Systems and methods for electrokinetic nanoparticle isolation and electrochemical quantification

Abstract

Disclosed herein are systems and methods for isolating and quantifying nanoparticles in high conductance media using a microfluidics-based dielectrophoresis (DEP) approach. Some embodiments employ a three-electrode geometry within a microfluidic environment that enables the system to first be run in DEP mode for nanoparticle collection, and then switched to a quantification mode employing the same three-electrode geometry. In embodiments, the three-electrode layout allows neutrally charged particles of a particular size and composition to be isolated onto the edge of a single, centrally positioned electrode, which facilitates downstream analysis and quantification. Importantly, in the disclosed systems and devices, the electrodes configured for DEP collection do not require the deposition of a protective layer, such as a hydrogel layer, to maintain functional or structural integrity during application of voltage signals during nanoparticle collection. Exemplary methods for quantification of DEP - collected nanoparticles may include immunofluorescent staining and imaging methods, colorimetric methods, and electrochemical immunostaining and sensing methods such as voltammetric analysis. The systems and methods disclosed herein are well-suited for analysis of fluid samples, including high conductance biofluid samples from which nano-sized constituent materials, or nanoparticles, are to be isolated, for example in a diagnostic or biomarker characterization context.

Description

SYSTEMS AND METHODS FOR ELECTROKINETIC NANOPARTICLE ISOLATION ANDELECTROCHEMICAL QUANTIFICATIONACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with government support under R37 CA258787 awarded by The National Institutes of Health. The government has certain rights in the invention.CROSS REFERENCE TO RELATED APPLICATION

[0002] The present application claims priority to U.S. Provisional Patent Application No. 63 / 569,688, filed March 25, 2024, the disclosure of which is hereby incorporated by reference.TECHNICAL FIELD

[0003] This disclosure relates to systems and methods for isolating and quantifying nanoparticles using dielectrophoresis-enabled devices and techniques.SEQUENCE LISTING

[0004] The Sequence Listing is submitted as an XML file in the form of the file named “Sequence.xml” (4,000 bytes), which was created on March 21, 2025, which is incorporated by reference herein.BACKGROUND INFORMATION

[0005] Dielectrophoresis (DEP) is an electrokinetic technique capable of isolating nanoparticles from a complex solution by utilizing a nonuniform electric field. This technique has been used in the past to quantify nanoparticle-bound biomarkers from undiluted plasma samples. However, the current gold standard analytical techniques for analyzing dielectrophoresis collected material lack the portability and scalability needed for clinical translation. For example, the immunofluorescent staining techniques that are frequently used to image and quantify DEP-collected nanoparticles also require microscopy systems and image analysis platforms that are cumbersome, expensive, and can suffer from low signal-to-noise ratio for quantification tasks.

[0006] A particle with a net zero permanent dipole in the presence of a nonuniform electric field, can be polarized and experience an induced electric dipole. An induced dipole can contribute to particle motion either towards regions of high electric field factors in a process termed “positive dielectrophoresis” or away high electric field factors in a process termed “negative dielectrophoresis”. The direction of particle motion can be determined based on thedielectric properties of the particle and media, while the magnitude of particle motion can be calculated by accounting for the radius of the particle and the magnitude of the electric field factor. The net force on the particle due to dielectrophoresis can thus be calculated using the classical dielectrophoresis equation:FDEP= CoW?WM*]V|f |2(1)

[0007] Where FDEPis the dielectrophoresis force, s0is the electric permittivity of free space, emis the electric permittivity of the media, R is the radius of the particle, IR[CM*] represents the real part of the complex Clausius-Mossotti factor, and V|£ |2is the electric field factor, which is equivalent to the gradient of the electric field squared. The increasing accessibility of microelectrode fabrication has led to many advancements in control over the electric field factor through precise configurations of microelectrodes. In some designs, microelectrode dielectrophoresis devices have a symmetrical electrode arrangement, which results in uniform collection around the edge of both electrode poles. Symmetrical dielectrophoresis is advantageous for applications in which recovery of larger volumes of nanoparticles is desired, and allows for more uniform collection of nanoparticles over an array of electrodes. Microelectrodes can also be designed in a manner in which one electrode pole has a smaller perimeter and thus a larger electric field factor, resulting in asymmetrical dielectrophoresis collection. Asymmetrical dielectrophoresis collection is advantageous in applications of electrode surface characterization on non-arrayed devices as a larger fraction of the desired nanoparticles will be isolated onto the surface of the higher field factor generating electrode. This is particularly advantageous for applications of electrochemical analysis described in this disclosure.

[0008] Despite advances in fabrication of microelectrodes for use in dielectrophoresis devices, challenges remain. For example, in some cases the size and material characteristics of nanoparticles targeted for isolation and the medium from which they are to be extracted necessitates the application of large voltages to drive collection. Such large voltages can have deleterious effects on the microelectrodes used to generate the dielectrophoresis forces in the sample medium, causing bubbling due to heat generation and electrochemical gas production, fouling the microelectrodes and compromising their structural and electrical integrity. These deleterious effects can be especially pronounced when the fluid medium from which nanoparticles are to be collected has a high conductivity, as is the case with biofluids such as blood or plasma. One strategy to mitigate these undesirable effects is to apply a protectivematerial over the over the microelectrodes, such as a hydrogel layer, so the sample fluid medium is not in direct contact with the energized electrodes during collection. However, addition of these protective layers adds complexity to the fabrication process and increases the electrical resistance of the electrodes thus coated. Another challenge is that after collection, many analytical techniques require that isolated nanoparticles be eluted from the microelectrode device for quantification with common analytical tools such as enzyme linked immunosorbent assays and mass spectrometry proteomics. However, immediately after collection, the highest concentration of the desired nanoparticles exists on the surface of the electrodes. Hence, rather than eluting and diluting the captured material, it would be preferable to be able to analyze and quantify the captured nanoparticles directly while they remain adhered to the electrode surface. This systems and methods described in this disclosure address these issues.SUMMARY OF THE DISCLOSURE

[0009] Systems and methods for isolating and quantifying nanoparticles in high conductance media using a microfluidics-based dielectrophoresis (DEP) approach.

[0010] In some embodiments, the systems and methods comprise a device, including a substantially planar substrate having first uninsulated sample manipulation region and an electronics interface region, but otherwise covered by a layer of insulating material. A set of three electrodes is formed upon the planar substrate in the sample manipulation region, comprising a working electrode, a first auxiliary electrode, and a second auxiliary electrode. A set of contact pads are positioned in the electronics interface region, and are in electrical continuity beneath the layer of insulating material with the set of electrodes in sample manipulation region.

[0011] Some embodiments employ a three-electrode geometry within a microfluidic environment that enables the system to first be run in DEP mode for nanoparticle collection, and then switched to a quantification mode employing the same three-electrode geometry.

[0012] Exemplary methods for quantification of DEP-collected nanoparticles may include immunofluorescent staining and imaging methods, colorimetric methods, and electrochemical immunostaining and sensing methods such as voltammetric analysis. The systems and methods disclosed herein are well-suited for analysis of fluid samples, including high conductancebiofluid samples from which nano-sized constituent materials, or nanoparticles, are to be isolated, for example in a diagnostic or biomarker characterization context

[0013] Additional aspects and advantages will be apparent from the following detailed description of preferred embodiments, which proceeds with reference to the accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Fig. 1A schematically shows an exemplary embodiment of a system configured for nanoparticle isolation and quantification.

[0015] Fig. IB shows an embodiment of a Quantitative DEP device disclosed herein.

[0016] Fig. 1C shows a detailed view of the set of electrodes in the embodiment of Fig. IB.

[0017] Fig. ID shows another embodiment of a device configured with a manifold having inlet and outlet ports to accommodate fluid loading.

[0018] Fig. 2 shows a flowchart of an exemplary method for isolating and quantifying nanoparticles present in a fluid sample.

[0019] Fig. 3A schematically shows a top view of an exemplary device wherein larger, 2 pm polystyrene beads are pushed away from the central working electrode, while smaller, 100 nm beads are collected along the edge of the central working electrode.

[0020] Fig. 3B schematically shows a side view of an exemplary device wherein larger, 2 pm polystyrene beads are pushed away from the central working electrode, while smaller, 100 nm beads are collected along the edge of the central working electrode.

[0021] Fig. 4 shows a plot comparing the normalized signals obtained using either an electrochemical immunosensing or an immunofluorescence staining approach and associated signal-to-noise ratio values.

[0022] Fig. 5 is a panel of graphs demonstrating validation of the electrochemical sensing method described herein.

[0023] Fig 6 schematically depicts an exemplary titanium / platinum conductor mask and silicon dioxide insulator mask used as part of the fabrication process for the embodiment of Fig. IB and Fig 1C.

[0024] Fig 7A shows a set of plots illustrating the use of the disclosed system to detect extracellular vesicles for use as biomarkers

[0025] Fig 7B shows a plot of a support vector machine-calculated decision boundary between cancer and healthy samples.

[0026] Fig 7C shows a plot of a receiver operator characteristic curve.

[0027] Fig. 7D shows candidate biomarker proteins that are overexpressed in PDAC cancer and are localized to exosomes.

[0028] Fig. 7E shows candidate biomarker proteins that are overexpressed in PDAC cancer and are localized to different organelles.

[0029] Fig. 7F shows candidate biomarker proteins that are localized to cellular organelles and are overexpressed by prolonged exposure to hypoxia.

[0030] Fig 8 schematically shows the elements of a handheld device for configured for nanoparticle isolation and quantification.

[0031] Fig. 9 shows an exemplary embodiment of a system for nanoparticle isolation and quantification wherein a plurality of Quantitative DEP devices are arranged in a linear array in communication with a function generator.

[0032] Fig. 10A shows an exemplary embodiment of a two dimensional array of Quantitative DEP devices wired in series.

[0033] Fig. 10B shows an exemplary embodiment of a two dimensional array of Quantitative DEP devices with interdigitated wiring.

[0034] Fig. 11A shows an exemplary embodiment of a DEP device arranged in a 96-well plate configuration.

[0035] Fig. 1 IB shows a detailed view of an exemplary embodiment of a DEP microdevice deployed in the wells of a 96-well plate configuration of a DEP device.

[0036] Fig. 12 shows an exemplary embodiment of an array of Quantitative DEP devices arranged in a 96-well plate CMOS configuration to allow control of individual wells.REFERENCE TO SEQUENCE LISTING

[0037] The nucleic acid and / or amino acid sequences described herein are shown using standard letter abbreviations, as defined in 37 C.F.R. §1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included in embodiments where it would be appropriate. The numbering of amino acid residues in each protein sequence starts from N-terminus (amino acid number 1) and goes to C-terminus.

[0038] SEQ ID NO: 1 is the amino acid sequence of the disclosed protein biomarker CD9 antigen: MPVKGGTKCIKYLLFGFNFIFWLAGIAVLAIGLWLRFDSQTKSIFEQETNNNNSSFYTGV YILIGAGALMMLVGFLGCCGAVQESQCMLGLFFGFLLVIFAIEIAAAIWGYSHKDEVIKE VQEFYKDTYNKLKTKDEPQRETLKAIHYALNCCGLAGGVEQFISDICPKKDVLETFTVKS CPDAIKEVFDNKFHIIGAVGIGIAVVMIFGMIFSMILCCAIRRNREMV. (UniProt Identifier - P21926)

[0039] SEQ ID NO: 2 is the amino acid sequence of the disclosed protein biomarker Glypican-1:MELRARGWWLLCAAAALVACARGDPASKSRSCGEVRQIYGAKGFSLSDVPQAEISGEH LRICPQGYTCCTSEMEENLANRSHAELETALRDSSRVLQAMLATQLRSFDDHFQHLLND SERTLQATFPGAFGELYTQNARAFRDLYSELRLYYRGANLHLEETLAEFWARLLERLFK QLHPQLLLPDDYLDCLGKQAEALRPFGEAPRELRLRATRAFVAARSFVQGLGVASDVVR KVAQVPLGPECSRAVMKLVYCAHCLGVPGARPCPDYCRNVLKGCLANQADLDAEWRN LLDSMVLITDKFWGTSGVESVIGSVHTWLAEAINALQDNRDTLTAKVIQGCGNPKVNPQ GPGPEEKRRRGKLAPRERPPSGTLEKLVSEAKAQLRDVQDFWISLPGTLCSEKMALSTASDDRCWNGMARGRYLPEVMGDGLANQINNPEVEVDITKPDMTIRQQIMQLKIMTNRLRS AYNGNDVDFQDASDDGSGSGSGDGCLDDLCSRKVSRKSSSSRTPLTHALPGLSEQEGQK TSAASCPQPPTFLLPLLLFLALTVARPRWR. (UniProt Identifier - P35052)

[0040] The corresponding nucleic acid sequences of each amino acid sequence provided above can be understood by one skilled in the art and are included herein without departing from the scope of this disclosure.DETAILED DESCRIPTION OF EMBODIMENTS

[0041] Disclosed herein are systems and methods for isolating and quantifying nanoparticles in high conductance media using a microfluidics-based dielectrophoresis (DEP) approach.Some embodiments employ a three-electrode geometry within a microfluidic environment that enables the system to first be run in DEP mode for nanoparticle collection, and then switched to a quantification mode employing the same three-electrode geometry. In embodiments, the three-electrode layout allows neutrally charged particles of a particular size and composition to be isolated onto the edge of a single, centrally positioned electrode, which facilitates downstream analysis and quantification. Importantly, in the disclosed systems and devices, the electrodes configured for DEP collection do not require the deposition of a protective layer,such as a hydrogel layer, to maintain functional or structural integrity during application of voltage signals during nanoparticle collection. Exemplary methods for quantification of DEP - collected nanoparticles may include immunofluorescent staining and imaging methods, or electrochemical immunostaining and sensing methods such as voltammetric analysis.

