urinary EV-DNA from healthy donors and LPS patients. Figures 13 A, 13B, 13C, and 13D show the quantitative PCR (qPCR) analysis of EV-DNA extracted from 10k pellet fractions showing significantly higher number of molecules of (Figure 13 A) MDM2 and (Figure 13B) CDK4 genes in LPS patient samples compared to healthy controls (p < 0.01). When expressed as (Figure 13C) MDM2 molecules / ng DNA and (Figure 13D) CDK4 molecules / ng DNA, representing the proportion of total EV-DNA corresponding to each gene, both markers remained significantly elevated in LPS patient samples (p < 0.01). Figures 13E, 13F, 13G, and 13H show the qPCR results from UC pellet fractions showed a similar pattern, with significantly higher (Figure 13E) MDM2 number of molecules (p < 0.05) and elevated (Figure 13F) CDK4 levels in LPS patient samples compared to healthy donors. When analyzed as a fraction of total EV-DNA, (Figure 13G) MDM2 remained significantly increased (p < 0.01), while (Figure 13H) CDK4 exhibited a consistent upward trend. Collectively, these results demonstrate enrichment of oncogenic MDM2 and CDK4 DNA fragments in urinary EVs from LPS patients, supporting their potential as non-invasive molecular biomarkers for liposarcoma. *p < 0.05, **p < 0.01.

[0035] Figures 14A and 14B show the NanoFCM-based surface protein phenotyping of uEVs. EVs were stained with PE-conjugated anti-CD81 and FITC-conjugated anti-CD63 antibodies. Figure 14A shows the CD81 analysis showed approximately 0.4% CD81+EV population.Docket No. 103362-085WO1 Figure 14B shows the CD63 analysis revealed approximately 1.8% CD63+EV population. These data confirm the presence of distinct tetraspanin-positive EV subpopulations in urine, which informed the selection of CD63 and CD81 antibodies for surface immobilization in the microfluidic EV capture device.

[0036] Figure 15 shows the comparison of EV-DNA yield between UC pellet EVs and deviceisolated EVs. EVs were isolated from LPS patient urine (n=ll) samples using both ultracentrifugation (UC) and the microfluidic EV isolation device. EV-associated DNA was extracted using the QIAamp DNA Mini Kit and quantified with the Qubit dsDNA HS Assay. The device-isolated EVs exhibited a markedly higher EV-DNA concentration (mean ~ 0.9 ng / mL urine) compared to UC pellet EVs (mean ~ 0.1 ng / mL urine), demonstrating the enhanced DNA recovery efficiency of the microfluidic device relative to conventional UC. ***p < 0.001.

[0037] Figures 16A, 16B, and 16C show the isolation protocol and characterization of saliva EVs. Figure 16A shows the modified urine-UC protocol for EV isolation from saliva. Figures 16B and 16C illustrate TEM images showing electron-dense, cup-shaped EVs isolated via UC.

[0038] Figures 17A, 17B, and 17C show the EV-DNA characterization. Figure 17A shows the total EV-associated DNA yield (ng / mL of saliva) in the 10 k and UC pellets for the three healthy saliva samples (HS). Figures 17B and 17C illustrates the number of molecules (MDM2 and CDK4) across the three healthy saliva samples (HS) for both the 10k and UC pellets.

[0039] Figures 18A and 18B show the EV-DNA comparison across biofluids. Figure 18A shows the total EV-associated DNA yield (ng / mL of biofluid) in the 10k and UC pellets for patients (LI and L3). Figure 18B illustrates the number of MDM2 molecules per mL of biofluid for both the 10 k and UC fractions.

[0040] Figure 19 shows the proposed microfluidic platform for on-chip DNA / RNA extraction. Figure 20 shows the integration of EV-DNA isolation chamber (enclosed in red box) with the exiting EV-isolation device (enclosed in black box).

[0041] Figure 21 shows the different versions of the EV-DNA isolation chamber for analysis. Figures 22A and 22B show the Spearman correlation analysis among the uEV-associated nucleic cargo levels in the LPS patient cohort for (Figure 22A) UC pellet uEVs and (Figure 22B) 10k pellet uEVs. Color represents Spearman coefficient and star denotes the significance value. *p < 0.05; **p < 0.01; ***p< 0.001.Docket No. 103362-085WO1 Figures 23 A and 23B show the differential expression of LPS-associated miRNAs in uEVs. Figure 23A shows the relative expression levels of selected miRNAs in uEVs isolated from device inlet-reject. Figure 23B shows the Ath spike-in concentration study.

[0042] Figure 24 shows a workflow diagram: biofluid (LCCM) is injected into the EV capture device to immunocapture the EVs from the population of the sub-200nm particles. The captured EVs (permeate EVs) are then sent to the DNA analysis module for on-chip EV lysis and DNA capture. The top row shows images of the fabricated devices, while the bottom row presents the corresponding schematics, including the dimensions of the serpentine and the micropillar array components of the DNA-analysis module. All dimensions are in mm.

[0043] Figures 25A, 25B, and 25C show the particle and DNA quantification. Figure 25A shows the Qubit DNA quantification of microfluidic device isolated LCCM-derived EV subpopulations for room temperature (RT) and 56°C lysis. Figures 25B and 25C show the particle concentration of permeate and flow-through EVs per mL of LCCM showing distinct sizes.

[0044] Figures 26A, 26B, and 26C show the lysis efficiency of the microfluidic device. Figure 26A shows the COMSOL simulation of the number of intact EVs (N) representative of the lysis efficiency of the serpentine channel, with Fluorescence microscopy images showing successful lysing and mixing of fluid streams in the serpentine channel (scale bar = 200pm). Figure 26B shows the lysis efficiency for EVs of different sizes at two different flow rates. Figure 26C shows the experimental evaluation of bench-top (BT) versus on-chip lysis (Device) performance for LCCM-EVs for 3 distinct samples showing inter-sample variability.

[0045] DETAILED DESCRIPTION

[0046] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof.Docket No. 103362-085WO1 Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0047] Terminology

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise.

[0049] The following definitions are provided for the full understanding of terms used in this specification.

[0050] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it isDocket No. 103362-085WO1 understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0051] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0052] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more increase so long as the increase is statistically significant.

[0053] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100%, or more decrease so long as the decrease is statistically significant.

[0054] "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.Docket No. 103362-085WO1 The term “cancer” is used to address any neoplastic disease and is not limited to epithelial neoplasms (surface and glandular cancers; such a squamous cancers or adenomas)). It is used here to describe both solid tumors and hematologic malignancies, including, but not limited to prostate cancer, urothelial cancers, epithelial (surface and glandular) cancers, soft tissue and bone sarcomas, angiomas, mesothelioma, melanoma, lymphomas, leukemias and myeloma.

[0055] As used herein, a “biomarker” refers to a measurable indicator of a normal biological process, a pathogenic process, or a pharmacological response to therapeutic interventions. Nonlimiting examples of a biomarker includes physiological indicators (such as blood pressure, heart rate, and / or blood glucose levels) and molecular indicators (such as proteins, nucleic acids, and / or metabolites) often found in tissues, blood, or urine.

[0056] A “nucleic acid” is a chemical compound that serves as the primary informationcarrying molecules in cells and make up the cellular genetic material. Nucleic acids comprise nucleotides, which are the monomers made of a 5-carbon sugar (usually ribose or deoxyribose), a phosphate group, and a nitrogenous base. A nucleic acid can also be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). A chimeric nucleic acid comprises two or more of the same kind of nucleic acid fused together to form one compound comprising genetic material.

[0057] A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.

[0058] Reference also is made herein to peptides, polypeptides, proteins, and compositions comprising peptides, polypeptides, and proteins. As used herein, a polypeptide and / or protein is defined as a polymer of amino acids, typically of length>100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). A peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length ofDocket No. 103362-085WO1 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110).

[0059] The term “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to interact with a chemerin polypeptide. The antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and / or prophylactic activities are tested according to known clinical testing methods. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. One skilled in the art would recognize the comparable classes for mouse. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

[0060] The terms “treat,” “treating,” and grammatical variations thereof as used herein, include partially or completely delaying, alleviating, mitigating or reducing the intensity of one or more attendant symptoms of a disorder or condition and / or alleviating, mitigating or impeding one or more causes of a disorder or condition. Treatments according to the disclosure may be applied preventively, prophylactically, palliatively or remedially. Treatments are administered to a subj ect prior to onset (e.g. , before obvious signs of tumor growth), during early onset (e.g. , upon initial signs and symptoms of tumor growth), or after an established development of a cancer.

[0061] As used herein, “fluidly connected” refers to a device or system with components that are connected by means that fluid is able to flow from one component to another with minimal restriction. In some embodiments, two or more components are not required to be physically connected to one another.

[0062] Disclosed are components that can be used to perform the disclosed methods and systems. These and other components are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these components are disclosed that while specific reference of each various individual and collective combinations and permutation of these may not be explicitly disclosed, each is specifically contemplated and described herein, for all methods and systems. This applies to all aspects of this application including, but not limited to, steps in disclosed methods. Thus, if there are a variety of additional steps that can beDocket No. 103362-085WO1 performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

[0063] As will be appreciated by one skilled in the art, the methods and systems may take the form of an entirely hardware embodiment, an entirely software embodiment, or an embodiment combining software and hardware aspects. Furthermore, the methods and systems may take the form of a computer program product on a computer-readable storage medium having computer-readable program instructions (e.g., computer software) embodied in the storage medium. More particularly, the present methods and systems may take the form of web-implemented computer software. Any suitable computer-readable storage medium may be utilized including hard disks, CD-ROMs, DVD-ROMs, optical storage devices, cloud-based storage systems, or magnetic storage devices.

[0064] Micro-fluidic Systems

[0065] Extracellular vesicles (EVs) are lipid-bilayer non-replicating particles released from most cell types that encompass the components of their parent cells, including but not limited to proteins, adhesion molecules, cytoskeleton molecules, cytokines, lipids, nucleic acids, metabolites, and other organelles. EVs can range in size from about 20nm to larger than 10pm. The present disclosure provides integrated micro-nanofluidic systems for collecting, extracting, harvesting, isolating, and / or analyzing cargo from EVs, including but not limited to nucleic acids (such as for example DNA and / or RNA) for further analysis. The present disclosure also provides a microfluidic system, such as a micro-nanofluidic system, for collecting, extracting, harvesting, isolating, and / or analyzing nucleic acid from EVs.

[0066] A microfluidic system, including a micro-nanofluidic system, refers to a small chip device comprising tiny channels (or microchannels) to manipulate small amounts of fluids. Such devices exploit forces, such as electrokinetics, capillary action, and vacuum to mix and separate liquids / fluids. Such devices are also optimal over conventional systems by using less sample volumes, fewer chemicals and reagents, and faster operation times. Non-limiting applications for using microfluidic systems include diagnosing diseases by analyzing bodily fluids containing cells or cellular organelles, synthesizing nanoparticles, detecting toxins, and analyzing nucleic acid sequences. It should be noted that “microfluidic” and “micro-nanofluidic” can be used interchangeably throughout the present disclosure.

