WO2024220553A2 - Système keyplex - Google Patents

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Publication number
WO2024220553A2
WO2024220553A2 PCT/US2024/025010 US2024025010W WO2024220553A2 WO 2024220553 A2 WO2024220553 A2 WO 2024220553A2 US 2024025010 W US2024025010 W US 2024025010W WO 2024220553 A2 WO2024220553 A2 WO 2024220553A2
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biosensor
dielectric
squares
nanowells
plasmonic
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WO2024220553A3 (fr
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Hyungsoon Im
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General Hospital Corp
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General Hospital Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons
    • G01N21/554—Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance

Definitions

  • Cancer is a worldwide public health issue with almost 2 million annual new cases in the U.S. Most cancers show devastating 5-year survival rates below 30% when found in a distant stage. Early detection can significantly improve the survival rate to over 80% if the cancer is detected in a localized stage.
  • Liquid biopsy tests analyzing circulating biomarkers (e.g., circulating tumor cells, cell-free DNA) in easily accessible biofluids, could enable safe, affordable, and frequent check-ups for early cancer detection, longitudinal monitoring, and long-term surveillance after treatment.
  • extracellular vesicle (EV) analysis represents a unique opportunity to detect cancers in a minimally invasive and repeatable manner.
  • EVs including microvesicles, exosomes, and other secreted vesicles, are cell-derived vesicles containing biomolecules in the form of nucleic acids, lipids, and proteins from originating cells. Because EVs are actively shed by cancer cells and can be easily accessible via various biological fluids, molecular EV analysis can be an effective means to access the presence of a tumor and its molecular status.
  • nPLEX nanoholebased nanoplasmonic EV
  • nPLEX sensors can detect tumor-derived EVs in human ascites and plasma samples obtained from patients with ovarian and pancreatic cancers.
  • Newer generations of nPLEX sensors have shown the capability to detect both surface and intravesicular markers in a single EV resolution. Because these sensors are made in substrates, detecting tumor-derived EVs at very low concentrations is often limited by EVs’ diffusion to the sensing surface, where the immunoaffinity capture of tumor-derived EVs occurs.
  • Microfluidic systems with micropatterns were subsequently developed to improve the diffusion-limited reactions, but these approaches are more applicable to capture EVs on the micropatterns rather than on plain plasmonic sensing substrates.
  • applying electrical fields or optical forces have been applied to manipulate and attract polystyrene nano- and micro-sized particles, cells, and EVs toward sensing surfaces.
  • the invention in general, features a device including: (a) a first electrode; (b) a biosensor including a second electrode; (c) a fluidic chamber between the first and second electrodes; (d) an alternating current (AC) signal generator electrically connected to both electrodes; and (e) a detector.
  • a device including: (a) a first electrode; (b) a biosensor including a second electrode; (c) a fluidic chamber between the first and second electrodes; (d) an alternating current (AC) signal generator electrically connected to both electrodes; and (e) a detector.
  • AC alternating current
  • the biosensor is a plasmonic biosensor, electrochemical biosensor, electrical biosensor, or a magnetic biosensor.
  • the plasmonic biosensor includes: (a) a base; and (b) an array of first and second regions on a surface of the base facing the first electrode, wherein the first region includes dielectric squares that are separated by the second region, which includes a reflective metal, wherein the second region further includes a plurality of nanowells.
  • the dielectric includes SiN.
  • each of the dielectric squares are between about 10 pm to about 100 pm by about 10 pm to about 100 pm. In some embodiments, each of the dielectric squares are about 40 pm by about 40 pm.
  • the dielectric squares are separated by about 10 pm to about 100 pm. In some embodiments, the dielectric squares are separated by about 40 pm.
  • the reflective metal includes gold.
  • each of the plurality of nanowells has a diameter of between about 10 nm and about 1000 nm. In some embodiments, each of the plurality of nanowells has a diameter of about 200 nm.
  • the plurality of nanowells have a periodicity of between about 10 nm and about 1000 nm. In some embodiments, the plurality of nanowells have a periodicity of about 500 nm.
  • the biosensor further includes a capture agent, wherein the capture agent specifically binds to a target biomarker.
  • the capture agent is an antibody or antigen-binding fragment thereof.
  • the first electrode includes a transparent planar surface.
  • the transparent planar surface is an indium tin oxide surface.
  • the chamber has a height of between about 1 pm to about 10 mm. In some embodiments, the chamber has a height of between about 50 pm to about 100 pm.
  • the invention features a plasmonic biosensor including: (a) a base; and (b) an array of first and second regions on a surface of the base facing the first electrode, wherein the first region includes dielectric squares that are separated by the second region, which includes a reflective metal, wherein the second region further includes a plurality of nanowells.
  • the dielectric includes SiN.
  • each of the dielectric squares are between about 10 pm to about 100 pm by about 10 pm to about 100 pm. In some embodiments, each of the dielectric squares are about 40 pm by about 40 pm.
  • the dielectric squares are separated by about 10 pm to about 100 pm. In some embodiments, the dielectric squares are separated by about 40 pm.
  • the reflective metal includes gold.
  • each of the plurality of nanowells has a diameter of between about 10 nm and about 1000 nm. In some embodiments, each of the plurality of nanowells has a diameter of about 200 nm.
  • the plurality of nanowells have a periodicity of between about 10 nm and about 1000 nm. In some embodiments, the plurality of nanowells have a periodicity of about 500 nm.
  • the biosensor further includes a capture agent, wherein the capture agent specifically binds to a target biomarker.
  • the invention features a method for concentrating a target biomarker, including: (a) applying a sample including the target biomarker to a device described herein; and (b) applying an AC pulse, wherein the target biomarker concentrates adjacent to the biosensor.
  • the method further includes detecting the target biomarker.
  • the AC pulse is between about 1 kHz and about 100 kHz and between about 1 Vpp to about 30 Vpp AC. In some embodiments, the AC pulse is about 10 kHz 10Vpp AC.
  • the target biomarker is detectable on an extracellular vesicle.
  • extracellular vesicles including the target biomarker are concentrated.
  • the sample is a plasma sample from a patient.