[0042] An aspect of the electrode geometry is that it allows for the use low voltages (for example, below 10 volts) to generate electric fields with steep electric field gradient magnitudes within a nanoparticle-containing, high conductivity fluid sample that are sufficiently strong to perform DEP isolation of the nanoparticles without engendering adverse electrochemical reactions at the electrodes. Accordingly, the disclosed systems eliminate the need for a protective insulative coating, such as a hydrogel layer, to be overlaid upon on electrode surfaces. Such protective hydrogel layers are often required to maintain the structural and electrical integrity of most electrode arrays operating in a high conducting media environment. By eliminating the need for an insulative layer over the exposed electrodes, manufacturing complexity and variability are reduced, and electrochemical sensing sensitivity is improved due to reduced electrical resistance.

[0043] The systems and methods disclosed herein are well-suited for analysis of fluid samples, in particular, for high conductance fluids including biofluid samples from which nanosized constituent materials, or nanoparticles, are to be isolated, for example in a diagnostic or biomarker characterization context. Exemplary biofluids include, but is not limited to, blood, plasma, serum, tears, urine, saliva, seminal fluid, pancreatic juice, bile, cerebrospinal fluid, and lymph fluid specimens. Exemplary nanoparticles contained in the biofluids that may be targeted for isolation and analysis include, but is not limited to, extracellular vesicles, cellular organelle fragments, cell free DNA nanoparticles, viruses, bacteria, and bacteria derived vesicles. The systems and methods disclosed herein may also be employed to analyze non-biologic fluid samples such as environmental fluid samples, including, but not limited to, sea water, lake water, river water, industrial synthesis and manufacturing fluids, sewage, mud, and rainwater. Exemplary nanoparticles contained in the environmental fluid samples that are targets for isolation and analysis from such samples may include, but are not limited to, plastic microparticle and nanoparticle pollutants, metallic nanoparticle pollutants, and ash particulates.Definitions

[0044] The terms “nanoparticle” or “biological nanoparticle” herein refer to naturally occurring molecules or atoms synthesized in or present in an organism or biological system with at least one dimension in the range of from about 1 nanometer (nm) to about 100 nanometer. Non-limiting examples of nanoparticles that may be isolated and / or quantified in the present systems and methods include extracellular vesicles, cellular organelle fragments, cell free DNA nanoparticles or cationic nanoparticles (e.g., nanoparticles that scavenge cfDNA to reduce inflammation / diseases), virus-like particles (VLPs) derived from viruses (e.g., Hepatitis B virus (HBV), Human Papillomavirus (HPV), Influenza virus, etc.), bacteria-derived vesicles, lipoproteins, biologically occurring metal-based nanoparticles (e.g., comprising metals such as, for example, iron, copper, zinc, silver, and gold), and circulating tumor cells.

[0045] The term “extracellular vesicles” herein refers to small membrane-bound structures that may contain as cargo, proteins, nucleic acids (DNA, mRNA, miRNA, rRNA, tRNA, etc.), polysaccharides, and / or lipids secreted by cells into the extracellular space. Non-limiting examples of extracellular vesicles include unilamellar liposomes, exosomes, autophagic extracellular vesicles, exophers, stressed extracellular vesicles, microvesicles, intraluminal vesicles, apoptotic bodies, oncosomes, matrix vesicles, migrasomes, exomeres, supermeres, macropinocytic vesicles, glial extracellular vesicles, non-vesicular particles, enveloped viruses, and membrane particles. In some examples herein, extracellular vesicles can serve as biomarkers of cellular activity, while in other examples, extracellular vesicles may serve in identifying the presence of a tumor and / or identifying tumor stage, based on their enclosed cargo and / or concentration.

[0046] The term “biomarker” herein refers to a measurable substance in sample collected from an organism or a natural system, such as bodies of water, whose presence is indicative of the state of the organism or natural system, which may include normal or abnormal states, conditions, or processes. In an organism, the presence or concentration of one or more biomarkers may be indicative of a physiological condition, disease, infection, or environmental exposure. In some instances, the presence or concentration of one or more biomarkers may be indicative of the level or progression of a physiological condition or disease, such as, but not limited to, the presence, type, or stage of a cancer, and may be utilized in determining a prognosis, diagnosis, and / or a course of treatment. A biomarker may be any molecular structure produced by a cell or organism. A biomarker may be expressed inside any cell or tissue; accessible on the surface of a tissue or cell; structurally inherent to a cell or tissue such as astructural component, secreted by a cell or tissue, produced by the breakdown of a cell or tissue through processes such as necrosis, apoptosis or the like; or any combination of these. A biomarker may be any protein, glycoprotein, carbohydrate, fat, nucleic acid, enzyme, catalytic site, or any combination of these forming a unimolecular or multimolecular structure. Biomarkers can be present on and / or within extracellular vesicles, biological nanoparticles, cell membrane, virus, cell, organ, organelles, and so on. In embodiments, biomarkers disclosed herein can be used to diagnose and / or stage pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC)) or to distinguish PDAC from benign or non-cancerous pancreatic disease such as benign cysts in the pancreas, Intraductal Papillary Mucinous Neoplasm, or pancreatitis. In other embodiments, the disclosed biomarkers can be used to diagnose and / or stage breast cancer, prostate cancer, lung cancer, colon cancer, liver cancer, ovarian cancer, bladder cancer, kidney cancer, thyroid cancer, or other diseases in a patient.

[0047] The term “cancer” refers to a disease or condition in which abnormal cells divide without normal regulation or control and are able to invade other tissues. Cancer cells spread to other body parts through the blood and lymphatic systems. Cancer is a term for many diseases. There are more than 100 different types of cancer in humans. Most cancers are named after the organ in which they originate. For instance, a cancer that begins in the pancreas can be termed a pancreatic cancer. However, the characteristics of a cancer, especially with regard to the sensitivity of the cancer to therapeutic compounds, are not limited to the organ in which the cancer originates. A cancer cell is any cell derived from any cancer, whether in vitro or in vivo. A tumor is characterized by abnormal or uncontrolled cell growth. The terms tumor and cancer are sometimes used interchangeably but can be distinguished from each other. Other features often associated with cancer include metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels and suppression or aggravation of inflammatory or immunological response, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.

[0048] The term “patient” or “subject” herein refers to a living multicellular vertebrate organism, a category that includes, for example, mammals and birds. A "mammal" includes both human and non-human mammals, such as mouse, rat, cow, sheep, pig, goat, dog, cat, or a non-human primate. In some embodiments of the present disclosure, a subject is a patient, such as a patient diagnosed with cancer. In some embodiments of the present disclosure, a subject is a patient yet to be diagnosed.

[0049] The term “tumor” herein is a lesion formed by an abnormal growth of cells (called neoplastic cells or tumor cells). A “tumor cell” is an abnormal cell divided by a rapid, uncontrolled cellular proliferation and continues to divide after the stimuli that initiated the new division ceases. Tumors show partial or complete lack of structural organization and functional coordination with the normal tissue. Usually, they form a distinct mass of tissue, either benign, pre-malignant, or malignant. Neoplastic cell growth and proliferation, whether malignant or benign, including all pre-cancerous and cancerous cells and tissues. Tumor markers include polynucleotides and polypeptides expressed by tumors to a greater extent than they are expressed by non-tumor cells, including cell surface or cytoplasmic or nuclear tumor antigens.

[0050] The term “polypeptide” herein refers to any chain of amino acids, regardless of length or posttranslational modification (such as glycosylation, methylation, ubiquitination, phosphorylation, or the like). The term polypeptide is used interchangeably with peptide or protein and is used to refer to a polymer of amino acid residues. The term residue refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic.

[0051] The term “nucleic acid molecule” as used herein refers to a polymeric form of nucleotides, which may include RNA, DNA, and synthetic forms and mixed polymers of the foregoing. The term includes single- and double-stranded forms of DNA and RNA. In some embodiments, a nucleic acid molecule may include cell-free DNA (cfDNA). A nucleic acid molecule can include either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages. A nucleotide may be a ribonucleotide, deoxyribonucleotide, or modified form of either. A “polynucleotide” refers to a physical contiguous nucleotide polymer, such as may be included in a larger nucleic acid molecule. By convention, the nucleotide sequence of a nucleic acid molecule is read from the 5' to the 3' end of the molecule. The “complement” of a nucleic acid molecule refers to a polynucleotide having nucleobases that may form base pairs with the nucleobases of the nucleic acid molecule (i.e., A-T / U, and G-C). The term “nucleic acid sequence” refers to both the sense and antisense strands of a nucleic acid as either individual single strands or in the duplex.

[0052] The term “CD9 antigen” as used herein is an integral membrane protein associated with integrins, which regulates different processes, such as sperm-egg fusion, plateletactivation and aggregation, and cell adhesion, cell motility and tumor metastasis. In pancreatic cancer, the tetraspanin CD9, a protein found on the surface of extracellular vesicles and cells, is associated with tumor aggressiveness and poor prognosis, potentially identifying pancreatic cancer stem cells and modulating glutamine metabolism to fuel tumor growth. In embodiments herein, CD9 is identified as a marker of pancreatic cancer, which can initiate and sustain pancreatic cancer growth. CD9 expression in human pancreatic ductal adenocarcinoma (PDAC) tumors negatively correlates with patient survival. Disclosed herein is the amino acid sequence of the protein biomarker CD9 antigen shown in the SEQUENCE LISTING as SEQ ID NO: 1. It is to be understood that any derivatives or modified forms of SEQ ID NO: 1 may also be included herein without departing from the scope of this disclosure.

[0053] The term “Glypican-1" as used herein refers to a protein that in humans is encoded by the GPC1 gene. It is a cell surface proteoglycan that bears heparan sulfate. Glypican-1 and its heparan sulfate (HS) chains play important roles in modulating many biological processes including growth factor signaling. The cell-surface heparan sulfate proteoglycan glypican-1 regulates growth factor action in pancreatic carcinoma cells and is overexpressed in human pancreatic cancer. In embodiments, Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Exosomes positive for the proteoglycan glypican-1 (GPC1) are expressed in serum of patients with pancreatic cancer since very early stages. Additionally, the GPC1 circulating exosomes correlate with tumor burden and could be used as prognostic biomarker in patients. Disclosed herein is the amino acid sequence of the protein biomarker Glypican-1 shown in the SEQUENCE LISTING as SEQ ID NO: 2. It is to be understood that any derivatives or modified forms of SEQ ID NO: 2 may also be included herein without departing from the scope of this disclosure.

[0054] The term “exosome” refers to nanometer-sized membrane-bound extracellular vesicles that carry proteins, lipids, metabolites, and genetic materials between cells. They are released by most cells and play a role in intercellular communication. Exosomes are involved in cancer development, including tumor growth and metastasis, and may contribute to the development of several diseases.

[0055] The term “label” herein refers to a substance capable of aiding a machine, detector, sensor, device, column, or enhanced or unenhanced human eye from differentiating a labeled composition from an unlabeled composition. Labels may be used for any of a number of purposes and one skilled in the art will understand how to match the proper label with theproper purpose. Examples of uses of labels include purification of biomolecules, identification of biomolecules, detection of the presence of biomolecules, detection of protein folding, and localization of biomolecules within a cell, tissue, or organism. Examples of labels include radioactive isotopes or chelates thereof; dyes (fluorescent or non-fluorescent), stains, enzymes, nonradioactive metals, magnets, protein tags, fluorescent proteins (e.g., GFP), any antibody epitope, any specific example of any of these; any combination between any of these, or any label now known or yet to be disclosed. A label may be covalently attached to a biomolecule or bound through hydrogen bonding, Van Der Waals or other forces. A label may be covalently or otherwise bound to the N-terminus, the C-terminus or any amino acid of a polypeptide or the 5' end, the 3' end or any nucleic acid residue in the case of a polynucleotide.