[0067] In one aspect, disclosed herein is a micro-nanofluidic system for collecting, extracting, and isolating an extracellular vesicle (EV) cargo from an EV, wherein the system comprisesDocket No. 103362-085WO1 four or more components including but not limited to a concentrator, one or more first microchannels, one or more second microchannels, and a capture channel, and wherein the concentrator, the one or more first microchannels, the one or more second microchannels, and the capture channel are fluidically connected to allow transport of the EV sample from the first component to the second component.

[0068] As used herein, a “concentrator component” or a “filtration component” refers to a component of the micro-nanofluidic device used to separate and focus one or more cells or one or more cellular components, such as for example an EV, into another region of the microfluidics device. In the present disclosure, the microfluidics device of any aspect comprises an electric field for separating EVs based on size, such that small EVs are collected and large EVs are stored elsewhere, or that large EVs are collected and small EVs are stored elsewhere. It should be noted that the terms concentrator component and filtration component can be used interchangeably. In some embodiments, the micro-nanofluidic system comprises 1, 2, 3, 4, 5, or more concentrator components for separating EVs.

[0069] As used herein, “microchannel(s)” refer to at least one tiny pipe within the micro-nanofluidics device that allows fluids to flow through said device. Consider to be the “heart and veins” of a device, microchannels function to guide fluids through the device. Microchannels can be as simple as a straight line of piping or a complex maze of piping, depending on the needs of the device. In the present disclosure, the micro-nanofluidic device of any aspect comprises at least one microchannel for transporting fluids through said device. In some embodiments, the micro-nanofluidic system comprises 1, 2, 3, 4, 5, or more microchannels for transporting fluids through said device.

[0070] As used herein, a “capture channel” refers to at least one tiny pipe, or microchannel, within the micro-nanofluidic device that directs fluids into a collection region or component of said device. In the present disclosure, the micro-nanofluidics device of any aspect comprises at least one capture channel for collecting a cell or a cell component, such as for example an EV or an EV nucleic acid. In some embodiments, the micro-nanofluidics device comprises 1, 2, 3, 4, 5, or more capture channels for collecting a cell or a cell component, such as for example an EV or an EV nucleic acid. In some embodiments, the capture channel is a nucleic acid capture channel, specific for collecting nucleic acids, including but not limited to DNA and RNA molecules. In some embodiments, the capture channel is an amino acid capture channel, specific for collecting amino acids, peptides, polypeptides, and / or proteins.Docket No. 103362-085WO1 In some embodiments, the concentrator comprises a first electric field for separating EVs into at least two groups. In some embodiments, the concentrator separates EVs based on size of EVs. In some embodiments, the micro-nanofluidic device comprises an electric field separation by size. As used herein, an “electric field separation by size” refers to the process of separating particles, such as for example a plurality of EVs, of different sizes within a mixture by applying an electric field, wherein smaller particles tend to be more readily affected by said electric field and move faster than larger particles, allowing for separation of the smaller particles from the larger particles.

[0071] In some embodiments, a first group of EVs is separated into a storage unit. In some embodiments, the first group of EVs are stored by any method or technique known in the art. In some embodiments, the first group of EVs are stored for later processing. In some embodiments, the first group of EVs are large EVs comprising more than lOOnm. In some embodiments, the first groups of EVs comprise a diameter of 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487,Docket No. 103362-085WO1 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, or more nanometers (nm). In some embodiments, the first groups of EVs comprise a diameter of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more micrometers (pm).

[0072] In some embodiments, a second group of EVs is directed into the one or more first microchannels. In some embodiments, the second group of EVs are small EVs comprisingDocket No. 103362-085WO1 lOOnm or less. In some embodiments, the second group of EVs comprise a diameter of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nanometers. In some embodiments, the one or more first microchannels comprise a second electric field for directing the second group of EVs into the center of the one or more first microchannels.

[0073] In some embodiments, the second group of EVs are further enriched by immunoaffinity capture comprising a surface-immobilized antibody. Immunoaffinity capture is an isolation technique using antibodies, often immobilized on magnetic beads or microfluidic chips to target specific surface proteins on EVs. Immunoaffinity capture also allows for enhanced purification and isolation of EV subpopulations, such as separating small EVs from large EVs, compared to conventional methods. In some embodiments, the surface-immobilized antibody targets any EV surface marker known in the art. In some embodiments, the surface-immobilized antibody includes, but is not limited to an anti-CD63 antibody, an anti-CD81 antibody, an anti-CD9 antibody, or a combination thereof.

[0074] In some embodiments, the one or more second microchannels comprises at least one agent for lysing, degrading, and / or dissolving away unwanted cellular components. In some embodiments, the one or more second microchannels comprise a lysis buffer and a proteinase K for lysing open the second group of EVs. In some embodiments, the lysis buffer and the proteinase K disrupt the EV membrane and release the EV cargo for further processing. It should be noted that further processing of the EV of any preceding aspect or the EV cargo of any preceding aspect comprises exposing the said EV or EV cargo to a post-processing device.

[0075] In some embodiments, the capture channel comprises one or more micropillars for selectively capturing the EV cargo from the second group of EVs. In some embodiments, the EV cargo is collected in a collection container prior to entering the post-processing device. In some embodiments, the EV cargo is immediately directed into the post-processing device. In some embodiments, the post-processing device includes, but is not limited to a thermal cycler, a PCR device, one or more biosensors, a microarray, a sequencer, a liquid chromatographer, a mass spectrometer, or any combinations thereof.

[0076] It should be understood that the micro-nanofluidic system of any aspect disclosed herein and the components thereof can be made any material known in the art. Non-limiting examples of said materials include elastomers (such as for example polydimethylsiloxane (PDMS),Docket No. 103362-085WO1 thermoplastics (such as for example COC and PMMA), glass, silicon, paper, and advanced nanomaterials like graphene oxide and hydrogels (such as for example an ionic hydrogel), and any combinations thereof. Further, the micro-nanofluidic system of any aspect disclosed herein can be designed using diverse materials for biocompatibility, transparency, and diagnostic / treatment methods.

[0077] In some embodiments, the system is further connected to a EV-cargo isolation chamber. In some embodiments, the EV-cargo isolation chamber includes but is not limited to a EV-DNA isolation chamber. In some embodiments, the system is further connected to a post-processing device (such as for example a thermal cycler, a PCR device, a biosensor, a microarray, a sequencer, a liquid chromatographer, a mass spectrometer, or any combinations thereof). In some embodiments, the EV cargo is collected in a collection container prior to entering the postprocessing device. In some embodiments, the EV cargo is immediately directed into the postprocessing device.

[0078] In some embodiments, the EV is extracted from a sample selected from tissue, cells, sweat, blood, urine, or saliva. In some embodiments, the micro-nanofluidic system is designed for collecting, extracting, harvesting, isolating, and / or analyzing EVs from urine. In some embodiments, the micro-nanofluidic system is designed for collecting, extracting, harvesting, isolating, and / or analyzing EVs from saliva. In some embodiments, the system of any preceding aspect is used for cancer diagnostics, infectious disease diagnostics, drug discovery, biomarker analysis, or a combination thereof.

[0079] Methods of using a micro-fluidic system

[0080] The present disclosure provides methods using the integrated micro-nanofluidic system to detect a nucleic acid biomarker and / or treat a subject having a disease or disorder. The present disclosure also provides non-limiting applications of methods for isolating, capturing, and analyzing the EV cargo.

[0081] In some aspects, disclosed herein is a method of detecting a DNA biomarker, the method comprising obtaining a biological sample from a subject, inserting the biological sample into the micro-nanofluidic system of any preceding aspect, wherein said system extracts at least one EV sample from other contents in the biological sample, and wherein said system further separates and isolates an EV cargo from the EV sample, and detecting a biomarker within the EV cargo, wherein the biomarker is a signature of a disease or disorder.Docket No. 103362-085WO1 In some aspects, disclosed herein is a method of treating a subject in need thereof, the method comprising obtaining a biological sample from the subject, inserting the biological sample into a micro-nanofluidic system of any preceding aspect, wherein said system extracts at least one EV sample from other contents in the biological sample, and wherein said system further separates and isolates an EV cargo from the EV sample, detecting a biomarker within the EV cargo, wherein the biomarker is a signature of a disease or disorder, and administering to the subject therapy when the biomarker is present or increased relative to a healthy control subject.

[0082] In some embodiments, the healthier condition comprises a healthy state or a state of health after receiving therapy. It should be understood that a patient previously treated for a cancer of any aspect disclosed herein can also be a control subject. A non-limiting example is a liposarcoma patient having a recurrence of liposarcoma, wherein a level of a biomarker within said patient over the course of the disease can be used as an appropriate control.

[0083] In some embodiments, the method of any preceding aspect detects one or more biomarkers. In some embodiments, the method of any preceding aspect detects 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more biomarkers. In some embodiments, the biomarker is a nucleic acid biomarker. In some embodiments, the nucleic acid biomarker comprises a genetic mutation, a non-coding RNA (ncRNA), such as for example a microRNA (miRNA), a short interfering RNA (siRNA), and a long non-coding RNA (Inc-RNA).

[0084] In some embodiments, the biomarker is an amino acid, peptide, polypeptide, or protein biomarker. In some embodiments, the amino acid, peptide, polypeptide, or protein biomarker comprises a genetic mutation.

[0085] In some embodiments, the method of any preceding aspect comprises detecting a biomarker panel within the EV cargo. In some embodiments, the biomarker panel comprises 2 or more biomarkers. In some embodiments, the biomarker panel comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more biomarkers.

[0086] In some embodiments, the biomarker comprises a MDM2 polypeptide. In some embodiments, the biomarker comprises a RNA encoding a MDM2 polypeptide. In some embodiments, the biomarker comprises a CDK4 polypeptide. In some embodiments, the biomarker comprises an RNA encoding a CDK4 polypeptide. In some embodiments, the biomarker or a panel thereof comprises MDM2, CDK4, miR-16-5p, miR-20a-5p, miR-25-3p, miR-92a-3p, miR-93-5p, or any combination thereof.Docket No. 103362-085WO1 In some embodiments, the biomarker or a panel thereof is detected using the postprocessing device of any preceding aspect, including but not limited to a thermal cycler, a PCR device, one or more biosensors, a microarray, a sequencer, a liquid chromatographer, a mass spectrometer, or any combinations thereof.

[0087] In some embodiments, the disease or disorder comprises a cancer including, but is not limited to liposarcoma cancer, prostate cancer (e.g., prostate adenocarcinoma), bladder cancer, acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma, glioblastoma; medulloblastoma), bronchus cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma), osteosarcoma, Ewing's sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., “Waldenstrom's macroglobulinemia”), hairyDocket No. 103362-085WO1 cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease), hemangioblastoma, inflammatory myofibroblastic tumors, immunocytic amyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e.g., Paget's disease of the penis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g., appendix cancer), soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g., seminoma, testicular embryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer), urethral cancer, vaginal cancer and vulvar cancer (e.g., Paget's disease of the vulva).Docket No. 103362-085WO1 In some embodiments, the therapy is a cancer therapy, including but not limited to immunotherapy, chemotherapy or a chemotherapeutic agent, radiation therapy, surgery, or any combinations thereof. In some embodiments, the immunotherapy comprises an anti-cancer agent including, but not limited to interferons, cytokines (e.g., tumor necrosis factor, interferon a, interferon y), vaccines, hematopoietic growth factors, monoclonal serotherapy, immunostimulants and / or immunomodulatory agents (e.g., IL-1, 2, 4, 6, or 12), immune cell growth factors (e.g., GM-CSF) and antibodies (e.g. HERCEPTIN (trastuzumab), T-DM1, AVASTIN (bevacizumab), ERBITUX (cetuximab), VECTIBIX (panitumumab), RITUXAN (rituximab), BEXXAR (tositumomab)). In some embodiments, the immunotherapy comprises a PD-L1 inhibitor, a PD-1 inhibitor, and / or a CTLA-4 inhibitor. In some embodiments, the PD-L1 inhibitor includes, but is not limited to Atezolizumab, Avelumab, Durvalumab, LY3300054 (Eli Lilly and Company), and monoclonal antibodies or monoclonal antibody conjugates that act as a PD-L1 inhibitor. In some embodiments, the PD-1 inhibitor includes, but is not limited to pembrolizumab, Nivolumab, Cemiplimab and monoclonal antibodies or monoclonal antibody conjugates that act as a PD-1 inhibitors. In some embodiments, the CTLA-4 inhibitor includes, but is not limited to Ipilimumab, AGEN1884 and monoclonal antibodies or monoclonal antibody conjugates that act as a CTLA-4 inhibitor.