  • compositions and methods significantly improve the assay time for analysis of cancer-derived EVs, such that the assay can be used for rapid screening while improving the detection sensitivity by 100-fold.
  • the key to the high sensitivity and fast assay is the ability to preconcentrate EVs near the sensing surface by generating electroosmosis and DEP forces that bring EVs closer to the sensing surface.
  • the compositions and methods provided herein can accurately detect cancer-derived EVs in 5 min with a 3-times greater difference between cancer and control groups than the conventional passive mode, and all cases were accurately classified. As many plasmonic sensors are made of gold films, the approach can be readily implemented in other plasmonic structures for sensitive EV detection.
  • the term “about” refers to a value that is within 10% above or below the value being described.
  • biomarker refers to an indicator, e.g., predictive, diagnostic, and/or prognostic, which can be detected in a sample. Exemplary biomarkers are described herein.
  • target biomarker refers to a biomarker that is bound by a capture agent.
  • the term “capture agent” refers to any molecule with the ability to bind to a target biomarker (e.g., a tumor biomarker found on an extracellular vesicle). Suitable capture agents include, but are not limited to antibodies or antigen-binding fragments thereof. In some embodiments, the capture agent may be immobilized on an array.
  • the terms “detecting” and “detection” include both qualitative and quantitative measurements of a target molecule. Detecting includes identifying the mere presence of the target molecule in a sample as well as determining whether the target molecule is present in the sample at detectable levels.
  • an “extracellular vesicle” refers to nano-sized secretory particles originating from the cellular endosomal trafficking system. EVs contain molecular cargo, including common and cell-specific proteins, nucleic acids, and lipids, reflecting the physiological characteristics of cells of origin. For example, a “tumor-derived EV” refers to an EV that is released from a tumor cell. EVs include a lipid bilayer membrane enclosing contents of the internal cavity.
  • An EV can include, but is not limited to, an ectosome, a microvesicle, a microparticle, an exosome, an oncosome, an apoptotic body, a liposome, a vacuole, a lysosome, a transport vesicle, a secretory vesicle, a gas vesicle, a matrix vesicle, or a multivesicular body.
  • peripherality refers to a recurrence or repetition of nanowells at regular intervals by their positioning.
  • sample means any biological or other fluids that may contain one or more target biomarkers (e.g., extracellular vesicles).
  • target biomarkers e.g., extracellular vesicles.
  • An exemplary sample is a plasma sample obtained from a subject (for example, a human patient).
  • specific binding refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target.
  • specific binding can refer to an affinity of the first entity for the second target entity that is at least 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times greater than the affinity for the third nontarget entity.
  • Fig. 1 shows the electrokinetically enhanced yield of plasmonic sensing (KeyPLEX) for rapid, label-free detection of extracellular vesicles (EVs).
  • KeyPLEX plasmonic sensing
  • B A photograph of the KeyPLEX system. A microfluidic channel was used to deliver samples to the plasmonic sensor and wash out unbound EVs.
  • Fig. 2 shows a KeyPLEX chip fabrication.
  • Chip fabrication schematic Starting material is a 4-inch Si wafer with a 200-nm thick, low-stress silicon nitride (SiN x ) layer deposited by low-pressure chemical vapor deposition (LPCVD). The nitride layer was patterned for periodic nanoholes with 200 nm diameter and 500 nm periodicity by interference lithography. After patterning nanowells, the wafer surface was coated with a photoresist, and micro patterns were made using direct laser writing. 100 nm-thick Au deposition with a 5-nm Ti adhesion layer followed by a lift-off process produces the KeyPLEX chips.
  • SiN x low-stress silicon nitride
  • LPCVD low-pressure chemical vapor deposition
  • Fig. 3 shows a numerical simulation of electrokinetic flows in the microchannel.
  • the field- induced vortex-like rotational velocity patterns by electroosmotic (A) and dielectrophoresis (B) forces are visualized by white arrows.
  • the colormaps represent the flow velocity.
  • Fig. 4 shows a numerical calculation of the DEP force on various sizes of EVs.
  • A Size distribution of EVs from CaOV3 ovarian cancer cell line measured by nanoparticle tracking analysis.
  • B Estimated DEP forces on different sizes of EVs located at different distances away from the sensing surface.
  • Fig. 5 shows a calculation of an optimal geometry size.
  • A, B Numerical calculation of electroosmotic (A) and dielectrophoretic (B) forces with different sizes of nitride squares.
  • Fig. 6 shows an experimental comparison of field-induced EV binding. Experimental comparison of field-induced EV binding to the sensing surface with different sizes of nitride squares for electroosmotic and dielectrophoretic forces.
  • A 10 pm.
  • B 20 pm.
  • C 30 pm.
  • D 40 pm.
  • E 50 pm.
  • F 60 pm.
  • G 70 pm.
  • H Fluorescence intensities of captured EVs.
  • Fig. 7 shows a characterization of electrokinetically enhanced EV detection.
  • A. EV binding kinetics to the anti-CD63 antibody-immobilized gold nanowell surface with (active) and without external potentials (passive).
  • B. Incubation time optimization. After applying AC fields for 1 min, additional EV incubation times between 0 and 9 min were added for EV binding to the nanowell surface. The spectral shift saturated after 4 min additional incubation and reached the maximum value after 9 min.
  • C Spectral shifts between active and passive modes were compared with titrating input EV counts.
  • Fig. 8 shows a molecular profiling of EVs from ovarian cancer and benign cell lines.
  • Spectral shifts shows higher levels of CD24 and EpCAM in EVs from ovarian cancer cell lines (OVCAR3, A; OV90, B; CaOV3, C) compared to those in EVs from benign cell line (TIOSE4, D).
  • the signal was normalized by the signals of CD63 (putative EV marker). Error bars represent standard deviation from triplicate measurements while individual values are shown as dots.
  • Fig. 9 shows a tumor-derived EV detection in human plasma samples.
  • A EVs isolated from plasma samples of 8 patients (P1 -5: ovarian cancer patients and N1 -3: 3 health donors) were analyzed for CD63, CD24, EpCAM signals via active and passive modes of KeyPLEX.