[0056] .

[0057] The term “high conductivity medium” or “high conductivity buffer” herein refers to solutions that readily conduct electricity due to a high concentration of ions, such as salts or acids. Non-limiting examples include phosphate buffered saline, such as solutions from O.IXphosphate buffered saline (PBS) to 10*PBS solutions, tris-acetate-EDTA (TAE), and phosphoric acid buffers. In some embodiments, the high conductivity buffer comprises from about 0.5x PBS to about lOx PBS. In other embodiments, the high conductivity buffer comprises from about l.Ox PBS to about lOx PBS. In the context of the applications and examples provided herein, biofluids from which biomarkers may be isolated using the disclosed systems and methods are considered to be “high conductance” or “high conductivity” media. For example, undiluted plasma, which has been measured to have a conductance of 11.75 ± 0.15 mS / cm by the inventors, is considered to be a high conductance fluid medium. By comparison, a low conductance fluid medium be would be 5 to 10 times lower.

[0058] The term “switching means” herein includes any physical, mechanical, electromechanical, or electronic means to alter the conductivity path between an electronic device, such as a voltage source, and the circuity of a connected microdevice. Examples include, but are not limited to, switches that can be operated by mechanical adjustment, electrical signal, logic controller under control of computing device such as a microcontroller, computer I / O interface. In its simplest form, a switching means can include wires or clips that may be physically repositioned to alter the conduction path, from one electronic device to another, or between electrodes of a microdevice as described herein.Systems, Devices and Methods

[0059] In the systems, devices, and methods disclosed herein, various microdevice configurations for use in DEP -based particle isolation and quantification applications are described The microdevices comprise an arrangement of electrodes with conduction paths to contact pads to allow communication with external devices such as, for example, a voltage or current sources. Before describing particular embodiments and electrode arrangements for exemplary microdevices and systems, it is helpful to introduce terminology related to the connectivity or electrical continuity of different components and regions arranged on the microdevice. Consider a microdevice having a single conduction path that forms an electrical connection between an electrode and its corresponding contact pad. This collective unit is described herein as an “electrode domain” and comprises a “node” (the contact pad) to which the electrode is “wired” via the conduction path. As used herein, an “electrode domain” may denote a single electrode or a group of separate electrodes connected to a common node. For an arrangement of two nodes (contact pads) that are wired each to its own separate electrode at the microdevice sample testing site, the resulting circuit comprises two separate electrode domains, or a “bi-domain” electrode arrangement in the context of the present disclosure. Similarly, a “tri-domain” electrode set has three separate nodes (contact pads) that are each independently connected to its own single electrode or to its own group of electrodes. Notably, a tri-domain set of electrodes may reconfigured into a bi-domain one by “shorting” or electrically connecting two nodes together, for example, connecting two of its nodes (conduction pads) together. A bi-domain configuration, however, is hard wired and cannot be reconfigured into a tri-domain arrangement because there are not three nodes available to do so. This distinction highlights that a hard wired tri-domain configuration can be made to go back and forth to a bi- domain configuration by shorting together or disconnecting two of its nodes, for example, by a switching means.

[0060] As fabricated on a microdevice, a bi-domain or tri-domain electrode arrangement establishes a hard wired connection from a set of contact pads to two or three, respectively, electrically independent electrodes at a sample test site where the electrodes reside. Using a bi- domain electrode arrangement as an example, when a high conductance fluid is placed in direct contact with and spanning the two electrodes at the sample site and voltage is applied across the associated nodes (i.e., conduction pads), a closed circuit is formed. Accordingly, an external device such as a function generator may be used to apply a voltage signal across thenodes (contact pads) to induce a nonuniform electric field within the high conductance fluid. When the electrode geometry in contact with the high conductance fluid is arranged in certain configurations and subjected to voltage signals having certain characteristics, the microdevice can be made to produce DEP forces within the fluid to preferentially attract or repel nanoparticles of various size and composition suspended in the fluid either to, or from, the electrodes. In the embodiments described herein, devices, microdevices, and systems may be configured to collect or isolate nano-sized particles (such as extracellular vesicles suspended within a biofluid) to the edge of one of the electrodes. In the embodiments described herein, the electrode to which such nanoparticles are collected is referred to as the “working electrode” or collection electrode. The other electrode of such a bi-domain arrangement is referred to herein as the “auxiliary electrode.”

[0061] Similarly, in a tri-domain electrode arrangement comprising a working electrode and a first and second auxiliary electrode, the two auxiliary nodes may be shorted or electrically connecting the two auxiliary electrodes together to operate as a single domain. In such a bidomain re-configuration, the working electrode may serve as the site of particle collection and the combined auxiliary electrodes as a ground electrode for DEP applications.

[0062] In the fabrication of the microdevices described herein and detailed in the examples below, an insulative material is deposited onto the surface of the microdevice and its electrical conduction paths to substantially cover and thereby electrically isolate all but two specific areas the electrode domains: the sample testing site and an external interface site. The sample testing site of a microdevice formed in this manner, thus, comprises an uninsulated (exposed) set of two or three electrodes and is the site at which a nanoparticle-containing fluid sample is to be deposited for nanoparticle collection. The external interface site comprises a set of two or three uninsulated (i.e., exposed) contact pads for interface with external hardware, wherein each of the electrical contact pads is in electrical continuity with a corresponding electrode at the sample testing site through the insulated conduction paths.

[0063] The application of an electrical signal, such as a time-varying voltage signal, to the contact pads of the external interface (for example, using a function generator connected the contact pads) will be conveyed through the insulated conduction paths to the set of electrodes at the sample testing site. An external switching means placed in electrical communication with the microdevice at the external interface site may be used to form (or interrupt) electrical continuity between, for example, two of the contact pads and thereby form (or interrupt)electrical continuity between the corresponding two electrodes at the sample testing site. When a high conductance fluid sample is placed in contact with the set of electrodes at the sample testing site while a voltage signal is applied across the contact pads, a nonuniform electric field is induced within the high conductance fluid at the sample testing site. This nonuniform electric field acts upon nanoparticles suspended within the fluid sample resulting in DEP collection of nanoparticles at a designated electrode (the working electrode) within the sample test site. Similarly, when a potentiostat is used to supply an input signal at the contact pads, electrochemical measurements may be made at the electrode set in contact with the fluid sample. Thus, by incorporating a switching means between the contact pads and two or more external devices (e.g., a function generator and a potentiostat), the electrode set can be made to alternate between a bi-domain and a tri-domain configuration. For example, the electrodes may be placed in a bi-domain configuration to perform in a DEP collection mode (wherein one of the electrodes - the working or collection electrode - becomes a site of nanoparticle collection) and subsequently switched to a tri-domain configuration to perform in quantification mode (wherein the set of electrodes is used to perform voltammetric measurements to characterize nanoparticles isolated onto the working electrode.

[0064] Fig. 1A shows a schematic drawing of a disclosed nanoparticle isolation and quantification system 100 configured in a first embodiment. Nanoparticle isolation and quantification system 100 includes a quantitative DEP microdevice 102, shown in one particular embodiment at exaggerated scale in Fig. 1A. DEP microdevice 104 is switchably connected for electrical communication by switching means 104 to a voltage source 106 and a current measurement device 108. In some embodiments, voltage source 102 may be a function generator and current measurement device 108 may be a potentiostat, as depicted schematically here. However, depending on the application and flexibility requirements of a specific use case, voltage source 106 may also comprise any device or circuit configured to provide the voltage signal required for the specific application. Similarly, current measurement device 16 may also comprise any suitable device or circuit suitable for the specific application. Nanoparticle isolation and quantification system 100 may also optionally comprise, in some embodiments, an imaging system (not depicted), such as a microscopy system, configured for viewing or imaging quantitative DEP microdevice 102 and any nanoparticle-containing fluid sample placed thereon.

[0065] Fig. IB and Fig. 1C depict in greater detail a specific embodiment of quantitative DEP microdevice 102, embodied here as a “lab on a chip” that has been microfabricated using layered deposition and etching techniques known in the art. Quantitative DEP microdevice 102 includes a set of electrodes 110 in a tri-domain electrode arrangement formed upon a substantially planar surface 112 of an underlying substrate material 114 and comprising a working electrode 120, a first auxiliary electrode 122 and a second auxiliary electrode 124. Working electrode 120 is centrally located within the set of electrodes 110 and, in the depicted embodiment, is circular in shape with a circumferential edge perimeter 126 along which nanoparticle collection occurs. First auxiliary electrode 122 and second auxiliary electrode 124 are positioned around, and offset from, centrally located working electrode 120.

[0066] In the embodiment depicted in Fig. 1C, first and second auxiliary electrodes 122 and 124 are semi-annular in shape and positioned on surface 112 on opposing sides of the working electrode 120. The semi-annular geometry of first auxiliary electrodes 122 is characterized by a radial width 128 bounded by an inner edge 130 and an outer edge 132. Inner edge 130 of first auxiliary electrodes 122 is offset from edge perimeter 126 of working electrode 120 by a gap distance 134. Similarly, the semi-annular geometry of second auxiliary electrodes 124 is characterized by a radial width 136 bounded by an inner edge 138 and an outer edge 140. The inner edges 138 of second auxiliary electrodes 124 is offset from edge perimeter 126 of working electrode 120 by a gap distance 142. In a typical embodiment of first and second auxiliary electrodes 122, 124, dimensioning and geometry of characteristic radial widths 128, 136, inner and outer edges 130, 138, 132, 140, and gap distances 134, 142 are equal or the same, as depicted in the specific embodiment shown in Fig. 1C. However, in alternate embodiments, the geometry, size, positioning, and spacing of first and second auxiliary electrodes 122, 124 relative to working electrode 120 may vary depending on specific requirements and restraints of the intended use case or context within which microdevice 102 is to be utilized without departing from the scope of this disclosure.

[0067] Working electrode 120, first auxiliary electrode 122, and second auxiliary electrode 124 each include an insulated conductive lead 150, 152, 154 extending outward the along surface 112 and terminating at and having electrical continuity with, respectively, a working contact pad 156, a first auxiliary contact pad 158, and a second auxiliary contact pad 160, positioned near the periphery of microdevice 102, the contact pads 156, 158, 160 sized and configured for interface with voltage source 106, current measurement device 108,switching means 104, or other electronic components or external hardware. It is noted that insulated conductive leads 150, 152, 154 (including the insulated portions in proximity of the working and auxiliary electrodes denoted by reference numbers 150a, 152a, 154a) are covered with a deposited layer of insulative material, for example silicon dioxide, at the time of fabrication. However, set of electrodes 110 (comprising working electrode 120 and auxiliary electrodes 122 and 124) and contact pads 156, 158, 160 are not covered with a protective or insulative material layer, and thus may be placed in direct fluidic contact with a fluid sample for analysis. Set of electrodes 110, contact pads 156, 158, 160, and the underlying conductive material of insulated conductive leads 150, 152, 154, 150a, 152a, 154a may be fabricated from any conductive material suitable amenable “lab on a chip” microfabrication techniques, including, for example, platinum, silver, graphene, and alloys thereof, or other suitable conductive materials known in the art.

[0068] Turning to Fig. ID, in some embodiments, quantitative DEP microdevice 102 may optionally further comprise a microfluidics manifold 170 positioned atop surface 112 and set of electrodes 110. In some embodiments, manifold 170 may include a microfluidic chamber 172 or reservoir formed above and enclosing a space about set of electrodes 110 such that a fluid sample introduced into chamber 172 will be placed in direct contact with set of electrodes 110. Manifold 172 may include an inlet channel 174 to introduce fluid into the microfluidic chamber 172 and an outlet channel 176 to remove fluid from microfluidic chamber 172.Manifold 170 may further include an inlet port 178 and an outlet port 180 for loading and rinsing a fluid sample into and from microfluidic chamber 172. Such loading and rinsing may be affected, for example using a motive means such as a syringe, pump, peristaltic pump, or other such device connected via tubing to inlet port 178 and an outlet port 180.

[0069] In some embodiments of quantitative DEP microdevice 102 a coverslip may be employed as a rudimentary manifold atop set of electrodes 110. In this embodiment the coverslip may be sized and positioned atop planar surface 112 such that one or more edges of the coverslip is aligned with or in proximity of the edges of substrate 114 of DEP microdevice 20, but leaving contact pads 156, 158, 160 substantially uncovered (i.e., exposed for connection with external electronics). In this configuration, a fluid sample placed in contact with the coverslip edge, but away from contact pads 156, 158, 160, will be drawn by capillary action beneath the coverslip and into contact with set of electrodes 110 where it may be analyzed using the methods disclosed herein. Such capillary loading may be advantageouslyemployed, for example, when using small volume of fluid samples that have been pre-labeled for analysis, or for immediate analysis of unprocessed biofluids such as, for example, blood expressed by a finger prick.