[0088] Exemplary chemotherapeutic agents include, but are not limited to, anti-estrogens (e.g. tamoxifen, raloxifene, and megestrol), LHRH agonists (e.g. goscrclin and leuprolide), antiandrogens (e.g. flutamide and bicalutamide), photodynamic therapies (e.g. vertoporfm (BPD-MA), phthalocyanine, photosensitizer Pc4, and demethoxy-hypocrellin A (2BA-2-DMHA)), nitrogen mustards (e.g. cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas (e.g. carmustine (BCNU) and lomustine (CCNU)), alkyl sulphonates (e.g. busulfan and treosulfan), triazenes (e.g. dacarbazine, temozolomide), platinum containing compounds (e.g. cisplatin, carboplatin, oxaliplatin), vinca alkaloids (e.g. vincristine, vinblastine, vindesine, and vinorelbine), taxoids (e.g. paclitaxel or a paclitaxel equivalent such as nanoparticle albumin-bound paclitaxel (ABRAXANE), docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin), polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2 bound to three molecules of paclitaxel), paclitaxel-EC-1 (paclitaxel bound to the erbB2-recognizing peptide EC-1), and glucose-conjugated paclitaxel, e.g., 2'-paclitaxel methyl 2-glucopyranosyl succinate; docetaxel, taxol), epipodophyllins (e.g. etoposide, etoposide phosphate, teniposide, topotecan, 9-aminocamptothecin,Docket No. 103362-085WO1 camptoirinotecan, irinotecan, crisnatol, mytomycin C), anti-metabolites, DHFR inhibitors (e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMP dehydrogenase inhibitors (e.g. mycophenolic acid, tiazofurin, ribavirin, and EICAR), ribonucleotide reductase inhibitors (e.g. hydroxyurea and deferoxamine), uracil analogs (e.g. 5 -fluorouracil (5-FU), floxuridine, doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosine analogs (e.g. cytarabine (ara C), cytosine arabinoside, and fludarabine), purine analogs (e.g. mercaptopurine and Thioguanine), Vitamin D3 analogs (e.g. EB 1089, CB 1093, and KH 1060), isoprenylation inhibitors (e.g. lovastatin), dopaminergic neurotoxins (e.g. l-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g. staurosporine), actinomycin (e.g. actinomycin D, dactinomycin), bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin), anthracycline (e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDR inhibitors (e.g. verapamil), Ca2+ATPase inhibitors (e.g. thap sigargin), imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g., axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN™, AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®, SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474), vatalanib (PTK787, PTK / ZK), trastuzumab (HERCEPTIN®), bevacizumab (AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib (NEXAVAR®), everolimus (AFINITOR®), alemtuzumab (CAMPATH®), gemtuzumab ozogamicin (MYLOTARG®), temsirolimus (TORISEL®), ENMD-2076, PCI-32765, AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and / or XL228), proteasome inhibitors (e.g., bortezomib (VELC DE)), mTOR inhibitors (e.g., rapamycin, temsirolimus (CCI-779), everolimus (RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502 (Pfizer), GDC0980 (Genetech), SF1126 (Semafoe) and OSI-027 (OSI)), oblimersen, gemcitabine, caminomycin, leucovorin, pemetrexed, cyclophosphamide, dacarbazine, procarbizine, prednisolone, dexamethasone, campathecin, plicamycin, asparaginase, aminopterin, methopterin, porfiromycin, melphalan, leurosidine, leurosine,Docket No. 103362-085WO1 chlorambucil, trabectedin, procarbazine, discodermolide, caminomycin, aminopterin, and hexamethyl melamine.

[0089] In some embodiments, the EV is extracted from a biological sample selected from tissue, cells, sweat, blood, urine, tears, nasal mucus, saliva, and / or related fluids. In some embodiments, the sample is urine. In some embodiments, the system is used for detection of liquid biomarkers for cancer diagnostics, infectious disease diagnostics, drug discovery, and biomarker analysis.

[0090] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0091] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

[0092] EXAMPLES

[0093] The following examples are set forth below to illustrate the compositions, devices, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

[0094] Example 1: A multi-layered, integrated micro-nanofluidic system for extracellular vesicle isolation, purification, and molecular extraction and analysis.

[0095] This invention disclosure describes an integrated surface modified, micro-nanofluidic system for isolation of extracellular vesicles (EVs) from biofluids to generate a purified sample enabling molecular extraction and analysis. This model system is a proof-of-concept on sarcoma-relevant cell conditioned media and patient biofluids (blood, urine, and saliva). As development proceeds, conceptual designs and supporting data are shown to achieve multifunctions on a chip for cancer diagnostics, identification of drug-targets, and a breadth of biomarker analysis delivering on the promise of a true- lab-on-chip technology for point-of-care biofluid analytics. The system is innovative and integrates multiple components, enabling the direct collection, isolation, purification, and analysis of patient biofluids to: (a) trackDocket No. 103362-085WO1 treatment progression by assessing the biomarker levels in body fluids and (b) provide a rapid and early diagnosis tool for cancer, particularly those that are difficult to diagnose, like sarcomas; (c) identify biomarkers that can serve as therapeutic targets, and (d) engineer vesicles as drug delivery targets for treatment of cancer. Based on current knowledge, while individual components of the multi-functional device exist and are under various stages of development, there are no integrated devices that perform all the functions. Furthermore, there are no point-of-care devices that can facilitate molecular analysis like the devices presented herein. Lastly, the integration of various components is not relying only on existing technologies, but brings together several intricate, non-obvious technological advances forward, thereby making the system entirely new and patentable.

[0096] The systems presented herein are operable with any biofluid and relevant to a pan-cancer molecular analysis. A brief background for Soft Tissue Sarcomas (STS) is presented next. STSs are an uncommon heterogeneous group of tumors of mesenchymal origin, among them the human liposarcoma (LPS) being the most common one. In the United States alone, there was an estimated 13,190 new cases of STS and 5,130 deaths annually in the year 2022. According to the 2024 report released by the American Cancer Society, the number of new cases of STS being reported is estimated to be -13,590 and the death toll has increased to 5,200 annually, with most of the deaths due to local recurrence and metastasis. Further classification of LPS based on histology and biological factors includes well-differentiated (WDLPS), de-differentiated (DDLPS), myxoid, and pleomorphic, among which WDLPS and DDLPS are most prevalent. Surgery remains the primary therapeutic approach for LPS. However, despite the addition of adjuvant treatments, over 50% of patients still develop recurrent or metastatic diseases. LPS is a lethal cancer for which there is a lack of means to unequivocally detect recurrence early when a cure may still be possible via adjuvant therapeutic interventions. Early detection of LPS recurrence is challenging as radiologic scans lack specificity, necessitating image-directed or open tissue biopsy resulting in time delays, significant costs, and patient discomfort. Alternatively, a liquid biopsy is minimally invasive but requires a specific and reliable biomarker. WD / DDLPS both have amplified 12ql3-15 locus on chromosome 12, which contains MDM2, the most commonly over- expressed LPS gene. WDLPS contains mutated p53, whereas DDLPS consistently has wild type (wt) p53. Both over-express MDM2 DNA, leading to MDM2 protein over-production that may override p53 tumor suppression, thereby serving as a key LPS driver; the underlying mechanisms remain uncertain.

[0097] In the quest to find a reliable biomarker for LPS recurrence, extracellular vesicles (EVs)Docket No. 103362-085WO1 have found a key niche. EVs are 30-1000 nm lipid bilayer membrane-enclosed nanoparticles that are secreted by all cells. EVs carry a diverse cargo, including DNA, RNA, and proteins, shielded from enzymatic destruction by the lipid bilayer, making them highly intriguing entities for biomarker analysis. It has been shown that LPS EVs are shed into the peripheral blood and have elevated levels oiMDM2 DNA compared to normal control EVs or peri-tumoral normal tissues. The critical technical challenge lies in isolating EVs with adequate purity and yield from a complex biofluid matrix to yield enough DNA for reliable diagnostic value, applicable within clinical settings. Moreover, quantification of total DNA and subsequent analysis of MDM2 DNA with sequencing is also required. Lastly, as research in the general field of biomarkers continues, emerging biomarkers are continually being identified. However, each biomarker requires a specialized analysis method. The device presented herein shows possible integration of distinct methods to allow not only identification of new biomarkers but also the possibility of the system to act as a biomarker screening tool. It is worth noting that identification of biomarkers is not simply for cancer diagnostics but biomarkers for an emerging area for therapeutic targets. In particular, for MDM2, one of the largest pharmaceutical companies (Boehringer-Ingelheim) is actively searching for therapeutics. Moreover, they are also seeking identification of additional biomarker targets for cancer therapeutics.

[0098] At present, the gold standard and most used method for extracellular vesicle isolation is differential centrifugation, which involves several centrifugation and ultracentrifugation steps. Moreover, this technique is time-consuming (4-5 hours), requires larger sample volumes (15 mL) and highly trained laboratory technicians. Yet, this method leads to inconsistencies in recovery of EVs mainly because of different biofluid viscosity, particle density, and results in low recovery rates (5-25%).

[0099] Recently, several alternative techniques were introduced to isolate exosomes, including antibody coated magnetic beads; precipitation technologies like ExoQuick and polyethylene glycol; microfluidic devices. However, each technique had its own disadvantages like lower yield, need for larger sample volume, and longer isolation time. For example, ExoQuick, a commercially available exosome isolation kit requires lengthy overnight incubations (12 hours), thereby making this technique completely challenging for use clinical settings that require a rapid response. Moreover, it has been shown that a decline in purity of exosomes using this ExoQuick kit may occur.

[0100] Emerging microfluidic systems have several advantages over conventional technologies (ultracentrifugation and commercial precipitation solution kit) like lower sampleDocket No. 103362-085WO1 volume (50-600 pL) and time (1-2 hours) for isolating exosomes and enhanced recovery rates (42-94%), each existing system has its own shortcomings. For example, the size-based microfluidic exosome separation device consists of ciliated micropillars forming a silicon “nanowire-on-micropillar” structure for selectively trapping exosome-like lipid vesicles. The recovery of the trapped vesicles was achieved by dissolving the micropillars with nanowires in phosphate buffer saline overnight. This non-continuous separation technique is restricted by exosome saturation limits, thereby limiting the number of exosomes that can be retrieved from the bio-fluid. On the other hand, even though one can achieve high exosome purity with immunoaffinity-based separation devices, these devices work only if the target protein of interest is present on the vesicle surface.