  • B Heatmaps showing ovarian cancer marker signals (CD24 and EpCAM) measured in active and passive modes.
  • Fig. 10 shows a comparison of tumor-derived EV detection between active and passive modes.
  • A Spectral shifts of EpCAM- or CD24-positive EV detection in human plasma samples of cancer patients and healthy control using active and passive modes. Only the active mode showed a significant difference between cancer and healthy control groups (* denotes P ⁇ 0.05).
  • B The area under the curve (AUC) of the receiver operating characteristic (ROC) between active (1 .0) and passive (0.73) modes.
  • Fig. 11 shows a comparison of coefficient of variance for CD63-positive EV detection.
  • A Coefficients of variance between active and passive modes for detecting CD63-positive EVs in clinical plasma samples.
  • B Estimation plot showing the mean differences between active and passive modes.
  • the disclosure provides compositions and methods for label-free plasmonic sensing of target biomarkers (e.g., target biomarkers detectable on tumor-derived EVs) powered by field- induced electroosmosis and dielectrophoresis forces.
  • target biomarkers e.g., target biomarkers detectable on tumor-derived EVs
  • This overcomes diffusion-limited reactions, enabling sensitive detection of target biomarkers, for example target biomarkers detectable on rare tumor-derived EVs in, for example human plasma samples of virtually any cancer, for example from ovarian cancer patients, in reduced assay time.
  • the disclosure provides compositions and methods for the electroki netically enhanced yield of plasmonic sensing (KeyPLEX) for rapid, label-free EV detection.
  • KeyPLEX electroki netically enhanced yield of plasmonic sensing
  • the inventors overcame the diffusionlimited reaction by applying a combination of alternative current (AC) electroosmosis and dielectrophoresis (DEP), which bring EVs toward the surface of a sensor chip and concentrate them on plasmonic sensing areas.
  • AC alternative current
  • DEP dielectrophoresis
  • the KeyPLEX approach showed that i) tumor-derived EVs can be sensitively detected in 5 min; ii) the electrokinetic enhancement yields a greater difference of signals between cancer and healthy control groups; iii) the active approach can accurately detect cancer patients that were near the borderline to a control group.
  • the KeyPLEX system will be a valuable tool for point-of-care rapid EV analysis.
  • plasmonic biosensors including (a) a base, (b) an array of first and second regions on a surface of the base facing the first electrode, wherein the first region includes dielectric squares that are separated by the second region, which includes a reflective metal, wherein the second region further includes a plurality of nanowells.
  • the plasmonic biosensor further includes a capture agent.
  • the plasmonic biosensors described herein include a base.
  • the base is a silicon wafer.
  • the base may be between about 0.1 inches and 3 inches (e.g., between about 0.1 inches and 0.5 inches, between about 0.5 inches and 1 inch, between about 1 inch and 1 .5 inches, between about 1 .5 inches and 2 inches, between about 2 inches and 2.5 inches, or between about 2.5 inches and 3 inches) by between about 0.1 inches and 3 inches (e.g., between about 0.1 inches and 0.5 inches, between about 0.5 inches and 1 inch, between about 1 inch and 1 .5 inches, between about 1 .5 inches and 2 inches, between about 2 inches and 2.5 inches, or between about 2.5 inches and 3 inches).
  • the base is about 1 inch by about 1 inch.
  • a dielectric layer may be deposited on the base.
  • the dielectric layer may include any dielectric material.
  • Exemplary dielectric materials include silicon nitride (SiNx), SiOa, AI2O3, and HfC>2.
  • direct patterning on Si may be employed.
  • the dielectric layer includes SiNx.
  • the thickness of the dielectric layer may be between about 10 nm and about 1000 nm thick (e.g., between about 10 nm and about 100 nm, between about 100 nm and about 200 nm, between about 200 nm and about 300 nm, between about 300 nm and about 400 nm, between about 400 nm and about 500 nm, between about 500 nm and about 600 nm, between about 600 nm and about 700 nm, between about 700 nm and about 800 nm, between about 800 nm and about 900 nm, or between about 900 nm and about 1000 nm).
  • the thickness of the dielectric layer is about 200 nm.
  • the dielectric layer is a 200 nm SiNx layer.
  • the plasmonic biosensors include an array of first and second regions on a surface of the base facing the first electrode, wherein the first region includes dielectric squares that are separated by the second region, which includes a reflective metal, wherein the second region further includes a plurality of nanowells.
  • the first region may include any dielectric material.
  • the dielectric material is SiN.
  • the dielectric material is SiC>2, AI2O3, and HfC>2.
  • direct patterning on Si may be employed.
  • each of the dielectric squares have a length of between about 10 pm to about 100 pm (e.g., between about 10 pm to about 20 pm, between about 20 pm to about 30 pm, between about 30 pm to about 40 pm, between about 40 pm to about 50 pm, between about 50 pm to about 60 pm, between about 60 pm to about 70 pm, between about 70 pm to about 80 pm, between about 80 pm to about 90 pm, or between about 90 pm to about 100 pm).
  • each of the dielectric squares have a length of about 40 pm. In some embodiments, each of the dielectric squares have a width of between about 10 pm to about 100 pm (e.g., between about 10 pm to about 20 pm, between about 20 pm to about 30 pm, between about 30 pm to about 40 pm, between about 40 pm to about 50 pm, between about 50 pm to about 60 pm, between about 60 pm to about 70 pm, between about 70 pm to about 80 pm, between about 80 pm to about 90 pm, or between about 90 pm to about 100 pm). In some embodiments, each of the dielectric squares have a width of about 40 pm. In some embodiments, each of the dielectric squares are about 40 pm by about 40 pm.
  • the first region includes dielectric squares that are separated by the second region.
  • the dielectric squares are separated by between about 10 pm and about 100 pm pm (e.g., between about 10 pm to about 20 pm, between about 20 pm to about 30 pm, between about 30 pm to about 40 pm, between about 40 pm to about 50 pm, between about 50 pm to about 60 pm, between about 60 pm to about 70 pm, between about 70 pm to about 80 pm, between about 80 pm to about 90 pm, or between about 90 irn to about 100 pm).