[0070] In some embodiments, the radial width of the first auxiliary electrode and second auxiliary electrodes is from about 100% of the working electrode diameter to about 150% of the working electrode diameter. In some embodiments, the radial width of the first auxiliary electrode and second auxiliary electrodes is from about 110% of the working electrode diameter to about 140% of the working electrode diameter. In some embodiments, the radial width of the first auxiliary electrode and second auxiliary electrodes is from about 120% of the working electrode diameter to about 130% of the working electrode diameter. In some embodiments, the radial width of the first auxiliary electrode and second auxiliary electrodes is aboutl25% of the working electrode diameter.

[0071] In some embodiments herein, the working electrode has a diameter of from about 10pm to about 1000pm. In other embodiments, the working electrode has a diameter of from about 10pm to about 800pm. In other embodiments, the working electrode has a diameter of from about 10pm to about 600pm. In other embodiments, the working electrode has a diameter of from about 10 pm to about 500 pm. In other embodiments, the working electrode has a diameter of from about 10 pm to about 400 pm. In other embodiments, the working electrode has a diameter of from about 10 pm to about 300 pm. In other embodiments, the working electrode has a diameter of from about 10 pm to about 250 pm. In other embodiments, the working electrode has a diameter of from about 10 pm to about 200 pm. In other embodiments, the working electrode has a diameter of from about 10 pm to about 150 pm. In other embodiments, the working electrode has a diameter of from about 10 pm to about 120 pm. In other embodiments, the working electrode has a diameter of from about 20 pm to about 120 pm. In other embodiments, the working electrode has a diameter of from about 20 pm to about 100 pm. In other embodiments, the working electrode has a diameter of from about 25 pm to about 100 pm. In other embodiments, the working electrode has a diameter of from about 30 pm to about 100 pm. In other embodiments, the working electrode has a diameter of from about 40 pm to about 100 pm. In other embodiments, the working electrode has a diameter of from about 50 pm to about 100 pm. In other embodiments, the working electrode has a diameter of from about 60 pm to about 100 pm. In other embodiments, the working electrodehas a diameter of from about 70 pm to about 90 pm. In other embodiments, the working electrode has a diameter of about 60 pm. In other embodiments, the working electrode has a diameter of about 70 pm. In other embodiments, the working electrode has a diameter of about 80 pm. In other embodiments, the working electrode has a diameter of about 90 pm. In other embodiments, the working electrode has a diameter of about 100 pm.

[0072] With respect to the gap distance between the working electrode and the auxiliary electrodes, in some embodiments, the gap distances are from about 5 pm to about 250 pm. In other embodiments, the gap distances are from about 5 pm to about 240 pm. In other embodiments, the gap distances are from about 10 pm to about 220 pm. In other embodiments, the gap distances are from about 10 pm to about 200 pm. In other embodiments, the gap distances are from about 10 pm to about 190 pm. In other embodiments, the gap distances are from about 10 pm to about 180 pm. In other embodiments, the gap distances are from about 10 pm to about 160 pm. In other embodiments, the gap distances are from about 10 pm to about 150 pm. In other embodiments, the gap distances are from about 10 pm to about 125 pm. In other embodiments, the gap distances are from about 10 pm to about 100 pm. In other embodiments, the gap distances are from about 10 pm to about 80 pm. In other embodiments, the gap distances are from about 20 pm to about 180 pm. In separate additional embodiments, the gap distances are, respectively, a) about 20 pm, b) about 25 pm, c) about 30 pm, d) about 35 pm, e) about 40 pm, f) about 45 pm, g) about 50 pm, h) about 55 pm, i) about 60 pm, j) about 65 pm, k) about 70 pm, 1) about 75 pm, m) about 80 pm, n) about 85 pm, o) about 90 pm, p) about 95 pm, q) about 100 pm, r) about 110 pm, s) about 120 pm, and t) about 125 pm.

[0073] In some embodiments, the radial width of the first auxiliary electrode and second auxiliary electrodes is between about 20 pm and about 1000 pm. In additional separate embodiments, the radial width of the first auxiliary electrode and second auxiliary electrodes is, respectively, a) from about pm mm and about 1000 pm, b) from about 20 pm and about 800 pm, c) from about 20 pm and about 600 pm, d) from about 20 pm and about 500 pm, e) from about 20 pm and about 400 pm, f) from about 20 pm and about 300 pm, g) from about 20 pm and about 200 pm, h) from about 40 pm and about 200 pm, i) from about 50 pm and about 200 pm, j) from about pm mm and about 180 pm, k) from about 80 pm and about 160 pm, 1) from about 90 pm and about 150 pm, m) from about 100 pm and about 150 pm, n) from about 110 pm and about 140 pm, and o) from about 1200 pm and about 130 pm.

[0074] It is further noted that while the working electrode depicted here is circular and the auxiliary electrodes are depicted as semi-annular, with associated dimensional ranges and positions, the disclosure is not intended to limit the electrode geometries to these shapes. Other shapes may be employed depending on the specific requirement of the particles to be collected, without departing from the intended scope of this disclosure.

[0075] In a specific embodiment of the three-electrode system shown in Fig. IB and Fig. 1C, a microdevice 110 was fabricated having a platinum central working electrode 120 diameter of 100pm, platinum auxiliary electrodes 122 and 124 having a radial width radial width 128, 136 of 125 pm , and a gap distance 134, 142 of 80 pm between working electrode 120 and auxiliary electrodes 122 and 124. In such a device having these dimensions, experiments showed that when a direct current voltage of 2.3Vdc was applied across the working an combined auxiliary electrodes, the device was a capable of generating electric field gradient magnitudes in excess of 1016. The inventors have observed that devices generating electric field gradient magnitudes less than 5xl015times the square of the applied peak to peak voltage are ineffective for isolating nanoparticles sized around lOOnm diameter from a high conductance buffer such as plasma or whole blood. It is important to note that 2.3Vdc is the point at which electrochemical oxidation of water will produce gas and disrupt collection of particles. The disclosed device is able to generate electric field gradients under direct current conditions strong enough to collect particles using DEP applying input voltages as small as 1 V across the electrodes with the electrodes in direct contact with the high conductance sample fluid. Accordingly, the large field gradient magnitude to operating voltage ratio of a device fabricated with the geometric dimensioning described here allows efficient nanoparticle trapping without the need for a protective hydrogel layer over the electrodes. The ability to successfully run DEP based collection of nanoparticles from a high conducting media without the need for a protective layer confers a significant advantage over other devices used in the electrokinetic field, where DEP collection historically has required either very low conducting buffers (which introduces a sample pre-preprocessing step which can alter physiochemical properties of materials suspended in the fluid sample ) or required a protective layer over the electrodes (which adds manufacturing complexity and increases resistance at the electrode surfaces, which increases resistance at the electrode surfaces and degrades sensitivity in electrochemical sensing techniques).

[0076] Because the systems and methods disclosed herein allow reconfiguration of the electrodes to a three-electrode setup after DEP-driven material capture, they are especially well-suited to electrochemical sensing modalities such as voltammetry, which can be employed using cost-effective and easily automatable instrumentation. Voltammetry describes a subset of electrochemical sensing techniques in which a potential is applied between a working and reference electrode, and the resulting electrochemical reactions are sensed by measuring the current between the working electrode and a counter electrode. Electrochemical sensing techniques are particularly well-suited for analysis of dielectrophoresis applications as the collected material is bound directly to the electrode surface. In particular, dielectrophoretic electrodes can be designed in such a way to isolate the diagnostically relevant particles onto the working electrode, as described herein, leaving the reference and counter electrodes unfouled.

[0077] Sensing of biological biomarkers from a complex solution, such as blood or plasma, without an inherent electrochemical reaction requires a method of labeling the collected biomarkers with electrochemical probes. Antibodies with electrochemical probes have previously been reported to show high sensitivity and specificity. While many reported probes require custom redox probe labeling of antibodies, some electrochemically active labels can be purchased commercially, as is the case with horse radish peroxidase and glucose oxidase antibodies. Electrochemical immunofluorescent sensing is well suited for multiplexed quantification of biomarkers on the electrode surface as the staining method itself is quite similar to the previously reported application of immunofluorescent antibodies, and the voltammetric sensing techniques span a window from around -2 V to +2 V in an aqueous solution, allowing for multiple probes with unique redox potentials to be quantified in a single scan.

[0078] Fig. 2 shows a high level flow chart of an exemplary method 200 for isolating and analyzing nanoparticles from a fluid sample using a microdevice such as disclosed quantitative DEP microdevice 102 using a setup such as the one depicted in Fig. 1A. Method 200 comprises, at step 205, isolating the nanoparticles from the fluid sample using DEP to collect them onto a working electrode. This step may be performed, in one embodiment, by placing the NP-containing fluid sample in contact with a set of electrodes comprising a working electrode and an auxiliary electrode and applying a voltage difference across them to generate a nonuniform electric field throughout the fluid sample. During application of voltage difference across the working and auxiliary electrodes, DEP forces act on particles suspended in the fluidsample, including the nanoparticles of interest. The working and auxiliary electrodes are arranged such that the nonuniform electric field produced by the applied voltage is incudes steep field gradients, as characterize by the electric field factor of Equation 1, in proximity of the working electrode. These steep field gradients are applied for a duration sufficient to collect nanoparticles onto the working electrode, where they remain adhered and thus isolated after application of the electric field is terminated. In a typical embodiment, the applied voltage difference across the working and auxiliary electrodes may be delivered as a time-varying potential (voltage) having a frequency and peak-to-peak voltage magnitude tuned to isolate the nanoparticles of interest based depending on their size, shape, and material properties.

[0079] At step 210 of method 200 the isolated nanoparticles adhering to the working electrode are labeled using an immunostaining method. In this step, nanoparticles may be labeled with an antibody or nanobody bound to an electrochemically active label (or tag) for subsequent electrochemical sensing, and optionally with an antibody or nanobody bound to a fluorescently active label (tag) for subsequent quantification or visualization by an imaging-based approach. Depending on the application and labeling agents available to tag the nanoparticles of interest, direct or indirect immunostaining methods may be employed at this step. For example, in an electrochemical sensing application, the electrochemical tag affixed to an antibody or nanobody may comprise an enzymatic agent such as horseradish peroxidase or glucose oxidase, or a small molecule agent, such as rhodamine. Because immunostaining methods typically entail numerous sequential processing steps such as rinsing, blocking, re-rinsing, primary staining, and secondary staining, it can be advantageous to configure microdevice 102 with a microfluidic manifold and associated fluid delivery and removal components as described above to carry out the procedures performed in step 210. At the conclusion of step 210, tagged isolated nanoparticles adhered to the working electrode are ready for quantification and / or visualization.

[0080] At step 215 of method 200 the tagged isolated nanoparticles adhered to the working electrode are analyzed using any or all of the following approaches: immunofluorescence imaging and quantification 215a, electrochemical sensing 215b, colorimetric quantification 215c, or other techniques known in the art.

[0081] For electrochemical sensing and quantification, the set of electrodes (including the working electrode to which the tagged, isolated NPs have been adhered) is placed in direct fluid contact with an electrochemical solution. For example, when horseradish peroixidase isused as tagging label, a solution containing hydrogen peroxide may be employed as the electrochemical solution. In this step the set of electrodes are further reconfigured for electrochemical sensing by splitting the auxiliary electrodes (that is, the first and second auxiliary electrodes that were configured in electrical continuity for DEP collection) into a reference electrode and a counter electrode. In this reconfiguration, the reference electrode and counter electrode have separate paths of electrical continuity, as does the working electrode. Such splitting of the auxiliary electrode into separate reference and counter electrodes may be effected by a switching means in communication with the set of electrodes to allow two electrodes to be “shorted” to function as a single auxiliary electrode, or “un-shorted” to serve as separate reference and counter electrodes. Once reconfigured in this manner, the three electrodes may be used to measure a signal in the fluid sample indicative of the quantity of tagged isolated NPs adhered to the working electrode employing an electrochemical sensing method. For example, a voltammetry method may be used wherein a voltage signal is applied across the working and reference electrodes, and the resultant current measured in the electrochemical solution between the working and counter electrodes. Examples of voltammetry methods that may be used include differential pulse voltammetry, cyclic voltammetry, square wave voltammetry, or other variants known in the art.

[0082] In some situations, it may be desirable to perform isolation and quantification of nanoparticles from a fluid sample using the disclosed systems and methods wherein the nanoparticles of interest are pre-labeled with an electrochemical and / or fluorescent tag. In such situations it may be unnecessary to configure the microdevice with a microfluidic manifold and associated channels, inlets and outlet ports, and external loading components and apparatus. Instead, the microdevice may be configured with the set of electrodes uncovered such that the fluid same may be placed in direct contact with the exposed electrodes.