[0101] To overcome the disadvantages of the existing microfluidic methods and also the challenges posed by conventional methods, an innovative micro-nanofluidic device was initially setup (Figure 1 A), which consists of a 5mmx5mm polycarbonate membrane (pore size: 220 nm; pore density: 3*108pores / cm2) (GE Water & Process Technologies, Catalog no.: K02CP02500) sandwiched between two microchannels, namely injection and separation channel (each 500 pm wide and 150 pm high). The microchannel features were first patterned on an n-type 4” silicon wafer using standard UV lithography to generate a negative mold for casting PDMS. Briefly, the silicon wafer was cleaned with a piranha (H2SO4:H2O2 (3:1)) solution for 10 minutes, rinsed with de-ionized water, dried with nitrogen, and baked at 200°C for 15 mins. Post dehydration bake, the silicon wafer was spin-coated with SU-82050 at 2500 rpm for 40 s, and soft baked at 65°C for 3 mins and at 95°C for 8 mins. The soft-baked wafer was exposed to 10 mW / cm2UV light for 17.5 s using a UV contact aligner (EV Group 620 contact aligner; wavelength: 365 nm), followed by a post-exposure bake at 65°C for 2 mins and at 95°C for 6 mins. The exposed pattern was then developed in SU-8 developer for 6 mins and hard baked at 200°C for 30 mins. The channels are made of PDMS (polydimethylsiloxane) (Ellsworth Adhesives, Catalog no.: 4019862). The silicone elastomer base and curing agent were combined at a 10: 1 ratio, then poured onto the silicon master, degassed, and cured at 70°C for 4 hours. The cured PDMS was peeled off the silicon master and cut into individual channels. Through-holes were punched with 1.5 mm biopsy punches on the PDMS with the injection channel to access the injection channel and the separation channel. The polycarbonate membrane served as the nanofluidic separator. The 5 mm x 5 mm membrane underwent silanization in a nitrogen glove bag with a 3% v / v (3 -Aminopropyl) triethoxysilane (APTES; Sigma Aldrich) solution in anhydrous ethanol for 1.5 minutes. Subsequently, both the silanizedDocket No. 103362-085WO1 NCAM and the PDMS monolith with the injection channel were subjected to oxygen plasma treatment at 600 mTorr for 15 seconds and bonded together, aligning the membrane over the middle of the injection channel along its length through visual inspection. Finally, the PDMS containing the monolith of the separation channel, and the NCAM-inj ection channel underwent another round of oxygen plasma treatment at 600 mTorr for 15 seconds. The treated surfaces were then bonded with their channels aligned perpendicular through visual inspection.

[0102] Use of PDMS enables visualization of flows and provide characterization of the proof-of-concept system. The 220 nm polycarbonate membrane ensures size-based filtration of exosome-like lipid vesicles (145±20 nm in diameter; based on electron microscopy images of exosomes derived from liposarcoma cell conditioned media) from the cell conditioned media. The separation channel was plasma activated and treated with APTES solution (2% v / v) in anhydrous ethanol (Decon Labs Inc.) 20 pL APTES solution was flushed in the channel 5 times at 3 min intervals and incubated for 2 h. After 2 hours, 20 pL of anhydrous ethanol was flushed through the separation channel to remove any physiosorbed APTES. Next, the separation channel was flushed with 5% glutaraldehyde solution in IX phosphate buffer saline (PBS; Coming) every 3 minutes for 15 minutes and left to incubate for 30 minutes at 4°C. Following the incubation, the channel was flushed with IX PBS three times to remove any physiosorbed glutaraldehyde molecules. Finally, 50 pg / mL of primary antibody anti-CD63 (Ancell) diluted in IX PBS was flushed through the separation channel and left to incubate at 4°C for 8 hours. The separation channel was flushed with IX PBS after the incubation to remove any physiosorbed antibody with the device stored at 4°C until further use.

[0103] The microfluidic device was perfused with cell conditioned media at 10 pL / min through the injection channel using a syringe pump (Harvard Apparatus) to ensure that the biological impurities do not block the pores of the polycarbonate membrane. After 30 minutes of perfusion, captured exosomes were eluted by perfusing 60 pL of pH ~2.4 glycine-HCl buffer solution followed by pushing air through the separation channel using pipette to ensure complete collection. After eluting the exosomes, a neutralization buffer was added to achieve a final pH of ~7.4. The final exosome solutions were stored at -80°C until further characterization or DNA isolation.

[0104] The system presents several advantages: (1) there is no longer an overnight unlike the size-based microfluidic exosome separation device that involves an overnight incubation step to recover the exosomes; (2) the isolation time is lowered by almost 90% - 95% compared to the current state-of-art technologies i.e. ultracentrifugation and ExoQuick respectively; (3)Docket No. 103362-085WO1 recovery of almost 3- times the amount of exosomes compared to ultracentrifugation with 500 pL of the cell conditioned media; and (4) unlike ultracentrifugation, the system presented herein doesn’t require expensive equipment to operate and the space occupancy is reduced by almost 95%.

[0105] The previously developed single-channel device was enhanced by integrating an electric field across the membrane to improve EV isolation. Fabricated using standard soft lithography in PDMS (polydimethylsiloxane), the microfluidic device features a 5 x 5 mm2nanocapillary array membrane (NCAM) with a nominal capillary diameter of 0.2 pm. Positioned between two microchannels, the vertically aligned configuration comprises an injection and separation channel, each measuring 500 pm wide and 60 pm high, perpendicular to one another (Figure 2A). Gold electrodes were inserted into the channel ports to provide electrical contact for bias application across channels and the NCAM. The separation channel was forward-biased because liposarcoma-derived EVs are negatively charged.

[0106] The surface of the separation channel was functionalized with anti-CD63 / anti-CD81 antibodies for EV capture, and an electric field was applied across the NCAM in conjunction with crossflow filtration for combined size and charge-based separation. Conditioned media from the human liposarcoma-derived Lipo246 cell line (LCCM) was perfused through the injection channel at 10 pL / min for 45 minutes, with isolation carried out at four voltage levels: 0 V, 1 V, 10 V, 50 V, 70 V, and 100 V. EV immunocapture was conducted using CD63 and CD81 antibodies.

[0107] The results indicate that the application of an electric field improved EV capture efficiency up to a threshold voltage of 50 V. As shown in Figure 2, the captured EVs increased progressively with voltage, reaching a peak at 50 V. Beyond this threshold, at 70 V, the total DNA yield from isolated EVs decreased significantly, likely due to Joule heating. Also, as applied potential was increased further to 100 V, Faradaic reactions reflecting the browning of the EV-enriched media in the separation channel and reservoirs was also noted. At higher voltage levels (100 V), the formation of bubbles near the electrodes disrupted the electric field and fluid flow, detaching captured EVs from the surface-bound antibodies.

[0108] In the next iteration of device development, both the design and operating conditions were modified to maximize the EV isolation per unit of sample volume used. This device consists of an injection microchannel (3 cm x 500 pm x 60 pm) which acts as the sample inlet path and is vertically integrated through 220 nm nanocapillaries (5 x 5 mm2) to a bottom layer with three separation microchannels (1.5 cm x 500 pm x 60 pm each). The functionalized deviceDocket No. 103362-085WO1 is now subjected to a cell-conditioned media perfusion of 10 pL / min for 45 minutes to process more volume in a test-run.

[0109] The performance of the multi-layer microfluidic device (Figure 3 A) was optimized for multiplex capture of extracellular vesicles (EVs) using CD63 and CD81 surface proteins, thus quantifying their relative surface protein abundance on EVs. The device was functionalized with capture antibodies for EV immunocapture. Antibodies were immobilized on the separation channel surface using silane chemistry, with glutaraldehyde (GA) as a linker between silane monolayer and antibodies. Initially, the surface was modified with (3 -Aminopropyl) triethoxysilane (APTES) to form a silane monolayer, followed by incubation with GA to optimize antibody immobilization. Using Lipo246 Cell Line Conditioned Media (LCCM) samples, the isolated EVs yield were evaluated for two GA concentrations (1.25% and 5% v / v in lx PBS) and two incubation temperatures (4°C and room temperature). The results show that 5% GA at 4°C provides significantly the highest total DNA yield from isolated EVs for CD63 and CD81 antibodies. Notably, EVs isolated with CD81 antibodies exhibited significantly higher DNA yield than those isolated using CD63, as illustrated in (Figure 4). This is the first identification of which antibody targets presents a more viable target for sarcoma-derived EVs.

[0110] Each data point contains pooled fluid from 4 microfluidic devices, each contributing -180 pL of fluid per test run. In the next iteration, the device performance was enhanced by applying electric field to the existing device keeping other parameters the same. The setup can be seen in (Figure 5).

[0111] EV-enriched conditioned media (LCCM) produced by the lipo246 cell line was used for the experiments. Separation channels modified with 5% GA at 4°C gave significantly higher yield and purity (260 / 280 absorbance between 1.6 and 2) of isolated EV-DNA from LCCM, with anti-CD81 giving the best EV capture efficiency. DNA yield was noted to increase as applied potential drop across the NCAM increased from 0-50V and then decreased again at 70 V, with damage to EVs noted at 100V. Therefore, 50 V was chosen as the maximum voltage bias for DNA quantification. Figure 6 demonstrates that, compared to UC, incorporating an electric field across the NCAM resulted in a 4.2- fold significant increase in the capture efficiency of the EVs, higher DNA purity (260 / 280 between 1.6- 2) and a 5-fold significant increase in the isolated EV-DNA / mL of LCCM, with a 75% reduction in isolation time. qPCR on isolated EV samples confirmed the detection of MDM2 DNA.

[0112] Finally, in another iteration the device setup where patient biofluid is first collected and processed using a mini centrifuge. The resulting supernatant is then introduced into theDocket No. 103362-085WO1 microfluidic device through the particle concentrator section. This section features an applied electric field across its inlet and outlet, which facilitates the separation of large and small particles by leveraging the combined effects of electrophoretic force and secondary flow induced by Dean vortices. Larger particles are directed toward the outer wall and are subsequently separated through a dedicated microchannel, leading to a storage unit for large particles.

[0113] The bottom portion of the stream exiting the particle concentrator is routed through a microchannel with an applied electric field to focus particles on the center of the stream. This enriched particle stream is channeled directly into the device's input channel, while the remaining portions are diverted to the large particle storage unit. The operation of the input channel, membrane, and separation channel remains consistent with the previously described multichannel device.

[0114] The output from the separation channel of the multichannel device integrates seamlessly with the extended system as shown in (Figure 7) further explained in (Figure 8). Here, the eluted extracellular vesicles (EVs) flow into a common microchannel, which tapers from 500 pm to 250 pm and converges at a junction with lysis buffer (Al buffer) and proteinase K. This mixture proceeds through a serpentine mixer with a mixing length of 21 cm, where the EVs are effectively lysed.

