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WO2025207694A1 - Systèmes et procédés d'isolement électrocinétique de nanoparticules et de quantification électrochimique - Google Patents

Systèmes et procédés d'isolement électrocinétique de nanoparticules et de quantification électrochimique

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Publication number
WO2025207694A1
WO2025207694A1 PCT/US2025/021422 US2025021422W WO2025207694A1 WO 2025207694 A1 WO2025207694 A1 WO 2025207694A1 US 2025021422 W US2025021422 W US 2025021422W WO 2025207694 A1 WO2025207694 A1 WO 2025207694A1
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Prior art keywords
electrode
electrodes
working electrode
nanoparticles
fluid sample
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Stuart IBSEN
Jason WARE
Samuel Olson
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Oregon Health and Science University
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Oregon Health and Science University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01L—SEMICONDUCTOR DEVICES NOT COVERED BY CLASS H10
    • H01L23/00—Details of semiconductor or other solid state devices
    • H01L23/48—Arrangements for conducting electric current to or from the solid state body in operation, e.g. leads, terminal arrangements ; Selection of materials therefor
    • H01L23/482—Arrangements for conducting electric current to or from the solid state body in operation, e.g. leads, terminal arrangements ; Selection of materials therefor consisting of lead-in layers inseparably applied to the semiconductor body (electrodes)
    • H01L23/485—Arrangements for conducting electric current to or from the solid state body in operation, e.g. leads, terminal arrangements ; Selection of materials therefor consisting of lead-in layers inseparably applied to the semiconductor body (electrodes) consisting of layered constructions comprising conductive layers and insulating layers, e.g. planar contacts
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0424—Dielectrophoretic forces

Definitions

  • 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.
  • Dielectrophoresis 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.
  • the current gold standard analytical techniques for analyzing dielectrophoresis collected material lack the portability and scalability needed for clinical translation.
  • 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.
  • 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 the dielectric 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:
  • F DEP is the dielectrophoresis force
  • s 0 is the electric permittivity of free space
  • e m is 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
  • 2 is the electric field factor, which is equivalent to the gradient of the electric field squared.
  • 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.
  • Fig. IB shows an embodiment of a Quantitative DEP device disclosed herein.
  • Fig. 1C shows a detailed view of the set of electrodes in the embodiment of Fig. IB.
  • Fig. ID shows another embodiment of a device configured with a manifold having inlet and outlet ports to accommodate fluid loading.
  • 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.
  • 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.
  • 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.
  • Fig 7C shows a plot of a receiver operator characteristic curve.
  • Fig. 7D shows candidate biomarker proteins that are overexpressed in PDAC cancer and are localized to exosomes.
  • Fig. 7F shows candidate biomarker proteins that are localized to cellular organelles and are overexpressed by prolonged exposure to hypoxia.
  • Fig 8 schematically shows the elements of a handheld device for configured for nanoparticle isolation and quantification.
  • 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.
  • Fig. 10A shows an exemplary embodiment of a two dimensional array of Quantitative DEP devices wired in series.
  • Fig. 11A shows an exemplary embodiment of a DEP device arranged in a 96-well plate configuration.
  • 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.
  • SEQ ID NO: 1 is the amino acid sequence of the disclosed protein biomarker CD9 antigen: MPVKGGTKCIKYLLFGFNFIFWLAGIAVLAIGLWLRFDSQTKSIFEQETNNNNSSFYTGV YILIGAGALMMLVGFLGCCGAVQESQCMLGLFFGFLLVIFAIEIAAAIWGYSHKDEVIKE VQEFYKDTYNKLKTKDEPQRETLKAIHYALNCCGLAGGVEQFISDICPKKDVLETFTVKS CPDAIKEVFDNKFHIIGAVGIGIAVVMIFGMIFSMILCCAIRRNREMV. (UniProt Identifier - P21926)
  • 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.
  • 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.
  • 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.
  • biofluid samples include, but is not limited to, blood, plasma, serum, tears, urine, saliva, seminal fluid, pancreatic juice, bile, cerebrospinal fluid, and lymph fluid specimens.
  • 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.
  • 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.
  • VLPs virus-like particles derived from viruses
  • viruses e.g., Hepatitis B virus (HBV), Human Papillomavirus (HPV), Influenza virus, etc.
  • bacteria-derived vesicles e.g.
  • extracellular vesicles 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.
  • 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.
  • biomarker 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.
  • the presence or concentration of one or more biomarkers may be indicative of a physiological condition, disease, infection, or environmental exposure.
  • 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 a structural 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a subject is a patient, such as a patient diagnosed with cancer.
  • a subject is a patient yet to be diagnosed.
  • tumor herein is a lesion formed by an abnormal growth of cells (called neoplastic cells or tumor cells).
  • 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.
  • polypeptide herein refers to any chain of amino acids, regardless of length or posttranslational modification (such as glycosylation, methylation, ubiquitination, phosphorylation, or the like).
  • polypeptide is used interchangeably with peptide or protein and is used to refer to a polymer of amino acid residues.
  • residue refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic.
  • 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.
  • Glypican-1 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 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.
  • 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.
  • label 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 the proper 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.
  • 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.
  • 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.
  • PBS O.IXphosphate buffered saline
  • TAE tris-acetate-EDTA
  • phosphoric acid buffers phosphoric acid buffers.
  • 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.
  • biofluids from which biomarkers may be isolated using the disclosed systems and methods are considered to be “high conductance” or “high conductivity” media.
  • 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.
  • a low conductance fluid medium be would be 5 to 10 times lower.
  • microdevices for use in DEP -based particle isolation and quantification applications
  • 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.
  • external devices such as, for example, a voltage or current sources.
  • 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.
  • 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.
  • an external device such as a function generator may be used to apply a voltage signal across the nodes (contact pads) to induce a nonuniform electric field within the high conductance fluid.
  • the two auxiliary nodes may be shorted or electrically connecting the two auxiliary electrodes together to operate as a single domain.
  • the working electrode may serve as the site of particle collection and the combined auxiliary electrodes as a ground electrode for DEP applications.
  • an electrical signal such as a time-varying voltage signal
  • an electrical signal such as a time-varying voltage signal
  • 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.
  • 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.
  • DEP collection mode wherein one of the electrodes - the working or collection electrode - becomes a site of nanoparticle collection
  • quantification mode wherein the set of electrodes is used to perform voltammetric measurements to characterize nanoparticles isolated onto the working electrode.
  • 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.
  • an imaging system such as a microscopy system, configured for viewing or imaging quantitative DEP microdevice 102 and any nanoparticle-containing fluid sample placed thereon.
  • 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.
  • 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.
  • insulated conductive leads 150, 152, 154 are covered with a deposited layer of insulative material, for example silicon dioxide, at the time of fabrication.
  • 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.
  • quantitative DEP microdevice 102 may optionally further comprise a microfluidics manifold 170 positioned atop surface 112 and set of electrodes 110.
  • 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.
  • 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.
  • 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.
  • the working electrode has a diameter of from about 60 pm to about 100 pm. In other embodiments, the working electrode has 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the isolated nanoparticles adhering to the working electrode are labeled using an immunostaining method.
  • 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.
  • an antibody or nanobody bound to an electrochemically active label (or tag) for subsequent electrochemical sensing
  • a fluorescently active label (tag) for subsequent quantification or visualization by an imaging-based approach.
  • direct or indirect immunostaining methods may be employed at this step.
  • 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.
  • an enzymatic agent such as horseradish peroxidase or glucose oxidase
  • a small molecule agent such as rhodamine.
  • 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.
  • 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.
  • an electrochemical solution For example, when horseradish peroixidase is used as tagging label, a solution containing hydrogen peroxide may be employed as the electrochemical solution.
  • 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.
  • 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.
  • 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.
  • Example 7 A more comprehensive and detailed example of extracellular vesicle isolation using the DEP device is presented below in Example 7.
  • SEM Scanning electron microscopy
  • 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 was performed 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.
  • ECIS electrochemical immunosensing
  • IF immunofluorescence
  • 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.
  • 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).
  • 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).
  • 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.
  • 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).
  • a 50 nm thick layer of titanium was sputtered onto the wafer surface to increase adhesion of the platinum layer.
  • 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.
  • 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 remove photoresist, revealing silicon dioxide.
  • the wafer was rinsed generously with acetone, methanol, and isopropanol.
  • 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.
  • 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.
  • PDAC pancreatic ductal adenocarcinoma
  • 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.
  • PDAC pancreatic ductal adenocarcinoma
  • IPMN intraductal papillary mucinous neoplasm
  • pancreatitis pancreatitis
  • 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 stages of 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
  • pancreatitis refers to a non-cancerous inflammatory disease.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Example 10 Example 10
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • Fig. 9 shows an example of a high throughput configuration 400 of system 100 employing multiple, linearly arrayed DEP microdevices 401 wired in parallel.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 nano
  • 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 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 second set of auxiliary electrodes gives rise to the spatial arrangement of the paired auxiliary electrodes flanking the rows of working electrode as shown.
  • 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.
  • 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.
  • 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.
  • only a single insulated wire lead is provided to interface the set of auxiliary electrodes with external electronics units.
  • 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.
  • the 96 well plate DEP device 500 comprises 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).
  • 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.
  • an external voltage source such as a function generator to allow DEP-driven nanoparticle collection at each of the 96 DEP microdevices 510.
  • 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.
  • 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.
  • 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.
  • Fig. 11B shows a detailed view of a single DEP microdevice 510 from Fig. 11A in enlarged form.
  • 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 working electrodes of the other DEP microdevices linearly arranged along the same column 504 and extends to working electrode “bus bar” 512.
  • 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.
  • each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component.
  • 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 embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment.
  • 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.