  • the dielectric squares are separated by about 40 pm.
  • the second region may include any reflective metal.
  • Exemplary reflective metals include gold, silver, copper, aluminum, platinum, and their alloys (e.g., silver/gold layers).
  • the reflective metal is gold.
  • the reflective metal is a noble metal (e.g., gold, palladium, platinum, rhodium, osmium, iridium, or silver), transition metal (e.g., titanium, aluminum, copper, or nickel), an alkali metal (e.g., lithium, sodium, potassium), metallic alloy, indium tin oxide, aluminum zinc oxide, gallium zinc oxide, titanium nitride, or graphene.
  • the thickness of the second region may be between about 10 nm and about 1000 nm (e.g., between about 10 nm and about 100 nm, between about 100 nm and about 200 nm, between about 200 nm and about 300 nm, between about 300 nm and about 400 nm, between about 400 nm and about 500 nm, between about 500 nm and about 600 nm, between about 600 nm and about 700 nm, between about 700 nm and about 800 nm, between about 800 nm and about 900 nm, or between about 900 nm and about 1000 nm). In some embodiments, the thickness of the second region is about 100 nm.
  • the second region further includes a plurality of nanowells.
  • each of the plurality of nanowells has a diameter of between about 10 nm and about 1000 nm (e.g., between about 10 nm and about 100 nm, between about 100 nm and about 200 nm, between about 200 nm and about 300 nm, between about 300 nm and about 400 nm, between about 400 nm and about 500 nm, between about 500 nm and about 600 nm, between about 600 nm and about 700 nm, between about 700 nm and about 800 nm, between about 800 nm and about 900 nm, or between about 900 nm and about 1000 nm).
  • each of the plurality of nanowells has a diameter of about 200 nm.
  • the plurality of nanowells have a periodicity of between about 10 nm and about 1000 nm (e.g., between about 10 nm and about 100 nm, between about 100 nm and about 200 nm, between about 200 nm and about 300 nm, between about 300 nm and about 400 nm, between about 400 nm and about 500 nm, between about 500 nm and about 600 nm, between about 600 nm and about 700 nm, between about 700 nm and about 800 nm, between about 800 nm and about 900 nm, or between about 900 nm and about 1000 nm). In some embodiments, the plurality of nanowells have a periodicity of about 500 nm.
  • the plasmonic biosensors may further include a capture agent that specifically binds to a target biomarker.
  • the capture agent may be any molecule with the ability to bind to a target biomarker (e.g., a target biomarker detectable on a tumor-derived EV). Suitable capture agents include, but are not limited to, antibodies or antigen-binding fragments thereof.
  • the capture agent may be an antibody that binds EGFR, EGFRvll I, EpCAM, EGFR, MUC1 , HER2, MUC1 , WNT2, GPC1 , Trop2, or MUC16.
  • the capture agent may be immobilized on an array.
  • the capture agent may be immobilized on the plasmonic biosensor via a layer of polyethylene glycol (PEG). Additional capture agents may include aptamers, streptavidin for biotinylated extracellular vesicles, and nucleic acid linkers for DNA-barcoded antibodies.
  • devices including (a) a first electrode; (b) a biosensor including a second electrode; (c) a fluidic chamber between the first and second electrodes; (d) an alternating current (AC) signal generator electrically connected to both electrodes; and (e) a detector.
  • the devices described herein include a first electrode.
  • the first electrode may include a transparent planar surface.
  • the transparent planar surface is made of a material that can transmit at least 70%, 80%, 85%, 90%, 95%, or 99% of light having a wavelength in the ultraviolet-visible-infrared range, which includes wavelengths from approximately 100 nm to 3000 nm.
  • Various materials e.g., glass, quartz, diamond, or a polymer
  • the skilled artisan can envision various combinations of such components that would produce a surface with the desired optical properties.
  • the surface can contain one or more of such materials in sufficient quantities to confer the necessary properties.
  • An exemplary transparent planar surface is an indium tin oxide surface.
  • the devices described herein include a biosensor including a second electrode.
  • the biosensor may be any biosensor that includes a conductive material, e.g., a plasmonic biosensor, electrochemical biosensor, electrical biosensor, or a magnetic biosensor.
  • the biosensor may be a plasmonic biosensor described herein.
  • Electrochemical sensors usually use enzymes that are reactive or attached to detection antibodies that induce redox reactions. Electrochemical sensors detect changes in an electrical signal from the redox reactions. Electrical sensors detect changes in an electrical signal or conductance of the sensor surface due to captured target molecules (e.g., ion-sensitive field-effect transistor (ISFET), nanowire sensors). Magnetic biosensors use magnetic nanoparticles conjugated to affinity ligands as detection probes and measures changes in a magnetic signal (e.g., T1 relaxation time). Exemplary methods for producing magnetic biosensors and electrochemical biosensors are found in Shao et al., Nat Med 18, 1835-1840 (2012) and Jeong et al., ACS Nano 2016, 10, 2, 1802-1809, respectively.
  • ISFET ion-sensitive field-effect transistor
  • the devices described herein include a fluidic chamber between the first and second electrodes.
  • the fluidic chamber has a height of between about 1 pm to about 10 mm (e.g., about 1 pm to about 50 pm, about 50 pm to about 100 pm, about 100 pm to about 500 pm, about 500 pm to about 1 mm, or about 1 mm to about 5 mm, or about 5 mm to about 10 mm).
  • the fluidic chamber typically has a height of between about 50 pm to about 100 pm.
  • the devices described herein include an alternating current (AC) signal generator electrically connected to the first and second electrodes.
  • the electrodes are connected to the signal generator by copper tapes.
  • the AC signal generator provides AC potentials to generate electrokinetic forces, pushing target biomarkers (e.g., target biomarkers detectable on EVs) toward the biosensor.
  • target biomarkers e.g., target biomarkers detectable on EVs
  • the electrokinetic forces generated by the AC signal generator push EVs comprising the target biomarker towards the biosensor. 5.
  • the devices described herein include a detector.
  • the detector may be used for reflective spectral measurements.
  • the detector may be used for surface plasmon resonance measurements.