[0115] The lysate is then directed to a DNA capture chamber equipped with a silanized micropillar array treated with 2% APTES in anhydrous ethanol. These micropillars are designed to selectively capture DNA from the lysed EVs. Following the capture, the chamber undergoes washing steps using AW1 and AW2 buffers. Finally, the DNA is eluted from the micropillars using water and collected via a side port on the collection chip. The collected DNA is transferred to a post-processing unit for subsequent characterization.

[0116] This invention represents a novel and valuable contribution to the field of diagnostics.

[0117] Example 2:

[0118] QI. Please clarify volume: say the EV concentration from samples is roughly the same between methods, how much volume is needed to do one experiment?

[0119] Response: See, Table 1.

[0120] Q2. For a 720uL recovered sample, how much volume do you require to start with to get to 720uL?Docket No. 103362-085WO1 Response:

[0121] After processing 450 pL of sample through one device, EVs are eluted using three 20 pL Glycine HC1 flushes, yielding 60 pL of eluted EVs. Neutralization with 120 pL Tris-Glycine HC1 buffer brings the total to 180 pL per device. Pooling outputs from four devices gives 720 pL, requiring a starting volume of 1.8 mL (4 x 450 pL).

[0122] Q3. Can the device handle a low EV concentration sample (is there a threshold)? Response: Yes, A single EV typically contains at least 0.1-lpg of DNA. In modem PCR kits, detecting 1-10 number of molecules of a gene of interest is enough. Hence, for our process (at least 10 number of molecules for detection, 2pL sample in 18pL of real-time mix, which is then aliquoted to 9pL per PCR plate well mixture (for DNA assessment this process is run in duplicate), lOObp MDM2 amplicon), 1.08 pg / pL DNA concentration is enough, a sample concentration of around 10^ EVs / mL is sufficient for detection considering our device efficiency of 10%.

[0123] Q4. The times for the steps can add up, so it looks like it'll still take about a day (one step is 8 hours), though some steps are sample or device prep such as antibody conjugation so can be done before hand. But realistically, how long does it take?

[0124] Response:

[0125] With cured PDMS is readily available, the fabrication process takes approximately 45 minutes. Including 12 hours for functionalization and 45 minutes for sample testing, the total time required is about 13.5 hours. However, multiple devices (specifically 16 devices) can be fabricated and functionalized simultaneously, maintaining the same precision as a single device, which reduces the per-device time to roughly 50 minutes. Since 4 devices are pooled to achieve meaningful results and testing can be conducted simultaneously for all 4

[0126] devices, the total time for a complete EV isolation process using this method is approximately 3 hours. Additionally, as you noted, preparatory steps like antibody conjugation can be done in advance, reducing the actual EV isolation process to just 45 minutes when 4 devices are tested simultaneously and pooled. Depending on the need for adjusting either inlet or final output sample volumes, the device pool size can be adjusted to operate either a single device or additional devices. In-laboratory set-up is limited currently to testing up to 7-devices in parallel. The limitation arises from work being done in a physical laboratory with finite space and related supporting paraphernalia.Docket No. 103362-085WO1

[0127] Q5. Which metric best highlight your device is it small sample volume required, yield, purity, specificity for soft sarcoma receptors, less time, small footprint?

[0128] Response:

[0129] While the device takes care of all the mentioned attributes, the metric which best highlights the device must be the reduced processing time and sample volume critical for point of care diagnostics.

[0130] Q6. I see BE has ongoing trials for Brigimadlin (MDM2-p53 Antagonist). They likely have a companion diagnostic. I wonder how this device could help or perform better than what they have. The extracellular vesicle angle is not clear to me.

[0131] Response:

[0132] The drug trial is off.

[0133] Q7. Why can't liquid biopsy work to detect the over expression of the MDM2 gene for sarcoma? Response:

[0134] The primary challenge in detecting MDM2 overexpression in sarcoma through liquid biopsy arises from the low concentration of tumor-derived material in circulation and the difficulty in isolating tumor-specific DNA. While cfDNA, CTCs, and EVs all fall under liquid biopsy approaches, EVs offer distinct advantages due to their stability and ability to preserve tumor-derived markers.

[0135] For a detailed understanding, the challenges are list below in detail:

[0136] cfDNA (Circulating Free DNA):

[0137] • lacks sensitivity due to lower concentrations of tumor-derived DNA in circulation, especially in sarcoma where cfDNA levels can be low.

[0138] • It can be harder to detect MDM2 overexpression accurately because non-tumor cfDNA may dilute the signal from tumor-derived DNA.

[0139] • cfDNA is highly susceptible to enzymatic degradation in circulation, which limits its stability and makes detection more challenging.

[0140] CTCs (Circulating Tumor Cells):

[0141] • Less effective for sarcoma due to their low presence in the bloodstream, making isolation and analysis more challenging.Docket No. 103362-085WO1 Pollock et al. (2015) has shown that sarcoma often has alow abundance of CTCs, which reduces their utility in liquid biopsy for detecting MDM2 overexpression.

[0142] EVs (Extracellular Vesicles):

[0143] • Most reliable option for detecting MDM2 overexpression in sarcoma.

[0144] EVs carry intact tumor-derived markers, including MDM2, and maintain their stability due to their protective bilipid membrane. This membrane shields genetic material and proteins from degradation.

[0145] This protection makes EVs more robust compared to other liquid biopsy components. Compared to UC, applying an electric field across the NCAM improved DNA purity (260 / 280: 1.6- 2), EV capture efficiency by 4.2-fold, and EV-DNA / mL of LCCM by 5-fold. (Figure 6).

[0146] Example 3: Further development of a multilayered, integrated micro-nanofluidic system. Establishing viability of urine for nucleic acid biomarkers.

[0147] Another target disease is liposarcoma (LPS). LPS is characterized by the amplification of MDM2 and CDK4 genes, which play critical roles in tumor progression by disrupting the p53 and Rb tumor suppressor pathways. Given their tumor-specific upregulation, these molecules represent promising biomarkers for LPS detection. These molecules are also used as diagnostic tools, in particular MDM2, with the analysis of tumor cells. It has been previously reported the upregulation of MDM2 in serum-derived extracellular vesicles (EVs) from LPS patients. The overall goal herein is to develop a small, portable, easy to operate “kit” that can enable surveillance of cancer (and other diseases) through a systematic detection of nucleic acid biomarkers. Towards this goal, any device or system must be non-invasive and easy to use.

[0148] Nearly all past work and existing FDA-approved devices use blood (and blood-derived materials like serum and plasma) as the primary diagnostic fluid. The present invention targets urine and saliva as a non-invasive fluids. Therefore, the first step is to establish the viability of urine as a fluid that can yield detectable levels of biomarkers.

[0149] However, there is limited evidence investigating the presence or upregulation oiMDM2 and CDK4 in EVs, particularly from urine. The present disclosure provides analysis of urine-derived EVs from LPS patients to evaluate the presence and potential upregulation oiMDM2 and CDK4 as non-invasive biomarkers for LPS detection and monitoring. Additionally, 6 microRNAs are also evaluated as circulating biomarkers. The use of CDK4 and the 6-additional biomarkers is new and has not been shown before for LPS. The experiments with urine requireDocket No. 103362-085WO1 the development of a new protocol to process urine. Moreover, initial studies have been conducted with saliva as another biofluid for LPS biomarkers.

[0150] The protocols noted next combine ideas from several published reports to yield processed urine for robust EV and subsequent biomarker yields. The following protocol was used for urine sample collection and initial processing: A clean-catch first-morning urine sample was obtained in a clean, dry, plastic cup, collecting 40 mL. The urine was maintained at 4 °C for a maximum of 3 hours and sent to the laboratory at the same temperature. Upon arrival, the sample was mixed by inverting it twice and transferred into a 50-mL conical-bottomed polypropylene tube. The tubes were centrifuged at 850 x g for 10 min at 4 °C. The urine supernatant was carefully transferred into another 50 mL conical bottom tube, leaving at least 3 mL of the supernatant above the cell pellet. The pellet was discarded, and 400 pL of 0.5 M EDTA and 5 ml of 5 x PBS were added to the supernatant to inhibit uromodulin aggregation and Ca-oxalate formation and pH neutralization, respectively. The samples were immediately stored at -80°C until EV isolation.

[0151] For the differential ultracentrifugation (UC) process, the frozen supernatant was thawed overnight at 4°C and vortexed well to homogenize the sample. The thawed sample was centrifuged at 10,000 x g for 30 minutes at 4 °C. The supernatant was again collected, and the pellet was resuspended in 500 pL lx PBS and stored at -80 °C. This resuspended pellet contains large EVs (ZEVs; EVs> 200 nm) as shown previously in published reports. The large EV pellet is distinct from the small EV pellet and the EVs in the large EV pellet (labelled as 10K) were analyzed separately.

[0152] For the small EVs (< 200 nm), the thawed supernatant was carefully layered over 4 mL of 30% sucrose cushion (prepared in 1 x PBS) and subjected to UC at 100,000 x g for 70 minutes at 4°C. Following UC, the supernatant was gently removed, leaving approximately 5 mL above the pellet. The remaining solution was washed by resuspending in lx PBS and centrifuging again at 100,000 x g for 70 minutes at 4°C. Finally, the supernatant was discarded, and the resulting EV pellet was resuspended in 500 pL of lx PBS. This pellet contained small EVs (sEVs; EVs< 200 nm). Both 10,000 x g (10k) and 100,000 x g (UC) pellets were aliquoted as follows for downstream analysis to meet the international standards guidelines such as those specified by MISEV 2023:

[0153] 1. Nanotrack (NT A) or NanoFCM analysis to evaluate particle sizes and concentrations - 50 pLDocket No. 103362-085WO1 2. Transmission electron microscopy (TEM) - 10 pL

[0154] 3. DNA extraction and quantification- 220 pL

[0155] 4. miRNA extraction and quantification- 220 pL

[0156] 5. Isolated EV-DNA was sequenced for MDM2 and CDK4 molecules using qPCR

[0157] For EV isolation from the device, the stored urine was thawed overnight at 4°C and vortexed well to homogenize the sample. Next, urine was infused into the injection channel via a syringe pump at 10 pL / min for 45 mins. sEVs were selectively enriched in the separation channels through size-based cross-flow filtration and electrokinetic transport across the NCAM. Captured sEVs bind to surface-immobilized antibodies (targets: CD63 and CD81) and are subsequently eluted using glycine HC1 (pH 2.2), then neutralization with Tris-HCl (pH 8.0). The isolated EVs are stored at -80°C for further analysis. The device flowthrough from the outlet of the injection channel is likely dominated by particles that need not go through the NCAM and so are equivalent to the pellet with ZEVs. As for UC, all processed and EV-enriched urine samples were collected and stored at -80°C for further downstream analysis. Isolated EVs were characterized using TEM imaging, NTA and NanoFCM, and DNA extraction and quantification, miRNA extraction and quantification, and qPCR for MDM2 and CDK4 molecules.

[0158] One key distinction between the UC isolated EVs and the microfluidic device isolated EVs is that the microfluidic device conducts a 2-stage isolation and capture of EVs. First, the NCAM “mechanically filters” sEVs vs. lEVs, and then the immunoaffinity captures a select population of EVs with CD63 and CD81 tetraspanins on the EV surface.