[0083] The disclosed systems and methods confer several advantages over existing DEP- based collection technologies, including:

[0084] On-chip electrochemical analysis enabled by dual purpose electrodes

[0085] The disclosed device is capable of performing electrochemical analysis of dielectrophoresis-isolated material on the same electrode surface used for collection. Electrochemical analysis allows for better signal to noise ratios, increasing the sensitivity of the disclosed technology over fluorescence quantification methods. Electrochemical analysis isalso more clinically translatable due to its high sensitivity with compact electronics compared to the standard fluorescence microscopy techniques used on similar devices.

[0086] Label free nanoparticle collection from high conducting media like plasma:

[0087] Dielectrophoresis allows for isolation of particles from complex solutions based on the dielectric properties of the particles, which can avoid the need for sample preparation. Such a label-free isolation technique also allows for a wide range of particles to be isolated, expanding potential targets for biomarker panels without the need for additional capture labels in the sample preparation.

[0088] Does not require a protective coating over the electrodes:

[0089] The ability of disclosed systems and methods to successfully run DEP-based collection of nanoparticles from a high conducting media without the need for a protective layer over the electrodes is an important innovation. Traditionally, DEP collection has required either the use of very low conducting buffers from which to collection material or required a protective layer be deposited over the electrodes.

[0090] The electrode configurations and geometries described in this disclosure have been specifically optimized to generate high electric field gradients from a low input voltage. This allows for stronger dielectrophoresis collection of biological nanoparticles at lower voltages compared to other electrode designs. Metal electrode-based devices typically require a protective insulative layer over the electrodes to reduce unwanted bubbling due to heat generation and electrochemical gas production when operating in a high conducting media.

[0091] Under direct current conditions in the systems disclosed herein, unwanted bubbling occurs at 2.3 V. The device configurations disclosed herein generate electric field gradients sufficiently strong for di electrophoretic collection of particles using DC voltages as small as 1 V without any additional electrode modifications or the need for a protective layer.Eliminating the need for a protective layer over the electrodes greatly reduces the manufacturing complexity and increases reproducibility compared to other similar devices.

[0092] Small sample volume requirement:

[0093] The volume of sample needed to practice the disclosed methods is typically less than 30 pL. In some cases, samples as small as 5 pL have been used, which is far less volume than is typically extracted by a finger prick. This makes it more cost effective to add tests using thedisclosed devices to preexisting diagnostic tests, and enables routine at-home or point-of-care testing, which is invaluable for longitudinal monitoring of progressive diseases.

[0094] Highly automatable:

[0095] Both the techniques of dielectrophoresis and electrochemical analysis can be fully automated, allowing for more repeatable testing and decreasing the need for advanced training. Using electrochemical analysis, there is no need to adjust light sources or focus microscopes, substantially simplifying the quantification of DEP-collected material.

[0096] Can be used with minimal equipment:

[0097] Dielectrophoresis and electrochemical analysis can be performed using instrumentation that can be packaged into a handheld device. This makes this technology well-suited for field use and in rural settings, where access to microscopes and advanced multifunctional power supplies may be limited

[0098] This above description and following examples and the accompanying figures explain possible embodiments of the disclosed systems & methods. They are not intended to limit the implementation or practice of the disclosed systems and methods to any particular form, but rather to illustrate the principles that may be applied in numerous ways without departing from the scope of the disclosure and claims.Examples

[0099] Example 1

[0100] Computer simulations were run to analyze the effect of electrode size and spacing for a three-electrode device configured according to the geometry shown in Fig. 1C. Simulations we performed using the multiphysics modeling software package COMSOL. Table E-l shows maximum and minimum electric field factor generated for applied peak-to-peak voltage values ranging from 1 V to 10V using an exemplary three-electrode model geometry. These simulations showed high electric field factor regions, (where particles are predicted to collect due to positive dielectrophoresis) about the perimeter of the edge of the central working electrode.

[0101] COMSOL simulations of the electric field gradient predict an electric field factor greater than IxlO22V2m'3over the voltage ranges tested, which is larger than theexperimentally observed electric field factor threshold needed to isolate extracellular vesicles from plasma using dielectrophoresis. These simulations also confirmed that the electric field factor favors the edge of the central working electrode over the inner edge of the auxiliary electrodes by a factor of 998: 1. Accordingly, these simulations predict that the disclosed three- electrode configurations will generate DEP forces sufficiently large to preferentially pull the particles towards the edge of the central working electrode.

[0102] Table E-l i

[0103] Example 2

[0104] To confirm that particle motion on the device was due to dielectrophoresis rather than other electrokinetic, electroosmotic, or thermally driven forces, two sizes of polystyrene beads, 10 pm and 100 nm, were manipulated simultaneously and observed under a microscope. As expected, the larger, 10 pm polystyrene beads experienced negative dielectrophoresis under a 14 kHz applied electric field, while the smaller, 100 nm polystyrene beads experienced positive dielectrophoresis. Fig. 3A and Fig. 3B show in schematic form top and side views, respectively, of the observed bead motion during DEP isolation and after DEP isolation. These diagrams illustrate that after 5 minutes of applying the 14 kHz electric field, the smaller beads were drawn centrally and downward where they collected around the working electrode edge (as observed under a light microscope). Conversely, the larger beads were repelled upwardly and outwardly away from the working electrode. Fluorescent imaging further confirmed that after DEP isolation the smaller beads were aggregated about the perimeter of working electrode while the larger beads were pushed away from the working electrode. These observations of simultaneous positive and negative dielectrophoresis confirmed that the primary driving force of the disclosed device is dielectrophoresis rather than electrophoresis, electroosmosis, or thermally driven convection.

[0105] .Example 3

[0106] To confirm the utility of the disclosed system for isolation of extracellular vesicles, a preliminary experiment was conducted using dielectrophoresis isolation followed by immunofluorescent staining. In this particular example, extracellular vesicles from an undiluted plasma sample were introduced into the microfluidic chamber of Fig. ID and successfully captured around the central working electrode edge. The captured extracellular vesicles were subsequently labeled using fluorescently labeled antibodies against CD9, a characteristic surface protein marker expressed in most extracellular vesicles. It was observed that after initial isolation of the vesicles from plasma, the collected vesicles were adhered to the electrode surface and remained in place after rinsing with buffer. The vesicles also remained adhered to the electrode surface through multiple rounds of immunofluorescence staining.

[0107] A more comprehensive and detailed example of extracellular vesicle isolation using the DEP device is presented below in Example 7.

[0108] Example 4

[0109] Scanning electron microscopy (SEM) was used to further confirm the isolation of extracellular vesicles using the disclosed system & methods. In this confirmatory example, purified vesicles from cell culture were used to avoid unwanted collection of protein and cellular debris typically present in biological samples, so that the extracellular vesicles could be readily distinguished in the SEM images. It was confirmed by SEM imaging that the collected vesicles adhered to the perimeter of the working electrode in this test. The adhered extracellular vesicles were found to have a diameter around 100 nm to 200 nm, which is within the expected size range of 50 to 2000 nm established for extracellular vesicles.

[0110] Example 5

[0111] Fig. 4 shows exemplary data for a comparison of quantification methods applied to DEP-isolated extracellular vesicles. Extracellular vesicles were collected on the central electrode from an undiluted plasma sample using dielectrophoresis as described in Example 3. The collected extracellular vesicles were subsequently stained for CD9 using a horseradish peroxidase conjugated antibody as a redox reagent. Quantification of the collected vesicles wasperformed using an electrochemical immunosensing (ECIS) and an immunofluorescence (IF) staining imaging-based measurement technique. The ECIS quantification in this specific example was performed using differential pulse voltammogram cyclic voltammetry. Collection of voltammograms (current-vs-potential curves) was performed using a substrate solution of 0.03% hydrogen peroxide. Voltammograms and IF measurements were obtained for five replicates (z.e., five microfluidic DEP devices manufactured to the same specifications were used), along with a measure of background signal with and without a substrate solution of 0.03% hydrogen peroxide.

[0112] The results for the five replicates are shown in bar graph form on the left in Fig. 4 and in tabular form on the right. The ECIS technique showed superior performance as quantified by signal-to-noise ratio (SNR), with an SNR of 533 using ESIS sensing versus an SNR of 42 for the IF technique. The coefficient of variation in SNR across five replicates was 18% for immunofluorescence, and 16% for electrochemical analysis. N = 5, error bars are mean ± standard deviation, **** ; p < 0.0001 by students T-test.

[0113] A more comprehensive and detailed example of extracellular vesicle quantification using the DEP device is presented below in Example 7.

[0114] Example 6

[0115] This example presents additional experimental data with more comprehensive experimental details on the materials and methods used for validating the performance of the electrochemical sensing approach described herein. While this example uses a Potassium Ferricyanide solution for demonstration purposes, practice of the disclosed systems and methods are not intended to be limited thereto.

[0116] Materials: Potassium ferricyanide was purchased from Millipore Sigma (Burlington, MA, Cat # 702587). Gibco Dulbecco’s phosphate buffered saline (PBS, IX) was purchased from Thermo Fischer Scientific (Waltham, MA, Cat. # 14190-136, lot # 2323601). PBS was diluted to 0.5X and filtered through a 0.1 pm syringe filter immediately before use.

[0117] Standard Addition of Potassium Ferricyanide: A stock of 15.2 mM potassium ferricyanide in 0.5X PBS was prepared and two-fold serial dilutions were performed to prepare samples with potassium ferricyanide of 15.2, 7.60, 3.80, 1.90, and 0.950 mM respectively along with three samples of 0.5X PBS to establish the background signals. Eight microelectrode devices were prepared as described in section 2.2 and 20 pL of each dilution was introduced toeach respective device. Cyclic voltammetry experiments were performed using a Wavenow wireless potentiostat from Pine Research (Durham, NC, Cat. # AFTP4). A sampling range of 5 pA was used for all differential pulse voltammetry measurements. Cyclic voltammograms were recorded using a 2-segment excitation waveform beginning at -1000 mV, inflecting at +1000 mV, and returning to -1000 mV with a 100 mV / s sweep rate. Baseline subtracted peak heights were determined using the peak height tool in the Aftermath software. Peak heights across three replicates were recorded in Excel and fit using linear regression via the linest function to determine the lower limit of detection, which was defined as 3 standard deviations above the background signals.

[0118] Scan Rate: Five microelectrode devices were prepared in the geometric configuration shown in Fig. IB and Fig. 1C and 20 pL of a solution of 15.2 mM potassium ferricyanide in 0.5X PBS was introduced to each device. Cyclic voltammetry was performed on each device using the parameters described in the previous paragraph with the excitation scan rates of 50, 100, 150, 200, and 300 mV / s respectively for each device. Baseline subtracted peak heights were determined using the peak height tool in the Aftermath software. Peak heights across three replicates were recorded in Excel and fit using linear regression via the linest function. The roughness factor of the device was calculated using the Randles-Sevcik equation.

[0119] Results: Fig. 5 shows exemplary results from the above-described experiment. A representative cyclic voltammogram of dilution series of potassium ferricyanide in 0.5X PBS is shown (only curves for concentrations of 15.2 mM and 1.9 mM are depicted for clarity). Linear fit of peak anodic currents (Ipa) revealed a roughness factor of 4.34 using the Randles-Sevcik equation (n=3, R2=0.9932). Extrapolation of a linear fit of the dilution series with an Ipanoise threshold (3o above background mean) of 4.35E-2 pA revealed a lower limit of detection of 0.143 mM potassium ferricyanide (n=3, R2=0.9999).

[0120] Example 7

[0121] The following paragraphs in this Example 7 section describe detailed materials and methods used to conduct the isolation and quantification experiments reported in Example 3 and Example 5 above. The specific materials and methods described here are not intended to limit the scope or practice of the methods, procedures, or materials set forth in this disclosure, but rather to provide an example that may be readily adapted for other use with different reagents, operating parameters, etc.

[0122] Materials: Gibco Dulbecco’s phosphate buffered saline (PBS, IX) was purchased from Thermo Fischer Scientific (Waltham, MA, Cat. # 14190-136, lot # 2323601). PBS was diluted to 0.5X and filtered through a 0.1 pm syringe filter each day before use. Powdered skim milk was purchased from Midland Scientific (Omaha, NE, Product # 232100, lot # 8211649).Al exafluor-488 conjugated goat anti-mouse IgG antibody was purchased from Invitrogen (Waltham, MA, Cat. # Al 1001). Hydrogen peroxide (30%) was purchased from Fisher Scientific (Waltham, MA, Cat # H325-100). An enzyme linked immunosorbent assay kit for human CD9 was purchased from AntibodiesOnline.com (Limerick, PA, Cat. # ABIN6954541). Pooled healthy plasma samples were purchased from Innovative Research (Novi, MI, Cat. # IPLAWBK2E).