  • the detector may be used to measure the intensity of reflected light, according to any standard procedure. For example, the detector may measure the intensity of reflected light at varying angles of incident light.
  • the detector may be used to measure a signal such as fluorescence signals, Raman signals, dark-field scattering signals, among others.
  • target biomarkers i.e. , target biomarkers detectable on EVs
  • This can be applied to biosensors made of conductive materials (e.g., plasmonic biosensors, electrochemical biosensors, electrical biosensors, and magnetic biosensors).
  • the methods include (a) applying a sample including the target biomarker to a device described herein; and (b) applying an AC pulse, wherein the target biomarker concentrates adjacent to the biosensor.
  • the AC pulse concentrates EVs comprising the target biomarker adjacent to the biosensor.
  • the methods provided herein improve detection sensitivity and reduce assay time by applying external electrical fields that generate alternative current electroosmosis and dielectrophoresis.
  • Application of the AC pulse concentrates the target biomarkers toward the surface of the biosensor.
  • Applying electric fields to concentrate extracellular vesicles on a sensing surface increases the detection sensitivity and assay time.
  • the method is particularly useful to detect rare tumor-derived EVs at extremely low concentrations.
  • the sample may be transported to the chamber at a flow rate of between about 1 pl/min to about 200 pl/min (e.g., about 1 pl/min to about 25 pl/min, about 25 pl/min to about 50 pl/min, about 50 pl/min to about 75 pl/min, about 75 pl/min to about 100 pl/min, about 100 pl/min to about 125 pl/min, about 125 pl/min to about 150 pl/min, about 150 pl/min to about 175 pl/min, or about 175 pl/min to about 200 pl/min).
  • the sample may be transported to the chamber at a flow rate of 25 pl/min for 1 minute.
  • the AC pulse may be between about 1 kHz to about 100 kHz (e.g., about 1 kHz to about 10 kHz, about 10 kHz to about 20 kHz, about 20 kHz to about 30 kHz, about 30 kHz to about 40 kHz, about 40 kHz to about 50 kHz, about 50 kHz to about 60 kHz, about 60 kHz to about 70 kHz, about 70 kHz to about 80 kHz, about 80 kHz to about 90 kHz, or about 90 kHz to about 100 kHz). In some embodiments, the AC pulse may be about 10 kHz.
  • the AC pulse may be between about 1 Vpp to about 30 Vpp (e.g., about 1 Vpp to about 5 Vpp, about 5 Vpp to about 10 Vpp, about 10 Vpp to about 15 Vpp, about 15 Vpp to about 20 Vpp, about 20 Vpp to about 25 Vpp, or about 25 Vpp to about 30 Vpp). In some embodiments, the AC pulse may be about 10 Vpp.
  • the AC pulse is between about 1 kHz and about 100 kHz and between about 1 Vpp to about 30 Vpp AC. In some embodiments, the AC pulse is about 10 kHz 10Vpp AC.
  • the AC pulse may be applied at an interval of between about every 1 second to about every sixty seconds (e.g., about every 1 second to about every 5 seconds, about every 5 seconds to about every 10 seconds, about every 10 seconds to about every 15 seconds, about every 15 seconds to about every 20 seconds, about every 20 seconds to about every 25 seconds, about every 25 seconds to about every 30 seconds, about every 30 seconds to about every 35 seconds, about every 35 seconds to about every 40 seconds, about every 45 seconds to about every 50 seconds, about every 50 seconds to about every 55 seconds, or about every 55 seconds to about every 60 seconds). In some embodiments, the AC pulse may be applied about every eight seconds.
  • the AC pulse may be applied for between about 30 seconds to about 5 minutes (e.g., about 30 seconds to about 1 minute, about 1 minute to about 1 .5 minutes, about 1 .5 minutes to about 2 minutes, about 2 minutes to about 2.5 minutes, about 2.5 minutes to about 3 minutes, about 3 minutes to about 3.5 minutes, about 3.5 minutes to about 4 minutes, about 4 minutes to about 4.5 minutes, or about 4.5 minutes to about 5 minutes). In some embodiments, the AC pulse may be applied for about 1 minute.
  • a pulse of about 10 kHz 10Vpp AC is applied about every eight seconds for about 1 minute.
  • the method further includes detecting the target biomarker.
  • the target biomarker is typically found on a extracellular vesicle (EV) and at times, is used interchangeably with particular markers found on the EV.
  • EV extracellular vesicle
  • the target biomarker is detectable on an extracellular vesicle (EV).
  • the EV is a tumor-derived EV.
  • the tumor-derived EV may be a glioblastoma multiforme, which may include the target biomarker EGFR or EGFRvlll.
  • the tumor-derived EV may be a breast cancer, which may include the target biomarker EpCAM, EGFR, MUC1 , or HER2.
  • the tumor- derived EV may be a pancreatic cancer, which may include the target biomarker EpCAm, EGFR, MUC1 , WNT2, or GPC1 .
  • the tumor-derived EV may be a cholangiocarcinoma, which may include the target biomarker EpCAM, EGFR, or MUC1 .
  • the tumor-derived EV may be an OvCA, which may include the target biomarker Trop2 or MUC16.
  • target biomarkers include viruses, bacteria, and lipid nanoparticles.
  • the sample is a plasma sample from a subject (e.g., a human patient).
  • extracellular vesicles including the target biomarker are concentrated.
  • the methods provided herein attract and concentrate extracellular vesicles (EVs) on sensing substrates to significantly improve the detection sensitivity by ⁇ 100-fold and reduce the assay time to 5 min.
  • the enhancement is achieved by applying external electrical fields that generate alternative current electroosmosis and dielectrophoresis. The enhancement leads to a greater difference in signals from tumor-derived EVs between cancer patients and healthy controls.
  • the method includes the following steps. First, prior to introducing the EV sample onto the KeyPLEX, the fluidic channels were filled with a working buffer, and the baseline reflection spectrum was measured. Next, an EV solution was perfused to the flowcell at a flow rate of 25 pl/min for 1 minute. Third, a pulse of 10 kHz 10Vpp AC was applied every eight seconds (periodic on/off for eight seconds) for 1 minute. Next, the samples were then incubated for an additional 9 minutes for EV from the cell line and 4 minutes for patient samples. Next, the flowcell was washed with a working buffer for 3 minutes at a flow rate of 50 pl/min, followed by spectra measurements. EXAMPLES
  • OV90, 0VCAR3, CaOV3, and Gli36 cell lines were purchased from American Type Culture Collection (ATCC).