[0159] Urine concentration can vary significantly between individuals and even within the same individual across different time points, depending on disease state, ongoing treatment, hydration status, diet, and kidney function. To account for this variability, creatinine concentration in urine (U-creatinine) is commonly used as a normalization factor in clinical and biomarker studies. Levels of U-creatinine were measured in the urine samples of both healthy donors and LPS patients using the Creatinine Urinary Detection Kit (Invitrogen). Each patient biomarker level (e.g., DNA) is normalized to the expression of U-creatinine.

[0160] Choice for current biomarker targets

[0161] Along with the DNA, a panel of microRNAs (miRNAs) was further evaluated as potential biomarkers for LPS recurrence in urine-derived EVs. Employing a multiplexedDocket No. 103362-085WO1 miRNA panel enhances diagnostic accuracy by capturing diverse and complementary molecular features of tumor biology. Unlike single biomarkers, which often reflect a narrow molecular feature such as cell cycle dysregulation, a biomarker panel allows simultaneous interrogation of pathways associated with genomic instability, apoptotic resistance, and tumor microenvironment remodeling. This strategy not only reduces the risk of false positives but also enhances cancer-type specificity and prognostic resolution. It is particularly advantageous in urine-based liquid biopsy, where miRNA levels may fluctuate due to dilution, renal clearance, or variable vesicle shedding, necessitating a more robust, multi-analyte framework.

[0162] Five relevant miRNAs were selected as initial targets to evaluate due to their use as targets for prognosis and diagnosis. However, as with most biomarkers reported in literature, these are based on internal heuristics.

[0163] The current 5 miRNA targets are: miR-16-5p, miR-20a-5p, miR-25-3p, miR-92a-3p, and miR-93-5p. These miRNAs have been implicated in various cancers and they have been reported to be significantly overexpressed in LPS plasma-EVs compared to healthy controls. For example, miR-16-5p is associated with cell death (apoptosis) and in cancer can impact tumor suppression genetic pathways, miR-25-3p promotes DDLPS progression by targeting tumor suppressors such as FBXW7 and PTEN, leading to activation of the PI3K / AKT and MYC pathways. It is highly enriched in exosomes (sEVs) from aggressive DDLPS cells and facilitates angiogenesis, immune evasion, and extracellular matrix remodeling by reprogramming recipient stromal and immune cells. miR-92a-3p, similarly exosome-enriched, enhances proliferation, invasion, and metastasis by downregulating PTEN, KLF4, and adhesion-related genes. It contributes to EMT, stromal activation, and vascular remodeling, underscoring its role as a key effector of DDLPS aggressiveness. Once internalized by surrounding macrophages at the tumor microenvironment level, both miR-25-3p and miR-92a-3p bind to the TLR8 receptor and stimulate the secretion of pro-inflammatory cytokine IL-6, promoting tumor growth. miR-93-5p, a member of the miR-106b~25 cluster that includes miR-25-3p, shares functional convergence in promoting PI3K / AKT activation and suppression of tumor suppressors like CDKN1A. While not yet fully characterized in DDLPS, its oncogenic roles in other cancers justify its inclusion as a candidate regulator of DDLPS progression and microenvironmental remodeling. miR-20a-5p, another cluster-associated onco-miR, inhibits pro-apoptotic and cell cycle checkpoint genes such as BIM, TGFBR2, E2F1, and p21, contributing to unchecked proliferation and therapy resistance across multiple cancers. Its inclusion enables the capture of cell cycle deregulation and anti-apoptotic phenotypes. Together, this miRNA panel enablesDocket No. 103362-085WO1 comprehensive assessment of tumor-derived EV content, facilitating non-invasive detection of molecular features relevant to LPS progression, and offering translational value for diagnostic and prognostic applications. Notably, some of these miRNAs are also impacted in other pathologies like Alzheimer’s disease.

[0164] Characterization of Urine-derived EVs

[0165] All biomarker work requires validation. As yet, it is unknown if urine can serve as a viable resource for LPS recurrence, even though urine is known to contain EVs. Therefore, as a starting analysis, the purity and integrity of urine-derived EVs isolated via ultracentrifugation (UC) was evaluated along with quantification of 7 biomarkers (MDM2, CDK4, + 5 miRNAs for nucleic acid markers). Additionally, we conducted Western blot (WB) analysis using a panel of EV and control markers to confirm that the specific biomarkers being quantified are indeed derived from EVs. Notably, the analysis also meets the growing list of standards for the field, currently defined in MISEV 2023.

[0166] The WB objective was to validate the presence of canonical EV markers and assess potential contamination by cellular components, with the data showing robustness of methods and protocols providing a high degree of confidence in the downstream analysis of biomarkers. Protein markers used to assess quality of EV samples:

[0167] • TSG101: exosomal marker

[0168] • CD63 and CD81 : tetraspanins commonly present on EV surfaces

[0169] • Calnexin: an endoplasmic reticulum protein used as a negative control for cellular contamination

[0170] • Uromodulin: a major urinary protein, used as a urine-specific control

[0171] Western blot results:

[0172] • TSG101 was detected in UC-isolated urine EVs, confirming successful isolation of EVs.

[0173] • Calnexin was absent in the EV samples, indicating minimal cellular contamination. In contrast, Lipo246 cell lysates showed a strong calnexin signal, validating the specificity of the assay.

[0174] • Uromodulin was strongly detected in the 10,000g urine pellet, serving as a positive control for urinary protein content. Uromodulin presence was also observed in the UC pellet, indicating partial co-isolation with EVs.Docket No. 103362-085WO1

[0175] • CD63 appeared as a broad, smeared band in UC pellet samples, consistent with the expected glycosylation profile of this tetraspanin.

[0176] • CD81 was not detected in UC-isolated urine EVs, which is unexpected given its frequent detection in EVs reported in the literature. However, literature also notes that CD81 expression in uEVs may be suppressed.

[0177] To further evaluate the CD81+ EV sub-population, NanoFCM-based fluorescence analysis was conducted. EVs isolated from 20 mL of pooled healthy donor urine were stained with CD81-fluorophore-conjugated antibodies. CD81+ particles comprised approximately 1.8% of the total EV population in healthy urine. For comparison, EVs isolated from 20 mL of Lipo246 cell line conditioned media (cancer cell line) showed a 4.2% CD81+ population as shown in Figures 10A and 10B. These results indicate that CD81+ EVs constitute a lower fraction of the EV sub-population in urine. Due to limited cancer patient urine availability, a comparison of CD81+ EVs is not yet available.

[0178] Validation of methods against actual human patient samples.

[0179] EVs were isolated from the urine of n=19 LPS patients and compared with those of n=6 healthy donor urine samples. Healthy donor urine was commercially bought. In the first stage of the analysis, ultracentrifugation-based isolated urine EVs were used for the validation of urine as a viable biofluid for LPS detection. Urine concentration and composition can vary significantly between individuals and even within the same individual, especially over time. Therefore, levels of U-creatinine were measured in each urine sample using the Urinary Creatinine Detection Kit (Invitrogen) to provide an in-sample reference for normalization of DNA allowing for a more coherent comparison across samples.

[0180] Nano flow cytometry (NanoFCM) was used to quantify the size distribution and concentration of isolated EV samples (Figure 11). High inter-donor variability was observed in the concentration, mean, and median size of urine-derived EVs across both healthy and patient cohorts. In the 10k pellet fraction (Figures 11 A, 11B, 11C, and 11D), the mean EV particle concentration was 7.79 x 107particles / mL of urine for patient samples and 1.92 x 107particles / mL of urine for healthy controls. Notably, when normalized to U-creatinine levels (Figure 1 IB), patient samples exhibited a significantly higher EV particle concentration than healthy donor samples (p < 0.01). Although no significant difference was observed in the mean particle size (Figure 11C), the median size was significantly larger in the patient samples (p < 0.01; Figure 1 ID).Docket No. 103362-085WO1

[0181] In the UC pellet fraction (Figures HE, 1 IF, 11G, and 11H), the mean EV particle concentration further increased to 9.41 x 108particles / mL of urine for patient samples and 3.66 x 108parti cles / mL of urine for healthy controls, with the difference being statistically significant (p <0.01; Figure 1 IE). Similarly, when normalized to urinary creatinine, LPS patient samples showed a significant elevation in EV concentration compared to healthy donors (p < 0.01; Figure 1 IF). In contrast, no statistically significant differences were observed in either mean (Figure 11 G) or median particle size (Figure 11H) between the two groups for the samples tested so far.

[0182] DNA was extracted from the isolated EVs samples (10k and UC pellet) using the QIAGEN QIAamp DNA kit and quantified using the Qubit dsDNA HS Assay Kit and Qubit Fluorometer (Invitrogen). In the 10k pellet, the mean EV-DNA concentration was approximately 0.34 ng / mL of urine for patient samples and 0.20 ng / mL of urine for healthy controls (Figure 12A). When normalized to urinary creatinine, the mean EV-DNA levels were -2990 ng / mmol U-creatinine for patients and 700 ng / mmol U-creatinine for healthy controls (Figure 12B). For the UC pellet, the mean EV-DNA concentration was approximately 0.36 ng / mL of urine for patients and 0.28 ng / mL of urine for healthy donors (Figure 12D). After normalization to urinary creatinine, the mean EV-DNA levels were 2080 ng / mmol U-creatinine for patients and 853 ng / mmol U-creatinine for healthy controls (Figure 12E).

[0183] In a new analysis, the DNA density per EV was calculated for both pellet types to assess DNA content relative to EV abundance and size, using Formula 1 :

[0184] DNA density per EV fg / nm

[0185] Total isolated EV DNA per unit urine volume (fg / mE) Particle concnetration (particles / ml) Xmean EV volume (nm3) Formula 1

[0186] 4 □ where the mean EV volume was estimated assuming spherical geometry as -nr , with r representing the mean particle radius of the respective pellet from NanoFCM analysis. Notably, the analysis assumes EVs are perfectly spherical and the natural packing of DNA within an EV scales with volume. At present, there is no clear guidance or consensus on uniformity of DNA packing within EVs, and so the analysis here presents a first-order estimate to enabling another metric to compare EVs.

[0187] Based on this calculation, the mean DNA density per EV in the 10k pellet was approximately 1.58 fg / nm3for patients and 1.17 fg / nm3for healthy donors, while in the UCDocket No. 103362-085WO1 pellet it was approximately 3.45 fg / nm3for patients and 2.13 fg / nm3for healthy donors (Figures 12C and 12F). With the limited samples sizes (both in number of samples and specific EVs sampled), no statistically significant differences were observed across groups, but patient-derived EV fractions consistently showed higher mean EV-DNA concentrations and DNA density per EV.

[0188] For each sample, all isolated EV-DNA from both 10k and UC pellet fractions was used for qPCR-based quantification of MDM2 and CDK4 molecules. In the 10k pellet, EVs isolated from LPS patient urine samples exhibited a significantly higher number of MDM2 and CDK4 molecules compared to healthy controls (p < 0.01; Figures 13 A and 13B). When expressed as MDM2 molecules / ng DNA and CDK4 molecules / ng DNA, which reflect the proportion of total EV-DNA corresponding to these genes, both targets remained significantly elevated in patient samples (p < 0.01; Figures 13C and 13D). This is the first demonstration of MDM2 levels elevated in LPS patient urine, which could be an expected finding based on past reported of elevated MDM2 expression in EVs-derived from blood serum. Moreover, the discovery of elevated CDK4 levels is new, demonstrating a new biomarker for evaluation of LPS. A key discovery here is the presence of both these molecules at detectable levels in the 10k pellet, which has not been evaluated previously and assumed to be only contaminants. As the isolation of the 10k pellet is significantly faster and easier than UC, the potential for using the 10k pellet for biomarker analysis presents a new opportunity.