[0123] Standard Addition Series of Purified Extracellular Vesicles in Plasma: The concentration of the extracellular vesicle surface marker CD9 on extracellular vesicles isolated from HeLa cell culture media was determined using an enzyme linked immunosorbent assay. The same assay was used to quantify the presence of CD9 in an undiluted plasma sample. Hela extracellular vesicles were diluted 1 : 10 into a plasma sample. A two-fold serial dilution into plasma was performed to create five dilutions with extracellular vesicle concentrations of 1 : 10, 1 :20, 1 :40, 1:80, and 1 : 160. Vesicles were isolated from each aliquot via dielectrophoresis using the protocol described in the next paragraph, after which they were stained using either immunofluorescent or electrochemically labeled secondary antibodies (see below for electrochemical labeling details).

[0124] Dielectrophoresis: After backfilling the microfluidic device with 0.5X PBS, a 20 pL sample was loaded into the inlet port and drawn into the device at a rate of 7 pL / min. A 14 kHz, 8 Vpp sine wave was applied between the central working electrode and the two auxiliary electrodes using a 33210A function generator from Keysight Technologies (Santa Rosa, CA) with impedance set to high Z mode. The alternating field was applied for 10 minutes, then the field was turned off and the channel was rinsed with 50 pL of fresh 0.5X PBS.

[0125] Immunostaining of Dielectrophoresis Isolated Material: After DEP isolation and rinsing, 20 pL of a blocking solution of 2% skim milk was introduced to the channel and incubated for 30 minutes at room temperature to reduce nonspecific binding. A solution of 20 pL of 1 : 100 unlabeled mouse anti-CD9 in 2% milk was introduced to the fluidic chamber and incubated at room temperature for 1 hour. The fluidic chamber was rinsed with 50 pL of 2% milk before secondary staining.

[0126] Immunofluorescent Staining and Imaging: A solution of 1.500 Al exafluor-488 conjugated goat anti-mouse IgG in 2% milk (20 pL) was introduced to the primary stained device prepared in 2.4.3 and incubated for 1 hour at room temperature. The device was rinsed with 50 pL of fresh 0.5X PBS and images using a Zeiss Axio Imager A2 equipped with a monochromatic Axiocam 506 (The Zeiss Group, Oberkochen, Germany). Reflectance illumination was provided by an X-Cite 120 LED Boost High-Power illumination system (Excelitas Technology, Waltham, MA). Brightfield images were collected with a 250 ps exposure time with 4% light source intensity. Fluorescence images were captured using an EGFP filter cube set with an exposure time of 2000 ms with 100% light source intensity.

[0127] Electrochemical Staining and Sensing: A solution of 1 : 500 horse radish peroxidase conjugated goat anti-mouse IgG in 2% milk (20 pL) was introduced to the primary stained device prepared in 2.4.3 and incubated for 1 hour at room temperature. The device was rinsed with 50 pL of fresh 0.5X PBS followed by 20 pL of a solution of 0.5X PBS containing 0.03% v / v hydrogen peroxide. Differential pulse voltammetry experiments were performed using a Wavenow wireless potentiostat from Pine Research (Durham, NC, Cat. # AFTP4) immediately after introduction of the hydrogen peroxide solution. A sampling range of 5 nA was used for all differential pulse voltammetry measurements. Differential pulse voltammograms were recorded using a 2-segment excitation waveform beginning at -1000 mV, inflecting at +1000 mV, and returning to -1000 mV. The excitation waveform increased at 10 mV per 10 ms period. Pulses of 200 mV for 50 ms were applied at the leading edge of the excitation waveform. A sampling period of 3 ms was used for both pre-pulse and post-pulse measurements.

[0128] Example 8

[0129] The following paragraphs in this Example 8 section describe detailed materials and methods used to fabricate a DEP device as described herein. The specific materials and methods described in this Example 8 are not intended to limit the scope or practice of the methods, procedures, or materials set forth in this disclosure, but rather to provide an example that may be readily adapted for other use with different reagents, operating parameters, etc.

[0130] Exemplary detailed materials and methods for microfabricaton of devices

[0131] Materials: Photomasks for resist patterning were purchased from ArtNet Pro Inc (San Jose, CA). Silicon wafers <100> precoated with a 100 nm thick layer of low-pressure chemical vapor deposited silicon nitride was purchased from University Wafer (ID: 1919, South Boston,MA). Negative resist NR9-1500PY (NR9) and tetramethylammonium hydroxide resist developer RD6 were purchased from Futturex, Inc. (Franklin, NJ). Acetone (cat # NC9909295), Methanol (cat. # NCI 689510), and Isopropanol (cat. # NC9869909) were purchased from High Purity Products (Portland, OR).

[0132] Physical Vapor Deposition of Platinum Microelectrodes: Concentric microelectrodes were fabricated using photolithography and physical vapor deposition in a class 100 cleanroom. A 2 pm thick layer of negative photoresist NR9 was spin coated onto the surface of the wafer. The photoresist was patterned using a MA6 mask aligner from Suss Microtech (Garching, Germany). The patterned photoresist was developed by submersion in RD6 developer solution for 12 seconds with gentle agitation followed by a thorough rinse with ultrapure water to remove excess developer. The wafer was dried using compressed air and loaded into a physical vapor deposition chamber from Kurt J. Lesker (Jefferson Hills, PA). After a descum plasma cleaning to remove photoresist imperfections, a 50 nm thick layer of titanium was sputtered onto the wafer surface to increase adhesion of the platinum layer. Immediately following titanium deposition, a 150 nm thick layer of platinum was deposited onto the wafer. Fig. 6 (left) shows a drawing of an exemplary titanium / platinum conductor mask used in this step. The wafer was removed from the process chamber and sonicated in an acetone bath for 10 minutes to remove masked the photoresist, revealing the patterned platinum electrodes. The wafer was rinsed generously with acetone, methanol, and isopropanol.

[0133] Physical Vapor Deposition of Silicon Dioxide: To reduce unwanted interactions with embedded wiring on the device, an insulative layer of silicon dioxide was deposited onto the wafer. Note that this layer does not cover the platinum electrodes, allowing for direct contact of the platinum microelectrodes with the sample where dielectrophoresis occurs, therefore this device is not an insulator dielectrophoresis device.

[0134] A second layer of NR9 was spin coated onto the wafer prepared above and patterned using a second photomask using a mask aligner followed by development via submersion in a bath of RD6 for 12 seconds. The wafer was sensed with deionized water and dried with compressed air. The wafer was loaded into the physical vapor deposition chamber. After photoresist descumming, a 300 nm thick layer of silicon dioxide was sputtered onto the wafer surface. Fig. 6 (right) shows a drawing of an exemplary silicon dioxide insulation mask used in this step. The wafer was removed from the chamber and sonicated in acetone to removephotoresist, revealing silicon dioxide. The wafer was rinsed generously with acetone, methanol, and isopropanol.

[0135] Wafer Dicing and Processing: The wafer was then coated in an unexposed layer of NR9 and diced using a DAD3221 dicer from Disco Hi-Tec (Tokyo, Japan). After Dicing, the individual devices were sonicated in acetone for 10 minutes to remove NR9 and rinsed generously with acetone, methanol, isopropanol, and water. The diced wafers were then plasma cleaned for 15 minutes using a Tergeo plasma cleaner from Pie Scientific (Union City, CA) to remove residual photoresist and increase electrode wettability. Custom fluidics were manufactured using double sided tape adhered to the bottom of a 19x19 mm, no. 1 glass cover slip with an inlet and outlet port allowing for an approximately 20 pL sample to be loaded into the device.

[0136] Example 9

[0137] The clinical diagnosis of pancreatic cancer is difficult. Pancreatic cysts are often found coincidentally in asymptomatic patients through computed tomography and magnetic resonance imaging. Pancreatic cysts can also be found by endoscopic ultrasound which is prescribed when patients present with clinical symptoms that are consistent with pancreatic cancer such as abdominal pain, jaundice, and weight loss. Patients found to have pancreatic cysts need further tests (e.g., an invasive biopsy procedure) to determine if the cyst is pancreatic ductal adenocarcinoma (PDAC). A blood test to reliably detect PDAC specific biomarkers could identify high-probability patients that require the invasive biopsy procedure as a diagnostic test. Currently, CA 19-9 is the standard blood-based pancreatic cancer biomarker; however, it is only used to characterize known PDAC tumors and is not recommended for PDAC screening. Thus, there exists an unmet clinical need to gain access to new sources of circulating PDAC biomarkers for cancer screening.

[0138] Patient Cohort. 52 patient plasma samples were collected from patients having either pancreatic ductal adenocarcinoma (PDAC) or benign pancreatic disease. PDAC patients were selected with cancer stages from 1-4. Healthy patients consisted of patients presenting to the clinic later diagnosed with non-cancerous intraductal papillary mucinous neoplasm (IPMN), pancreatitis, or benign pancreatic cysts. The selected cohort consisted of 52 patients with an average age of 64.5 and a 50:50 sex balance. The cohort comprises the following disease types: 8 patients with benign pancreatic cysts, 8 patients with non-cancerous IPMN, 8 patients with pancreatitis, and 28 patients with PDAC. PDAC patients comprise patients with different stagesof the cancer: 6 patients with Stage 1 PDAC, 6 patients with Stage 2 PDAC, 8 patients with Stage 3 PDAC, and 8 patients with Stage 4 PDAC. The non-cancerous pancreatic diseases may include benign cysts, IPMN, and pancreatitis. The benign refers to benign cysts in the pancreas, IPMN refers to a pre-cancerous lesion called Intraductal Papillary Mucinous Neoplasm, and pancreatitis refers to a non-cancerous inflammatory disease.

[0139] Methods'. All samples were drawn into EDTA vacutainers and underwent identical plasma isolation protocols. The samples were blinded by the OHSU CEDAR Repository. 30 pL of each undiluted plasma sample was introduced to the electrode device, and a 14 kHz, 8 Vpp sine wave was applied across the working and auxiliary electrodes to drive DEP collection of extracellular vesicles to the working electrode edge. The field was applied for 8 minutes and turned off. A rinse of 50 pL of 0.5X PBS was used to remove uncollected material. The remaining EVs on the electrode surface were stained with a 1: 100 solution of either mouse anti CD9, rabbit anti GPC1 , or mouse anti cfDNA antibodies diluted 1 : 100 in 2% milk and incubated for 1 hour. After rinsing with 50 pL of 0.5X PBS, a respective goat anti rabbit or goat anti mouse rhodamine conjugated secondary antibody diluted 1:500 in 2% milk was introduced to the chamber and incubated for 1 hour. Finally, the chamber was rinsed with phosphate buffer and square wave voltammetry from 0-2V with a frequency of 17 Hz was used to determine the presence of the rhodamine labeled secondary via linear baseline peak height extraction around 1.5V.

[0140] The resulting values for GPC1, CD9, and cfDNA were plotted against each other and a decision boundary was determined using a support vector machine using the SVC function of the sklearn (vl.6.1) package in python (vl3.3.1). The SVC used an equal weighted second order polynomial kernel with a tolerance of IxlO'3, C value of 50, coefO of 0.7, gamma value of 0.1, and a one verses rest decision function shape.

[0141] Results'. FIG 7A shows the performance of each biomarker individually. Both the GPC1 and CD9 biomarkers show significant differences between cancer and noncancerous controls. The cf-DNA biomarker does not show a significant difference, but it does contribute to improved performance of the combined panel of all three biomarkers when analyzed using SVM. The nanoparticles released by PDAC tumors into circulation carry with them biomarkers that can be used to differentiate PDAC from benign pancreatic disease and would allow physicians to stratify patients into categories of high-probability and low-probability for the presence of a PDAC tumor.

[0142] FIG 7B: shows the decision boundary drawn between cancer and non-cancerous pancreatic disease control samples based on a bivariate analysis of GPC1 and CD9 using a support vector machine learning model. The cancer is specific to PDAC and relates to specific stages of PDAC (6 Stage 1, 6 Stage 2, 8 Stage 3, and 8 Stage 4).

[0143] FIG 7C: This ROC analysis shows the performance for Stage 1 and 2 PDAC patients compared to non-cancerous pancreatic disease controls using the combined biomarker panel of CD9, Glypican 1, and cf-DNA showing an AUC of 0.93 which is a better performance than the traditional endoscopic ultrasound procedure used to diagnose pancreatic cancer clinically (0.79).