  • the ovarian benign cell line (TiOSE4) was generated by transfection of hTERT into NOSE cells (Zorn et al., Clin. Cancer Res. 9, 4811-4818, 2003), maintained in 1 :1 Media 199:MCDB 105 with gentamicin (25 pg/mL), 15% heat-inactivated serum, and G418 (500 pg/mL).
  • OV90 and TiOSE4 cells were maintained in RPMI-1640 (Hyclone), and OVCAR3, CaOV3, and Gli36 cells were grown in DMEM (Hyclone).
  • fetal bovine serum FBS, ThermoFisher Scientific
  • 100 U/mL penicillin 100 pg/mL streptomycin (Cellgro)
  • 100 pg/mL streptomycin Cellgro
  • a Gli36-CD63-EGFP cell line was generated by transiently transfecting a CD63-EGFP expressing plasmid, CD63-pEGFP C2, a gift from Paul Luzio (Addgene plasmid # 62964) into Gli36 cells using FuGENE HD transfection reagent (Promega) by the manufacturer’s instruction.
  • the antibiotic selection was performed by treating 500 pg/mL of G418 (ThermoFisher Scientific) for 4 weeks with changing medium every 2-3 days. The expression efficiency was measured by fluorescence microscopy.
  • Plasma samples were obtained from patients with informed consent according to a protocol approved by the Dana Farber Cancer Institute (IRB Protocol Number: 12-238, PI: Cesar M.cer). Blood samples were collected from patients and processed per procedures at Massachusetts General Hospital (MGH). Briefly, 10 ml peripheral blood was collected into polypropylene tubes containing sodium citrate (Vacutainer System; BD Biosciences). Whole blood samples were centrifuged at 1 ,500 x g for 15 min at 4°C, and plasma layers were obtained. Plasma samples were aliquoted and frozen at -80°C until further analysis. Plasma samples from healthy controls were obtained from the MGH Biobank.
  • EVs were isolated as previously reported (Van Deun et al. Adv Biosyst 4, e1900310, 2020, Min et al., Adv Biosyst 4, e2000003, 2020).
  • cells were incubated with RPMI- 1640 or DMEM with 1% Exosome-depleted FBS (ThermoFisher Scientific), 100 U/mL penicillin, and 100 pg/mL streptomycin for 48 hrs, and the cell culture supernatant was then collected.
  • frozen plasma samples were first thawed on ice and centrifuged at 10,000 x g for 20 min at 4°C to remove apoptotic bodies and platelet.
  • the concentrated supernatant 0.5 mL was loaded on the size exclusion chromatography (SEC) column prepared with Sepharose® CL-4B (GE Healthcare) (Van Deun et al. Adv Biosyst 4, e1900310, 2020).
  • the protease/phosphatase inhibitor was added into the EV concentrates and stored until use at -80°C.
  • EV solutions were diluted into a working buffer (0.1 mM PBS).
  • the isolated EVs were characterized by western blot and nanoparticle tracking analysis (Nanosight LM10 microscope, Malvern) for molecular markers, sizes, and concentrations.
  • KeyPLEX chips were fabricated using interference lithography on SiN-deposited 4-inch Si wafers. Periodic circular nanowells with 200 nm diameter and 500 nm periodicity were patterned in the SiN layer by interference lithography (LumArray, Inc.) and subsequent reactive ion etching. Then, square micropatterns with a 40 pm width and 40 pm gap were defined by direct light writing lithography (MLA150 Maskless Aligner, Heidelberg). A 100-nm thick gold film with a 5-nm thick titanium adhesion layer was deposited by e-beam evaporation (FC-2000, Temescal). A subsequent lift-off process produces a hybrid structure in the KeyPLEX chips.
  • interference lithography Li array, Inc.
  • FC-2000 e-beam evaporation
  • Microfluidic flow channels were formed by using an approximately 60 pm thick acrylic spacer (3M) covered by an indium tin oxide (ITO)-coated glass slide (IT10-111 -25, Nanocs) as a counter electrode.
  • the electrodes were connected to an arbitrary signal generator (AFG3011 C, Tektronics).
  • Inlet and outlet holes were pierced on the glass substrate using a high-speed rotary tool with a diamond-coated tip.
  • EV solutions were injected using a syringe pump (NE-1000, New Era pump systems).
  • the gold nanowell surface was first coated with a mixture of thiolated polyethylene glycol (PEG) containing 1 kDa carboxylated and 0.35 kDa methylated thiol-PEG in a 1 :3 ratio (Nanocs, 1 mM in PBS) that was previously optimized for EV capture (Im et al., Nat. Biotechnol. 32, 490-495, 2014).
  • PEG polyethylene glycol
  • the monoclonal antibodies against CD63 (215-820, Ancell), EpCAM (MA5-12436, Invitrogen), CD24 (MAS- 11828, Invitrogen), and IgG isotype control (14-4714-82, eBioscience) were conjugated by a 1 -ethyl-3-(3- dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/Sulfo-NHS) activation using MES buffer (pH 4.7, 28390, Thermo).
  • Antibodies 50 pg/ml
  • a KeyPLEX chip was placed on an upright microscope (Eclipse Ci, Nikon).
  • a spectral dip position around 750 nm was measured using a custom-built Matlab program by fitting the reflection dip to a second polynomial curve.
  • a minimum detection level (0.03 nm) was determined by three times the standard deviation of background signals measured at a steady state for 10 min.
  • the fluidic channels Prior to introducing the EV sample onto the KeyPLEX, the fluidic channels were filled with a working buffer, and the baseline reflection spectrum was measured.
  • EV solution was perfused to the flowcell at a flow rate of 25 pl/min for 1 minute.
  • a pulse of 10 kHz 10Vpp AC was applied every eight seconds (periodic on/off for eight seconds) for 1 minute while no voltage was applied in passive capture.