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Abstract

L'invention concerne des systèmes et des procédés pour isoler et quantifier des nanoparticules dans des milieux à conductance élevée à l'aide d'une approche de diélectrophorèse basée sur la microfluidique (DEP). Certains modes de réalisation utilisent une géométrie à trois électrodes dans un environnement microfluidique qui permet au système d'être d'abord mis en route en mode DEP pour la collecte de nanoparticules, puis commuté vers un mode de quantification utilisant la même géométrie à trois électrodes. Dans des modes de réalisation, l'agencement à trois électrodes permet d'isoler des particules de charge neutre d'une taille et d'une composition particulières sur le bord d'une seule électrode positionnée centralement, ce qui facilite l'analyse et la quantification en aval. De manière importante, dans les systèmes et dispositifs divulgués, les électrodes configurées pour la collecte DEP ne nécessitent pas le dépôt d'une couche de protection, telle qu'une couche d'hydrogel, pour maintenir l'intégrité fonctionnelle ou structurelle pendant l'application de signaux de tension pendant la collecte de nanoparticules. Des exemples de procédés de quantification de nanoparticules collectées par DEP peuvent comprendre des procédés de coloration et d'imagerie à immunofluorescence, des procédés colorimétriques et des procédés d'immunocoloration et de détection électrochimiques tels que l'analyse voltamétrique. Les systèmes et les procédés de l'invention sont bien adaptés à l'analyse d'échantillons de fluide, notamment des échantillons de fluide biologique à conductance élevée à partir desquels des matériaux constitutifs de taille nanométrique, ou nanoparticules, doivent être isolés, par exemple dans un contexte de diagnostic ou de caractérisation de biomarqueurs.
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