[0144] Disclosed herein are the amino acid sequences of the protein biomarkers CD9 antigen and Glypican- 1 shown in the SEQUENCE LISTING as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. It is to be understood that any derivatives or modified forms of SEQ ID NO: 1 and SEQ ID NO: 2 may also be included herein without departing from the scope of this disclosure. Cancer derived nanoparticles carry with them protein biomarkers that can be used to differentiate patients with PDAC from those with benign pancreatic disease. This embodiment of the present disclosure characterizes the ability of the DEP technology to recover these nanoparticles enabling immunostaining detection and direct comparison between patients for PDAC biomarkers. Also, DEP-based recovery method allows study of biomarkers carried by many different particle types from a single volume-limited patient sample.

[0145] FIG 7D shows a table for known biomarker candidates from the literature and their ability to differentiate PDAC from healthy patients. These biomarkers are overexpressed in PDAC cancer and are localized to exosomes. These biomarkers were identified using conventional methods to recover exosomes from plasma samples. The volume requirements, in case of conventional methods, prevent multiple nanoparticle types (such as EVs and organelle fragments) from being evaluated in a single patient sample. The present embodiment evaluates known PDAC biomarkers carried by extracellular vesicles for compatibility with DEP collection and detection.

[0146] FIG 7E shows a table for biomarker proteins that are overexpressed in PDAC cancer and are localized to different organelles (e.g., endoplasmic reticulum, Golgi apparatus, mitochondria, centrosomes, and nucleus). This set of biomarkers carried by organelles are proteins derived from biochemical pathways overexpressed by PDAC and are distinct from the biomarkers carried by the actively secreted EVs and exosomes. The present embodimentevaluates circulating nanoparticles derived from cellular organelle fragments for DEP-based collection and detection of known PDAC biomarkers to differentiate PDAC from benign pancreatic disease. This set of biomarkers disclosed in FIG 7E are known to be overexpressed in PDAC through oncogenic pathways. Additionally, the present disclosure indicates that the level of the candidate PDAC biomarker UQCRC1, carried by the mitochondria fragments, is significantly higher in the PDAC patients compared to the non-cancerous controls

[0147] FIG 7F shows a table for biomarker proteins that are localized to cellular organelles and are overexpressed by prolonged exposure to hypoxia. This set of protein biomarkers are proteins related to unique adaptations acquired by cancer cells to survive prolonged exposure to hypoxia and necrosis in the tumor environment. Cells eventually necrotically die, lyse open, and release organelles that carry these protein biomarkers. Many of these hypoxia and necrosis related biomarkers overexpress in PDAC including DRP1, AURKA, PLK4, Cyclin E, ERO IL, ARG2, and HMGB1 . Biomarkers related to tumor hypoxia and necrosis are distinct from nontumor tissue and can differentiate PDAC from benign pancreatic disease. It is to be noted that the tumor hypoxia biomarkers shown in FIG 7F would apply to any type of cancer that has a hypoxic core, while the biomarkers shown in FIG 7D and FIG 7E are known to be overexpressed in pancreatic cancer.

[0148] Overall, the data indicates that DEP with cyclic voltammetry biomarker quantification can distinguish PDAC from benign pancreatic diseases very well and it is also capable of distinguishing early stage PDAC (stages 1 and 2) from benign pancreatic diseases with an AUC of 0.93. This is compared to the traditional invasive endoscope ultrasound guided fine needle aspiration biopsy with an AUC of 0.79 (the stages 1 and 2 of PDAC are combined to get this value). The test may not be able to indicate the difference between stage 1 and stage 2 of PDAC, however, it can certainly indicate whether an early-stage tumor is present in a subject.

[0149] Although the present disclosure includes a patient cohort related to pancreatic cancer and that there is an unmet need for reliable detection of PDAC specific biomarkers, the disclosed device and DEP-based technique can be used for any cancer type with known biomarkers. For example, stage 2 liver cancer can be detected using DEP technology with Glypican-1 and cfDNA. The systems and methods of the present disclosure may easily be adapted to other cancer types, that may include without limitation, breast cancer, prostate cancer, lung cancer, colon cancer, liver cancer, ovarian cancer, bladder cancer, kidney cancer, thyroid cancer, or other diseases in a patient.

[0150] Example 10

[0151] Fig. 8A shows an exemplary schematic wiring diagram 300 of a combined handheld device 302 capable of performing dielectrophoresis isolation and subsequent electrochemical analysis using embodiments of the DEP microdevices described herein. The portable device is powered by a rechargeable 3.7V battery pack 304 and is equipped with a primary microcontroller 306 which can activate a second microcontroller programmed to act as a function generator 308 to output a 14 kHz, 7.4 Vpp sine wave to drive dielectrophoresis collection of particles on a microelectrode device 310. After dielectrophoresis isolation, primary microcontroller 306 activates a micro potentiostat 312 to run voltammetric quantification of the collected material. The status of the device is displayed by an indicator LED light 314 attached to primary microcontroller 306. Data is collected and processed on primary microcontroller 306 and can be exported via a USB output port 316.

[0152] Fig. 8B shows a line drawing of an exemplary combined handheld device 302, depicted here in a housing resting in the palm of a hand. A microelectrode device 310 is inserted into a receiving slot 318 for interface with hardware components housed within and configured according to schematic wiring diagram 300. In the embodiment depicted here, sample fluid can be loaded into an inlet tube 320 and introduced into the fluidic chamber using a syringe 322 connected to an outlet tube 324. Once the sample is loaded, the automated dielectrophoresis isolation and subsequent electrochemical quantification process can be started by turning on device 302 via a switch 326 on the top surface of the housing. Data can be exported from device 302 using a USB port on the back of the device (not depicted).

[0153] Example 11

[0154] Fig. 9 shows an example of a high throughput configuration 400 of system 100 employing multiple, linearly arrayed DEP microdevices 401 wired in parallel. In this linear array configuration, when current or voltage is applied by function generator 304, all of the linearly arrayed DEP microdevices 402 having electrodes sets 410 exposed to a fluid sample will be activated in parallel to induce dielectrophoretic action within the fluid sample. Such a linear array configuration may be adapted in some embodiments to include, for example, a multi-reservoir type manifold to partition the electrode sets 410 of each of DEP microdevices 402 into separate open-topped “wells.” In other embodiments, the linear arrayconfiguration may be adapted to incorporate a closed manifold design comprising a set of microfluidic chambers partitioning and enclosing each DEP microdevice’s 402 electrode set 410 separately, and wherein the manifold may be configured with either a shared inlet and outlet port to load all chambers from a common fluid sample source, or with each chamber having its own inlet and outlet ports for individualized fluid sample loading. In both of the manifold partitioning embodiments described here (i.e., the open well or microfluidic chamber partitioning schemes), it is noted that contact pads 456, 458, 460 of each of DEP microdevices 402 would typically be positioned outside the reservoir wells or microfluidic chambers, away from fluid contact, and positioned to allow interface with external electronic equipment.

[0155] It should also be noted that although the configuration of Fig.9 depicts a linear array of microdevices 402 only in communication with a function generator 404, alternate embodiments may further incorporate a potentiostat and switching means, analogous to the configuration of system 100 shown in Fig. 1A. Such a configuration would allow the practice of previously described methods of DEP isolation of nanoparticles from a fluidic sample employing function generator 404 followed by (i.e., switched to) voltammetric measurement employing a potentiostat to quantitate the captured nanoparticles.

[0156] Example 12

[0157] In some embodiments, the tri-domain electrode arrangement (as depicted in Fig. 1C) of a central working electrode flanked on opposing sides by two auxiliary electrodes may be replaced by a plurality of such electrodes. Fig. 10A shows one such exemplary embodiment, where an array of six working electrodes (arranged three per row) are each flanked by a pair opposing auxiliary electrodes. In such a configuration, the size, spacing, and arrangement of the plurality of working electrodes and their respective flanking auxiliary electrodes may be reduced to accommodate the desired fluid sample volume while providing a plurality of working electrodes on whose perimeters the nanoparticles of interest can be collected. In the embodiment depicted in Fig. 10A, the routing of the insulated lead wire joining the plurality of working electrodes in series traverses a snaking or zig-zag pattern through the arrayed layout. Such a series conduction path with multiple particle collection sites, despite its complexity, still represents a tri-domain electrode configuration. The routing and branching of the insulated lead wire joining the first set of auxiliary electrodes and the insulated lead wire joining the secondset of auxiliary electrodes (labeled Auxl and Aux2, respectively, in the figure) gives rise to the spatial arrangement of the paired auxiliary electrodes flanking the rows of working electrode as shown. When the number, size, and spacing of the working and auxiliary electrodes are configured to optimize the voltage required for collection, it was observed that different sized particles were collected on different rows of the array. It is also noted that because this configuration employs three electrode domains (one working and two auxiliary electrode domains), it is suitable for both DEP collection and electrochemical chemical measurement (e.g., voltammetry) applications. Further, the arrayed electrode arrangement of Fig 10A may be deployed, for example, in systems comprising a single microdevice as shown in Fig. 1A, a handheld device as described in Example 10, or a linearly arrayed system as described in Example 11.

[0158] Fig. 10B shows another exemplary embodiment of an array of six working electrodes, arranged three per row, and each again flanked by a pair opposing auxiliary electrodes. However, in this embodiment the routing and branching of the insulated lead wire joining the plurality of working electrodes and the insulated lead wire joining the plurality of auxiliary electrodes form an interdigitated pattern. As depicted here, only a single insulated wire lead is provided to interface the set of auxiliary electrodes with external electronics units.Accordingly, the arrayed pattern as depicted essentially employs two electrode domains (a bidomain electrode set comprising one working and one auxiliary electrode domain), and as such, is suitable for DEP collection but not electrochemical quantification by voltammetry. Quantification may, however, be performed on particles collected at the working electrodes using immunofluorescent, colorimetric, or other techniques known in the art. It is noted that the interdigitated arrangement of working and auxiliary electrodes allows for a more uniform collection of particles across all working electrodes. It is further noted that in an alternate embodiment of the array depicted in Fig. 10B, a reference electrode may be placed in close proximity, outside of the main electrode array, but still in contact with the fluid sample, to allow for electrochemical measurements.

[0159] Example 13

[0160] With reference to Figures 11 A and 1 IB, Fig. 11A shows an example of a DEP device 500 configured in the form of a 96 well plate for use in high throughput applications, in accordance with this disclosure. As shown in the figure, the 96 well plate DEP device 500comprises an arrayed layout of eight rows 502 by twelve columns 504 of DEP microdevices 510 (one DEP microdevice 510, of which, is identified with a dashed circle in Fig. 11 A). In each of the twelve columns 504, eight individual DEP microdevices 510 are linearly aligned and spaced apart, with their working and auxiliary electrodes sharing electrical continuity along a common insulated conductive path, and terminating, respectively, at working electrode “bus bar” 512 and auxiliary electrode “bus bar” 514. Accordingly, each of the 96 DEP microdevices 510 has its working electrode in electrical continuity with working electrode “bus bar” 512, and its auxiliary electrode in electrical continuity with auxiliary electrode “bus bar” 514. The conductive paths of working electrode “bus bar” 512 and auxiliary electrode “bus bar” 514 each further terminates, respectively, at uninsulated working electrode contact pad 516 and auxiliary electrode contact pad 518. These contact pads 516 and 518 serve as an interface for electrical communication between DEP device 500 an external voltage source (not pictured) such as a function generator to allow DEP-driven nanoparticle collection at each of the 96 DEP microdevices 510. When DEP device 500 is configured as a 96 well plate such that each of its 96 DEP microdevices 510 are partitioned into independent fluidically-segregated open-topped wells, the device 500 is particularly well-suited for use with standard laboratory high-throughput measurement devices and systems known in the art, such as, but not limited to robotic fluidic handling systems, automated plate reader instruments for fluorescence and absorption measurements, and automated microscopy stages. It is to be understood that while the specific embodiment of a 96 well plate configuration is presented here, the number of arrayed DEP microdevices 510 to be partitioned into wells may be varied for other configurations, including, for example, standardized configurations having 24 wells, 384 wells, 1536 wells, etc. In yet further embodiments, non-standardized well number configurations may be produced, or configurations wherein only a subset of the wells in the partitioned array contain DEP microdevice electrodes.

[0161] Fig. 11B shows a detailed view of a single DEP microdevice 510 from Fig. 11A in enlarged form. In the specific configuration depicted here, an array of six circular working electrodes (arranged three per row) are connected in series in a manner similar to the snaking or zig-zag pattern shown in Fig 10A of Example 12. These serially-connected working electrodes have electrical continuity though insulated conductive lead wire 520 to working electrode conduction path 522. Working electrode conduction path 522 further merges with a common working electrode conduction path 524 to form electrical continuity with the workingelectrodes of the other DEP microdevices linearly arranged along the same column 504 and extends to working electrode “bus bar” 512.