  • the samples were then incubated for an additional 9 minutes for EV from the cell line and 4 minutes for patient samples, and the flowcell was washed with a working buffer for 3 minutes at a flow rate of 50 pl/min, followed by spectra measurements.
  • the mesh size was set to be ⁇ 200 nm, which is smaller than the measure of minimum particle displacement; this approach was found to be reliable in microscale observations in our previous study. ⁇ Kwak et al., 2021 , The Journal of Physical Chemistry C, 125, 6278- 6286 ⁇ Park et al., 2016, ACS Nano, 10, 4011 -9 ⁇
  • FDTD finite-difference time-domain simulations were used to obtain electromagnetic field distribution around gold nanowells.
  • a unit cell of a single nanowell was 200 nm diameter with 500 nm periodicity in a 100-nm thick Au thin film. Periodic boundary conditions in x- and y-directions were used to simulate an infinite array of periodic nanowells.
  • the KeyPLEX system is composed of a nanoplasmonic substrate with periodic gold nanowell structures made on a Si wafer with a SiN layer and an indium tin oxide (ITO) electrode to apply alternating current (AC) potentials between the two substrates (Fig. 1A).
  • ITO indium tin oxide
  • AC alternating current
  • the plasmonic gold surface of sensing arrays was functionalized with different capture antibodies (e.g., CD63 as an EV marker, EpCAM and CD24 for ovarian cancer markers, and IgG isotype negative control) via a polyethylene glycol (PEG) linker, which passivates the gold sensing surface to reduce nonspecific EV binding.
  • the EV detection was done by measuring a spectral shift of the reflectance spectrum upon EV binding to the sensing surface in a label-free manner.
  • a microfluidic channel was used to deliver EV samples to the sensing area and wash out unbound EVs after incubation (Fig. 1B).
  • a KeyPLEX chip was made in a hybrid structure; SiN micropatterns (40 pm square with 40 pm gap between squares, Fig. 1C) were made to generate and direct electroosmosis and DEP forces toward gold sensing areas made of periodic nanowell arrays (Fig. 1D).
  • Interference lithography was used for periodic nanowell patterns (200 nm well diameter and 500 nm periodicity) and conventional optical lithography for micropatterns. This allowed for the fabrication of KeyPLEX chips in a wafer scale (Fig. 2).
  • fluorescent EVs were prepared with green fluorescence proteins (GFPs, see methods for the sample preparation) and monitored their binding upon applying AC potentials. Without an external potential, EVs were dispersed randomly throughout the microfluidic channel, and thus, only a few EVs were captured on the surface (Fig. 1E). In contrast, when an electric potential was applied between the plasmonic substrate and the ITO electrode, EVs were attracted towards the gold nanowell surfaces and accumulated on areas between square micropatterns (Fig. 1F) by the field-induced electroosmosis and DEP forces. The attraction occurred almost instantly, and unbound EVs can be removed from the surface with a washing fluid when the applied potential was off.
  • GFPs green fluorescence proteins
  • F DEP 2nr 3 E m Re[f CM ]V ⁇ E ⁇ 2 (3)
  • r is the particle radius
  • £ m is the medium permittivity
  • fcwi denotes the Clausius-Mossotti factor, a frequency-dependent function of the complex permittivities of particle and medium.
  • s‘ p and E* m denote the complex permittivities of the particle and the medium, respectively
  • the Stokes drag force on the particle in the medium is estimated as
  • UEO is the Stokes drag force generated by electroosmotic flow, assuming that the particle's initial velocity is zero.
  • the strongest electrokinetic movement occurs at the micropattern edges and decreases towards gold sensing areas by the AC electroosmosis force, visualized as a set of vortex-like rotational velocity patterns around the edges of micropatterns (Fig. 3A).
  • the DEP force also attracts EVs to the edges of micropatterns (Fig. 3B).
  • the DEP force becomes more dominant for larger-size EVs as the DEP force is proportional to the particle volume (Fig. 4).
  • the square micropattern size was optimized first by finite element analysis, comparing the integrated forces across the channels with different geometry sizes; 40 pm was found to have the highest efficiency between 10 and 70 pm (Fig. 5).
  • Fig. 7A shows the binding kinetics of EVs with an external potential (active) and without it (passive).
  • the gold nanowell surface was functionalized with anti-CD63 antibodies, and 6 x 10 3 EVs from OVCAR3 ovarian cancer cells were introduced onto the chip. This is a relatively low amount of EVs as 1 mL of human plasma sample typically contains >10 10 EVs.
  • active capture a pulse of AC 10 kHz 10Vpp was applied every eight seconds (periodic on/off for eight seconds) for a 1 min total, whereas no voltage was applied in passive mode. Then, the sample was left for an additional 9 min.
  • AA CD63 relative spectral shifts of anti-CD63 functionalized nanowell substrates with titrating EVs counts were measured with (active) and without external potentials (passive). We determined the cut-off shift as 0.03 nm, three times the standard deviation of background signals.
  • the limit of detection (LOD), the lowest EV count generating a shift above the cut-off value, was determined in ⁇ 30 EVs in the active mode, which corresponds to the concentration of 1 EV/pL in a 30 pl solution and ⁇ 3 x 10 3 EVs (100 EVs/pL x 30 pl) in the passive mode.
  • the LOD of the passive mode was similar to the previously reported number (Im et al., Nat. Biotechnol. 32, 490-495, 2014; Yang et al., Sci. Transl. Med. 9, eaal3226, 2017) while the active mode improves it by 100-fold.
  • Example 4 Tumor-derived EV detection in human plasma samples
  • the KeyPLEX assay was applied to detect different surface markers on EVs derived from ovarian cancer (OVCAR3, OV90, CaOV3) and benign (TIOSE4) cell lines (Fig. 8).
  • the gold nanowell surface was functionalized with antibodies against CD63, CD24, EpCAM, and IgG.
  • CD63 was used as a universal EV marker.