[0189] Similarly, in the UC pellet, patient-derived EVs contained a significantly higher number of MDM2 molecules (p < 0.05; Figure 13E) and showed an increasing trend for CDK4 molecules (Figure 13F) compared to healthy donors. In another new analysis, the proportion of MDM2 and CDK4 as a fraction total EV-DNA is estimated on a mass basis. MDM2 remained significantly elevated (p < 0.01; Figure 13G), whereas CDK4 also showed an increase (Figure 13H). Overall, these findings demonstrate that urinary EVs from LPS patients are enriched for MDM2 and CDK4 DNA fragments, reflecting tumor-specific molecular signatures within EV cargo.

[0190] Microfluidic device-based EV isolation and EV characterization for uEVs

[0191] The surface of the separation channels was functionalized with antibodies against EV tetraspanin markers CD63 and CD81, selected based on NanoFCM-based EV surface protein phenotyping results (Figures 14A and 14B). NanoFCM analysis on healthy urine isolated EVs revealed that approximately 0.4% of urine EVs were CD81+and 1.8% were CD63+, indicatingDocket No. 103362-085WO1 the presence of distinct tetraspanin-positive EV sub-populations. These findings guided the antibody cocktail (CD81 and CD63) immobilization strategy for enhanced EV capture efficiency in the separation microchannels. Electric potentials are applied across the PDMS channels and the NCAM junction as reported previously.

[0192] Microfluidic-based EVs analysis is a work-in-progress. So far, uEVs were isolated from 11 LPS patients using the microfluidic device. For EV isolation, the urine was infused into the injection channel via a syringe pump at 10 pL / min for 45 minutes (for a total volume of 0.45 mL per device, compared to 20 mL used for UC) with an applied voltage AV across NCAM of 50 V. For each patient, EVs isolated from four devices were pooled and subjected to EV-DNA extraction and quantification (Figure 15).

[0193] EV-associated DNA yield was compared between urinary EVs isolated using ultracentrifugation (UC) and those captured by the microfluidic EV isolation device (Fig. 16). UC isolation using 20 mL of urine produced a mean EV-DNA concentration of approximately 0.1 ng / mL, while the microfluidic device, operating with only 1.8 mL of urine, achieved a mean EV-DNA yield of ~0.9 ng / mL. The UC process required approximately 4 hours for isolation (not including other purification and preparation steps), whereas the microfluidic device completed EV capture within 1 hour (including set-up time), demonstrating a markedly higher recovery efficiency from a smaller urine volume and in significantly less time.

[0194] Further DNA analysis from the remaining 8 LPS patient samples, analysis for the miRNAs and the CDK4 presence is also being conducted. Notably, due to the finding of elevated CDK4 (and M / )M2) in the 10k pellet, the reject fluid stream from the microfluidic device, which contains EVs not filtered through the NCAM, is also being analyzed.

[0195] Protocol Development for Salivary EV Isolation

[0196] In addition to urine, saliva presents another non-invasively collected biofluid. At present, use of saliva for cancer biomarkers is minimal and no reports exist for use of saliva for sarcoma or LPS (sarcoma-sub-type). Therefore, the characterization of EVs isolated from saliva was initiated to test if saliva-derived EVs from LPS patients exhibit overexpression of MDM2 DNA, and other biomarkers. Overall, a multi-fluid, multi-biomarker analysis system for cancer diagnostics is being designed and if used with treatment protocols, for cancer prognostics.

[0197] Although ultracentrifugation (UC) is widely regarded as the gold standard for EV isolation from biofluids such as urine, serum, and cell-conditioned media, its application to saliva has been limited due to challenges posed by the fluid’s high viscosity, mucin content, andDocket No. 103362-085WO1 cellular debris. Therefore, there are also no reliable protocols to use for salivary EV isolation to meet the standards already set through the blood serum and urine analysis. Furthermore, there are no saliva analysis reports from a microfluidic device for LPS.

[0198] The first step, therefore, is to develop a reliable protocol to isolate and analyze EVs from UC and validate presence of adequate total DNA and then subsequently MDM2 DNA. To evaluate the feasibility of using UC for salivary EV isolation, it was examined whether previously validated urine UC protocol could be effectively applied to saliva. As shown in Figure 17A, the adapted workflow omitted the EDTA pre-treatment and sucrose cushion steps employed in the urine protocol, given the absence of uromodulin in saliva.

[0199] As an initial proof of concept, the modified UC protocol was applied to healthy control saliva samples, followed by morphological and molecular characterization of the isolated EVs. For the pilot study to validate our protocol, whole saliva samples from three healthy single donors (aged 50-70 years) were commercially acquired from Innovative Research, USA (SKU: IRHUSLS5ML). From each donor sample, 0.5 mL was used for EV isolation. Samples were thawed on ice and processed within two hours of opening to minimize degradation.

[0200] EVs were isolated following the modified UC workflow (Figure 17A), which applied the same centrifugal forces and durations as the urine protocol but omitted the EDTA and sucrose cushion steps. After sequential centrifugation and washing, the EV pellets were resuspended in phosphate-buffered saline (PBS) and imaged via transmission electron microscopy (TEM). The micrographs (Figures 17B and 17C) revealed nanosized vesicles with the characteristic cup-shaped morphology of exosomes and small microvesicles, confirming successful EV isolation from low saliva volumes (0.5 mL input) for both the 10k and UC pellets.

[0201] EV-DNA Extraction and Molecular Detection

[0202] DNA was extracted and quantified from the isolated EVs using the same workflow previously established for urine-derived EVs, ensuring methodological consistency across biofluids for comparative evaluation. Figure 17A shows that 10k pellet contained a higher total DNA yield than the UC pellet across the three healthy saliva samples (HS). The EV-DNA served as a template for quantitative PCR (qPCR) analysis targeting two tumor-associated genes, MDM2 and CDK4.

[0203] qPCR analysis confirmed the presence of both MDM2 and CDK4 in at least two of the three saliva-derived EV samples (Figures 17B and 17C), demonstrating the successful isolation and downstream molecular characterization of EV-DNA from small saliva volumes.Docket No. 103362-085WO1 As with urine, the analysis of the saliva samples also shows the importance of retaining and analyzing the 10k pellet. EV-DNA from urine, serum, and saliva samples collected from two liposarcoma (LPS) patients (LI and L3) were analyzed to compare DNA yields across biofluids. As shown in Figures 18A and 18B, the total DNA concentration normalized per milliliter of biofluid was highest in saliva, followed by serum, and lowest in urine. The comparative MDM2 number of molecules profile for these samples are presented in Figure 18B. Notably, MDM2 exists at detectable levels in EVs obtained from saliva samples. However, there are currently not enough samples analyzed to draw any definitive conclusions.

[0204] On-chip DNA extraction and analysis

[0205] In addition to biological analyses, there is continued development of physical device hardware. The EV-DNA isolation chamber shown in Figure 21 is currently being integrated into the existing device to advance towards the proposed microfluidic platform shown in Figure 20. The EV-DNA isolation chamber requires careful design, which has been a critical focus recently.

[0206] To enable systematic evaluation, multiple designs have been finalized into four distinct versions of the EV-DNA isolation chamber as shown in Figure 21. These designs are tested to evaluate efficiency of DNA extraction on-chip followed by DNA quantification. Versions 1 and 2 share a total serpentine mixer length of 13 cm, differing only in their turn geometry. Version 1 incorporates rectangular turns, whereas Version 2 uses rounded fillets to reduce flow perturbations that can arise at sharp comers. Versions 3 and 4 extend the serpentine length to 26 cm, doubling the effective mixing path. As with the shorter designs, these versions are identical except for the turn geometry, with Version 3 using rectangular comers and Version 4 incorporating rounded fillets. This design set enables a controlled comparison of how both serpentine mixer length and turn geometry influence EV lysis efficiency. Importantly, all four versions interface with an identical downstream DNA-capture chamber, ensuring that differences in performance can be attributed solely to the upstream mixing architecture.

[0207] Example 4: Evaluation of uEV-associated miRNAs

[0208] Analysis to evaluate nucleic acid biomarker correlations

[0209] Within the LPS cohort, Spearman correlation analysis among uEV-miRNA expression levels revealed coordinated expression patterns among several miRNAs (Figures 22Aand 22B). In UC-derived EVs, miR-25-3p correlated strongly with miR-93-5p (p = 0.78, p = 0.0011) andDocket No. 103362-085WO1 moderately with miR-92a-3p (p = 0.66, p = 0.013) and miR-16-5p (p = 0.57, p = 0.048). MiR-93-5p showed moderate correlations with miR-20a-5p (p = 0.55, p = 0.048) and miR-92a-3p (p = 0.50, p = 0.048). A particularly strong correlation was observed between miR-20a-5p and miR-16-5p (p = 0.85, p < 0.001).

[0210] In contrast to the UC fraction, no statistically significant differences in miRNA expression were observed between LPS patients and healthy donors in the TenK-EV fraction. Consistent with these findings, none of the tested miRNA showed meaningful discriminatory performance in this fraction, with AUC values ranging from 0.46 to 0.66, with wide confidence intervals.

[0211] miRNA analysis in Device protocol development to reliably assess miRNAs

[0212] A similar analysis was carried out for the EV fractions isolated through the device (Inlet-Reject and Device isolated). Figure 23 A, shows that for the Inlet-Reject, miR-25-3p, miR-92a-3p, and miR-20a-5p were significantly enriched in patient-derived uEVs compared to healthy controls.

[0213] A study was further conducted to obtain comparable Ct values for the reference spikein Ath-miR-159a in the Inlet-Reject (IR) fraction relative to those observed in the UC and 10k pellet fractions (Figure 23B). Initially, a significant difference in Ct values was observed between the IR fraction and the UC and 10k pellet fractions when the same amount of spike-in (25 ng) was added to each sample.

[0214] This could be attributed to column losses due to additional spin step, as the spike-in was introduced into a larger sample volume (400 pL) for the IR fraction compared to 240 pL for the other fractions, leading to a loss of detectable Ath signal. To address this issue, the spike-in amount was scaled proportionally to the sample volume, and a total of 47 ng was added to the IR fraction. Under these conditions, no significant difference in Ct values was observed relative to the UC and 10k pellet fractions, thereby achieving comparable Ct levels suitable for normalization of EV fractions isolated using the device.