[0162] The auxiliary electrode arrangement of DEP microdevice 510 departs, in this embodiment shown in Fig. 11B, from the dual semi-annular configurations depicted in Fig. 10A, and is instead configured as a single large, contiguous auxiliary electrode 526 that surrounds, but is not in electrical continuity with, the arrayed set of working electrodes. Contiguous auxiliary electrode 526 merges with a common auxiliary electrode conduction path 528 to form electrical continuity with the auxiliary electrodes of the other DEP microdevices along the same column 504 and extends to auxiliary electrode “bus bar” 514.

[0163] Note that in this specific configuration, because the conduction paths of all the working electrodes and all the auxiliary electrodes of the 96 DEP microdevices 510 join their respective working or auxiliary electrode “bus bars” 512 and 514, these bus bars each constitute a node such that the well-plate device 500 is functionally a bi-domain system. Accordingly, well-plate device 500 as depicted here is DEP-capable only and any well holding a fluid sample will be active when current is applied. Once DEP collection is completed, standard laboratory devices and techniques that are configured for, or amenable to, well plate processing may be employed for further analysis.

[0164] It is to be understood that alternate embodiments of DEP device 500 may utilize different configurations of DEP microdevice 510 within each well than the specific arrangement depicted in Fig. 11B without departing from the scope of this disclosure. Similarly, the well-to-well wiring of DEP device 500 may employ different configurations in continuity and connectivity, also without departing from the scope of this disclosure.Concluding Paragraphs

[0165] Each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of’ excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of’ limits the scope of the embodimentto the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment.

[0166] Unless otherwise indicated, all numbers expressing quantities of ingredients, agent / drug / inhibitor concentrations, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0167] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0168] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0169] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0170] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0171] t is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0172] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard,no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0173] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the example(s) or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 11th Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology, 2nd Edition (Ed. Anthony Smith, Oxford University Press, Oxford, 2006).

Claims

CLAIMS\What is claimed is:

1. A device, comprising a substantially planar substrate having first uninsulated region and a second uninsulated region but otherwise covered by a layer of insulating material, a set of electrodes formed upon the planar substrate in the first uninsulated region, the set of [three] uninsulated electrodes comprising a working electrode, a first auxiliary electrode, and a second auxiliary electrode; and a set of contact pads formed upon the planar substrate in the uninsulated second region, wherein the contact pads are in electrical continuity beneath the layer of insulating material with a corresponding electrode in the set of electrodes in the first uninsulated region.

2. The device of claim 1, wherein the working electrode is centrally located within first uninsulated region and the first and second auxiliary electrodes are positioned around and offset from the working electrode.

3. The device of claim 1, wherein the working electrode has a circular shape with a circumferential edge, and wherein the first and second auxiliary electrodes are positioned on opposing sides of the working electrode and offset from the circumferential edge of the working electrode by a gap distance.

4. The device of claim 3, wherein the first auxiliary electrode and a second auxiliary electrode, are each semiannular in shape.

5. The device of any of claims (l)-(4), further comprising: a microfluidic chamber enclosing a space above the set of electrodes in the first uninsulated region, with the microfluidic chamber configured to retain a fluid sample in contact with the set of electrodes.

6. The device of claim 5, further comprising: an inlet channel to introduce fluid into the microfluidic chamber; andan outlet channel to remove fluid from the microfluidic chamber.

7. The system of claim 5, wherein the microfluidic chamber is sized to retain a volume of fluid sample between 5pl and 350pl in contact with the set of electrodes.

8. The system of claim 5, wherein the microfluidic chamber is sized to retain a volume of fluid sample between 5pl and 30pl in contact with the set of electrodes.

9. The system of claim 5, wherein the set of electrodes comprise a conductive material.

10. The system of claim 9, wherein the conductive material comprises a layer material selected from the group of platinum, gold, graphene, and indium tin oxide.

11. The device of any of claims 1, 2, 3, 4, 9, and 10, further comprising an open-topped well positioned upon the planar substrate and surrounding the first uninsulated region within which the set of electrodes are positioned; wherein the open-topped well is configured to retain a fluid sample in contact with the set of electrodes.

12. A multi-well device comprising a plurality of open-topped wells, wherein each open-topped well comprises a bottom surface on which the device of Claim 1 is configured.

13. A system for isolating nanoparticles from a fluid sample, the system comprising: a microfluidic chamber enclosing a space above a surface onto which a set of electrodes is formed, the microfluidic chamber configured to retain the fluid sample in contact with the set of electrodes and wherein the set of electrodes comprise: a working electrode centrally located on the surface within the fluidic chamber and having a circular shape with a circumferential edge; a first auxiliary electrode and a second auxiliary electrode, each having a semiannular shape with a radial width, the first auxiliary electrode and the second auxiliary electrode positioned on the surface on opposing sides of the working electrode, and each of the auxiliary electrodes offset from the circumferential edge of the working electrode by a gap distance; and a function generator in electrical communication with the set of electrodes and configured to generate electric field gradients within the fluid sample sufficient to preferentially isolate the nanoparticles onto the circumferential edge of the working electrode.

14. The system of claim 13, further configured for electrochemical quantification of nanoparticles isolated upon the circumferential edge of the working electrode, the system further comprising: a potentiostat; and a switching means to allow the function generator to be removed from electrical communication with the set of electrodes and replaced by the potentiostat, wherein the potentiostat is configured for electrochemical quantification of nanoparticles isolated upon the circumferential edge of the working electrode by taking voltammetric measurements.

15. The system of claim 14, wherein the potentiostat is configured for electrochemical quantification by utilizing the first auxiliary electrode as a counter electrode and the second auxiliary electrode as a reference electrode, and voltammetric measurements are performed by applying a potential between the working and reference electrodes and sensing the resulting electrochemical reactions by measuring the current between the working electrode and the counter electrodes.

16. A system for isolating and analyzing nanoparticles from a fluid sample, the system comprising: a voltage source; a voltammetry device; a substantially planar substrate having first uninsulated region and a second uninsulated region, but otherwise covered by a layer of insulating material; a set of three electrodes formed upon the planar substrate in the first uninsulated region, the set of three uninsulated electrodes comprising a working electrode, a first auxiliary electrode, and a second auxiliary electrode; and a set of three contact pads formed upon the planar substrate in the uninsulated second region, wherein the three contact pads are in electrical continuity beneath the layer of insulating material with a corresponding electrode in the set of electrodes in the first uninsulated region; and a switching means to allow the first and second auxiliary electrodes to be electrically connected to and disconnected from one another, such that: when the first and second auxiliary electrodes are electrically connected to one another, the voltage source may be used to isolate nanoparticles from a fluid sample; andwhen the first and second auxiliary electrodes are electrically disconnected from one another, the voltammetry device may be used to analyze the nanoparticles isolated from a fluid sample.

17. A method of isolating and analyzing nanoparticles (NPs) from a fluid sample, the method comprising: collecting the NPs by dielectrophoresis (DEP) from the fluid sample onto a working electrode con, thereby isolating NPs; immunolabeling the isolated NPs with a tag; thereby generating tagged isolated NPs; and; analyzing the tagged isolated NPs.

18. The method of claim 17, wherein collecting / isolating the NPs is performed by: placing the NP-containing fluid sample in contact with a set of electrodes, the set of electrodes configured as comprising the working electrode and an auxiliary electrode through which a voltage difference is applied to generate in the fluid sample a nonuniform electric field with DEP forces acting on NPs contained therein, the nonuniform electric field having a gradient in proximity of the working electrode sufficient to adhere and thereby isolate the nanoparticles onto the working electrode.

19. The method of claim 17, wherein immunolabeling the isolated NP is performed by affixing an electrochemical tag to the NPs isolated onto the working electrode.

20. The method of claim 18, wherein analyzing the tagged isolated NPs is performed by: placing the set of electrodes, including working electrode to which the tagged isolated NPs are adhered, in contact with an electrochemical solution; reconfiguring the set of electrodes by splitting the auxiliary electrode into a reference electrode and a counter electrode, the reference electrode and counter electrode having separate paths of electrical continuity; and measuring by an electrochemical, immunochemical, immunosensing, or voltammetric method a signal indicative of the quantity of tagged isolated NPs adhered to the working electrode byapplying a voltage across the working and reference electrodes, and measuring the resultant current between the working and counter electrodes, thereby quantifying the NPs.

21. A method of isolating nanoparticles from a fluid sample, the method comprising: introducing a fluid sample comprising nanoparticles into a microfluidic chamber through an inlet channel, the microfluidic chamber having a surface on which a set of electrodes is formed, the set of electrodes comprising: a working electrode centrally located on the surface within the fluidic chamber and having a circular shape with a circumferential edge; and a first auxiliary electrode and a second auxiliary electrode, each having a semiannular shape with a radial width, wherein the first auxiliary electrode and the second auxiliary electrode are positioned on the surface on opposing sides of the working electrode, and each of the auxiliary electrodes offset from the circumferential edge of the working electrode by a gap distance; connecting the first auxiliary electrode and the second auxiliary electrode to act as a combined ground electrode; and applying an alternating electric current across the working electrode and the combined ground electrode to produce a time-varying electric field within the fluid sample retained in the microfluidic chamber, the time-varying electric field inducing a nonuniform electric field gradient in proximity of the set of electrodes which induces dielectrophoretic (DEP) forces within the fluid sample, wherein the electric field gradients generated within the fluid sample by the alternating electric current are sufficient to cause DEP forces to preferentially direct and isolate a subset of_the nanoparticles onto the circumferential edge of the working electrode.

22. The method of claim 21, wherein the fluid sample is a high conductance medium.

23. The method of claim 22, wherein the high conductance medium is plasma or whole blood.

24. The method of claim 23, wherein the high conductance medium has a conductivity greater than 5 mS / cm.

25. The method of claim 22, wherein the alternating voltage signal has a frequency between 5Hz and 12MHz, a peak-to-peak voltage between IVpp and 20Vpp, and a duration ofapplication between 30seconds and 30minutes depending on the type and size of the nanoparticle to be isolated.

26. The method of claim 21, wherein the alternating voltage signal has a sinusoidal frequency of 14kHz, a peak-to-peak voltage of 8 volts, and a duration of application of 10 minutes to isolate extracellular vesicle nanoparticles.

27. A method of electrochemically quantifying nanoparticles present in a fluid sample, the method comprising: introducing the fluid sample comprising nanoparticles into a microfluidic chamber through an inlet channel, the microfluidic chamber further comprising an outlet channel and having a surface on which a set of electrodes is formed, the set of electrodes comprising: a working electrode centrally located on the surface within the fluidic chamber and having a circular shape with a circumferential edge; a first auxiliary electrode and a second auxiliary electrode, each having a semiannular shape with a radial width, wherein the first auxiliary electrode and the second auxiliary electrode are positioned on the surface on opposing sides of the working electrode, and each of the auxiliary electrodes offset from the circumferential edge of the working electrode by a gap distance; connecting the first auxiliary electrode and the second auxiliary electrode to act as a combined ground electrode; applying an alternating electric current across the working electrode and the combined ground electrode to produce a time-varying electric field within the fluid sample retained in the microfluidic chamber, the time-varying electric field inducing a nonuniforn electric field gradient in proximity of the set of electrodes which induces di electrophoretic (DEP) forces within the fluid sample, wherein the electric field gradients generated within the fluid sample by the alternating electric current are sufficient to cause DEP forces to preferentially direct and isolate the nanoparticles onto the circumferential edge of the working electrode; terminating the alternating current;introducing a rinsing solution into the microfluidic chamber through the inlet channel to displace the fluid sample through the outlet channel, leaving the isolated nanoparticles adhered to circumferential edge of the working electrode and bathed in the rinsing solution; performing immunostaining of the isolated nanoparticles adhered to circumferential edge of the working electrode, thereby labeling the isolated nanoparticles with an antibody or nanobody bound to a fluorescently active or electrochemically active label; introducing an electrochemical sensing solution into the microfluidic chamber through the inlet channel; disconnecting the first auxiliary electrode from the second auxiliary electrode, and reconfiguring them for use with a potentiostat for voltammetric measurements, with the first auxiliary electrode serving as a counter electrode and the second auxiliary electrode as a reference electrode; and performing voltammetry by applying a potential between the working electrode and the reference electrode and sensing the resulting electrochemical reactions by measuring the current between the working electrode and the counter electrode, thereby electrochemically quantifying the nanoparticles isolated from the fluid sample.

28. The method of claim 27, wherein the rinsing solution comprises 0.5X PBS.

29. The method of claim 27, wherein the immunostaining of the isolated nanoparticles labels the nanoparticles electrochemical solution comprises 20 pL of a solution of 0.5X PBS containing 0.03% v / v hydrogen peroxide.

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