  • CD24 and EpCAM were previously identified as ovarian cancer EV biomarkers (Im et al., Nat. Biotechnol. 32, 490-495, 2014; Park et al., ACS Photonics 5, 487-494, 2018; Zhang et al., Sci. Transl. Med. 12, eaaz2878, 2020).
  • Isotype IgG was used to estimate nonspecific EV binding.
  • the results showed that i) CD63 was detected for all EV samples regardless of their cellular origins; ii) CD24 and EpCAM signals were higher for EVs from ovarian cancer cell lines (OVCAR3, OV90, CaOV3) than the signal in the IgG controls, while the signals of EVs from benign cells (TIOSE4) were comparable to IgG signals; Hi) nonspecific bindings on the IgG-coated surfaces were negligible for all EV samples even with applying the electrokinetic forces.
  • the area under the curve (AUC) of the receiver operating characteristic (ROC) increased from 0.73 with the passive mode to 1 .0 with the active mode (Fig. 10B). This is because the CD24/EpCAM signals were increased for cancer patients in the active mode (1 .81 nm for active vs.
  • the active mode showed significantly lower coefficients of variance for CD63-positive EV signals (12.1% for active and 48.8% for passive, Wilcoxon test p ⁇ 0.01 , Fig. 11). The result indicates that the active mode can detect cancer- derived EVs even with a short assay time of 5 min. In contrast, the short assay time was insufficient in the conventional passive mode.
  • a device comprising: (a) a first electrode; (b) a biosensor comprising a second electrode; (c) a fluidic chamber between the first and second electrodes; (d) an alternating current (AC) signal generator electrically connected to both electrodes; and (e) a detector.
  • AC alternating current
  • E2 The device of E1 , wherein the biosensor is a plasmonic biosensor, electrochemical biosensor, electrical biosensor, or a magnetic biosensor.
  • the plasmonic biosensor comprises: (a) a base; and (b) an array of first and second regions on a surface of the base facing the first electrode, wherein the first region comprises dielectric squares that are separated by the second region, which comprises a reflective metal, wherein the second region further comprises a plurality of nanowells.
  • E4 The device of E3, wherein the dielectric comprises SiN.
  • E5. The device of E3 or E4, wherein each of the dielectric squares are between about 10 pm to about
  • E6 The device of E5, wherein each of the dielectric squares are about 40 pm by about 40 pm.
  • E7 The device of any one of E3-E6, wherein the dielectric squares are separated by about 10 pm to about 100 pm.
  • E8 The device of E7, wherein the dielectric squares are separated by about 40 pm.
  • each of the plurality of nanowells has a diameter of between about 10 nm and about 1000 nm.
  • E11 The device of E10, wherein each of the plurality of nanowells has a diameter of about 200 nm.
  • E12 The device of any one of E3-11 , wherein the plurality of nanowells have a periodicity of between about 10 nm and about 1000 nm.
  • E13 The device of E12, wherein the plurality of nanowells have a periodicity of about 500 nm.
  • E14 The device of any one of E1 -E13, wherein the biosensor further comprises a capture agent, wherein the capture agent specifically binds to a target biomarker.
  • E15 The device of E14, wherein the capture agent is an antibody or antigen-binding fragment thereof.
  • E16 The device of any one of E1 -E15, wherein the first electrode comprises a transparent planar surface.
  • E17 The device of E16, wherein the transparent planar surface is an indium tin oxide surface.
  • E18 The device of any one of E1 -E17, wherein the chamber has a height of between about 1 pm to about 10 mm.
  • E19 The device of E18, wherein the chamber has a height of between about 50 pm to about 100 pm.
  • a plasmonic biosensor comprising: (a) a base; and (b) an array of first and second regions on a surface of the base facing the first electrode, wherein the first region comprises dielectric squares that are separated by the second region, which comprises a reflective metal, wherein the second region further comprises a plurality of nanowells.
  • E21 The plasmonic biosensor of E20, wherein the dielectric comprises SiN.
  • E22 The plasmonic biosensor of E20 or E21 , wherein each of the dielectric squares are between about 10 pm to about 100 pm by about 10 pm to about 100 pm.
  • E23 The plasmonic biosensor of E22, wherein each of the dielectric squares are about 40 pm by about 40 pm.
  • E24 The plasmonic biosensor of any one of E20-E23, wherein the dielectric squares are separated by about 10 pm to about 100 pm.
  • E25 The plasmonic biosensor of E24, wherein the dielectric squares are separated by about 40 pm.
  • E26 The plasmonic biosensor of any one of E20-E25, wherein the reflective metal comprises gold.
  • E27 The plasmonic biosensor of any one of E20-E26, wherein each of the plurality of nanowells has a diameter of between about 10 nm and about 1000 nm.
  • each of the plurality of nanowells has a diameter of about 200 nm.
  • E29 The plasmonic biosensor of any one of E20-E28, wherein the plurality of nanowells have a periodicity of between about 10 nm and about 1000 nm.
  • E30 The plasmonic biosensor of E29, wherein the plurality of nanowells have a periodicity of about 500 nm.
  • E31 The plasmonic biosensor of any one of E20-E30, wherein the biosensor further comprises a capture agent, wherein the capture agent specifically binds to a target biomarker.
  • a method for concentrating a target biomarker comprising: (a) applying a sample comprising the target biomarker to the device of any one of E1 -E19; and (b) applying an AC pulse, wherein the target biomarker concentrates adjacent to the biosensor.
  • E33 The method of E32, further comprising detecting the target biomarker.
  • E34 The method of E32 or E33, wherein the AC pulse is between about 1 kHz and about 100 kHz and between about 1 Vpp to about 30 Vpp AC.
  • E35 The method of E34, wherein the AC pulse is about 10 kHz 10Vpp AC.
  • E36 The method of any one of E32-E35, wherein the target biomarker is detectable on an extracellular vesicle.
  • E37 The method of any one of E32-E36, wherein extracellular vesicles including the target biomarker are concentrated.

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Abstract

L'invention concerne des procédés et des compositions pour concentrer des biomarqueurs cibles par application d'une combinaison d'électro-osmose en courant alternatif (AC) et de diélectrophorèse (DEP), qui amènent des biomarqueurs cibles vers la surface d'un biocapteur.
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