[0215] Example 5: Integrated microfluidic system for extracellular vesicle DNA analysis Herein, an innovative, integrated microfluidic device that actively captures and the analyses of nanoscale particles in biofluids to determine their DNA content is reported. In particular, extracellular vesicles (EVs) are analyzed, which are nanoscale particles generated by nearly every cell type in the mammalian world. Those particles contain molecular cargo (e.g.,Docket No. 103362-085WO1 DNA, RNA, and proteins) encased in a lipid bilayer. The molecular cargo is packaged within the EVs through a variety of fundamental biological processes, leading to a size-dependent cargo composition. Moreover, in a biofluid, EV populations are highly heterogeneous (30-1000 nm sizes are common). Additionally, the EV-cargo carries a fingerprint of the parent cell, with the cargo protected in biofluids from enzymatic degradation due to the lipid bilayer envelope. At HH2024 and Traducers 2025, a novel micro-nanofluidic device to selectively sort and capture EVs with diameters < 200 nm was reported. A unique challenge for EVs due to their small size is the low biomass quantity per EV, making quantification of EV-cargo extremely challenging. Herein, for a significant advance, a new microfluidic device was designed, fabricated, and tested that can integrate with a previous microfluidic device EV-capture device to lyse and subsequently analyze DNA quantity on-chip (i.e., a new DNA-analysis module), thereby overcoming the challenge of low biomass analysis through bulk methods. By this approach, biomass is able to be quantified to as low as 0.2 ng / pL DNA lysed from ( (106) EVs, in contrast to bulk methods which require ( (1O10) EVs for equivalent analysis. The DNA-analysis module incorporates a serpentine micromixer to mix a commercial lysis buffer with an EV-enriched stream.

[0216] In a new result, Figure 25 shows for EVs derived from human liposarcoma cell-conditioned media (LCCM), that the DNA content within EVs is distinct in EVs with a mean size of -120 nm compared to a mean size of -160 nm. Thus, this is the first report that DNA content is distinct within EVs separated by a mean size of -40 nm while both sizes being within the conventionally classified small EV size range (i.e., EVs < 200 nm). Qubit DNA quantification showed higher DNA content in the flow-through EV fraction than in the permeate EV fraction. In a methodological advance, the difference persists under two different EV-lysis conditions at both room temperature (RT, useful for resource-limited conditions) and 56°C (recommended by the kit manufacturer; Figure 25 A), thus enabling for modeling on-chip lysis under RT conditions and eliminate the need for external heating. The lysis process (also verified by fluorescence imaging; Figure 26A) within the micromixer was also modelled using a convection-diffusion-reaction equation implemented in COMSOL Multiphysics to determine the number of intact EVs after lysis (Figure 26A) and subsequently estimate the DNA quantity. Briefly, using the APTES monolayer packing density (-3.15 molecules nm'2), accessible site fraction (facc= 0.7), and the total pillar surface area (A - 0.94 cm2), accounting for steric interactions between bound 150 bp dsDNA molecules, predicts a static DNA capture capacity of -1.7 pmol. The effective capture capacity was estimated to be -0.8 - 1.0 pmol, representingDocket No. 103362-085WO1 a ~7x enhancement over the -0.15 pmol binding capacity of a conventional silica spin column used for bulk DNA analysis. Numerical simulations based on this model predict greater than 85% EV lysis efficiency across multiple relevant flow conditions for different EV sizes (Figure 26B), with the lysis efficiency for this module compared to conventional bench-top methods (Figure 26C). To achieve this high sensitivity in on-chip DNA quantification, the microchannel contains a high-density PDMS micropillar (20 pm x 70pm x 60pm) array (Figure 24).

[0217] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.Docket No. 103362-085WO1

[0218] TABLES

[0219] Table 1. A comparison between various prototypes of the present device and ultracentrifugation. _

[0220] Prototype DNA Volume Time Additional notes Specificity isolated required required

[0221] (ng) / ml of per for the

[0222] starting Experiment experiment

[0223] sample

[0224] Single Channel 30-40ng 1.8mL 45min Employs an electric High EV- with EF (Pool of 4 field (EF) for DNA DNA devices) isolation, offering Specificity moderate yield in

[0225] minimal time.

[0226] Multi-channel 40-50ng 1.8mL 45min Multi-channel setup High EV- Device OV (Pool of 4 without EF DNA devices) increases yield Specificity slightly owing to

[0227] the increased

[0228] surface area for

[0229] capture.

[0230] Multi-Channel 130-140ng 1.8mL 45min Enhanced DNA High EV- with EF 50V (Pool of 4 yield due to the DNA devices) application of an Specificity electric field (50V)

[0231] in a multi-channel configuration.

[0232] Ultracentrifugation 20-30ng 20mL 200min Traditional method Lower EV- (Pool of 4 using centrifugal DNA devices) force; lower yield Specificity

[0233] (Co

[0234]

[0235] isolation)

[0236] Table 2. Current methods employed for detecting various types of cancer.

[0237] Test Galleri OncoExtra Shield Cancer Seeking Cologuard Name (GRAIL) (Guardant (Guardant SEEK Care

[0238] Health) Health) (Seekin)

[0239] Detection Multi-cancer Liquid biopsy Liquid biopsy Blood test Liquid Stool DNA Method early using cfDNA & using cfDNA detecting biopsy Test detecting detection tumor-specific and mutations mutations detecting genetic using DNA mutations and methylation mutations and methylation proteins patterns blood patterns biomarkers

[0240] Specimen Blood Blood Blood Blood Blood Stool Used (plasma or (plasma) (plasma) (plasma (plasma or

[0241]

[0242] serum) or serum) serum)Docket No. 103362-085WO1 Types of Over 50 Primarily Focuses on 8 types, Multiple Colorectal Cancer types, focuses on multiple including cancers, cancer and Detected including detecting cancers, ovarian, focusing on precancerous breast, mutations primarily liver, early polyps lung, linked to solid tumors breast, detection

[0243] prostate, cancers, colorectal,

[0244] ovarian, including lung, and

[0245] etc. multiple more

[0246] types

[0247] Time 1-2 days 1-2 weeks 1-2 weeks 1-2 1-2 weeks 1-2 weeks for Required for result for result for result weeks for result result for reporting reporting reporting for result reporting reporting Detection reporting

[0248] Volume ~10mL of ~10mL of -lOrnLof ~10mL ~10mL Stool sample Required blood blood blood of of (single sample blood blood required)

[0249]

Claims

1. Docket No. 103362-085WO1 CLAIMSWhat is claimed is:

1. A micro-nanofluidic system for collecting, extracting, and isolating an extracellular vesicle (EV) cargo from an EV sample, wherein the system comprises four or more fluidly connected components comprising a concentrator, a first microchannel, a second microchannel, and a capture channel, and wherein the concentrator, the first microchannel, the second microchannel, and the capture channel are fluidically connected to allow transport of the EV sample from one component to a second component.

2. The micro-nanofluidic system of claim 1, wherein the concentrator comprises a first electric field for separating the EV sample into at least two groups.

3. The micro-nanofluidic system of claim 1 or 2, wherein the concentrator separates EVs based on size of EVs.

4. The micro-nanofluidic system of any one of claims 1-3, wherein a first group of EVs is separated into a storage unit.

5. The micro-nanofluidic system of any one of claims 1-4, wherein a second group of EVs is directed into the first microchannel.

6. The micro-nanofluidic system of claim 5, wherein the second group of EVs are further enriched by immunoaffinity capture comprising a surface-immobilized antibody.

7. The micro-nanofluidic system of claim 6, wherein the surface-immobilized antibody comprises an anti-CD63 antibody, an anti-CD81 antibody, or a combination thereof.

8. The micro-nanofluidic system of any one of claims 1-7, wherein the first microchannel comprises a second electric field for transporting the second group of EVs into the center of the first microchannel.

9. The micro-nanofluidic system of any one of claims 1-8, wherein the second microchannel comprises a lysis buffer and a proteinase K for lysing open the second group of EVs.Docket No. 103362-085WO110. The micro-nanofluidic system of any one of claims 1-9, wherein the capture channel comprises one or more micropillars for selectively capturing the EV cargo from the second group of EVs.

11. The micro-nanofluidic system of any one of claims 1-10, wherein the EV cargo comprises a nucleic acid, peptides, or other cellular components.

12. The micro-nanofluidic system of any one of claims 1-11, wherein the system is further connected to a EV-cargo isolation chamber.

13. The micro-nanofluidic system of claim 12, wherein the EV-cargo isolation chamber comprises a EV-DNA isolation chamber.

14. The micro-nanofluidic system of any one of claims 1-13, wherein the system is further connected to a post-processing device.

15. The micro-nanofluidic system of any one of claims 1-14, wherein the EV cargo is collected in a collection container prior to entering the post-processing device.

16. The micro-nanofluidic system of any one of claims 1-14, wherein the EV cargo is immediately directed into the post-processing device.

17. The micro-nanofluidic system of any one of claims 14-16, wherein the post-processing device comprises at least one of a thermal cycler, a PCR device, a biosensor, a microarray, a sequencer, a liquid chromatographer, a mass spectrometer, or any combinations thereof.

18. The micro-nanofluidic system of any one of claims 1-17, wherein the EV is extracted from a biological sample selected from tissue, cells, sweat, blood, urine, or saliva.

19. The micro-nanofluidic system of any one of claims 1-18, wherein the system is used for detection of liquid biopsy biomarkers for cancer diagnostics, infectious disease diagnostics, drug discovery, biomarker analysis, or a combination thereof.Docket No. 103362-085WO120. A method of detecting a nucleic acid biomarker, the method comprising:a) obtaining a biological sample,b) inserting the biological sample into the micro-nanofluidic system of any one of claims 1-19, wherein said system extracts at least one EV sample from other contents in the biological sample, and wherein said system further separates and isolates an EV cargo from the EV sample, andc) detecting a biomarker within the EV cargo, wherein the biomarker is a signature of a disease or disorder.

21. The method of claim 20, wherein the biomarker comprises MDM2, CDK4, miR-16-5p, miR-20a-5p, miR-25-3p, miR-92a-3p, miR-93-5p, or a combination thereof.

22. The method of claim 20 or 21, wherein the disease or disorder comprises a cancer.

23. The method of claim 22, wherein the cancer comprises liposarcoma (LPS) cancer, prostate cancer or bladder cancer.

24. The method of any one of claims 20-23, wherein the biological sample comprises tissue, cells, sweat, blood, urine, or saliva.

25. A method of treating a subject in need thereof, the method comprising:a) obtaining a biological sample from the subject,b) inserting the biological sample into a micro-nanofluidic system of any one of claims 1-19, wherein said system extracts at least one EV sample from other contents in the sample, and wherein said system further separates and isolates an EV cargo from the EV sample,c) detecting a biomarker within the EV cargo, wherein the biomarker is a signature of a disease or disorder, andd) administering to the subject a therapy when the biomarker is detected or the biomarker levels are significantly increased compared to a subject having a healthier condition.

26. The method of claim 25, wherein the biomarker comprises MDM2, CDK4, miR-16-5p, miR-20a-5p, miR-25-3p, miR-92a-3p, miR-93-5p, or a combination thereof.Docket No. 103362-085WO1 27. The method of claim 25 or 26, wherein the disease or disorder is cancer.

28. The method of claim 27, wherein the cancer comprises liposarcoma (LPS) cancer, prostate cancer or bladder cancer.

29. The method of any one of claims 25-28, wherein the healthier condition comprises a healthy state or a state of health after receiving therapy.

30. The method of any one of claims 25-29, wherein the cancer therapy comprises an immunotherapy, chemotherapy, radiation therapy, surgery, or any combinations thereof.

31. The method of any one of claims 25-30, wherein the biological sample comprises tissue, cells, sweat, blood, urine, or saliva.

32. The method of any one of claims 25-31, wherein the system is used for detection of liquid biopsy biomarkers for cancer diagnostics, infectious disease diagnostics, drug discovery, and biomarker analysis.

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