EP4638010A1

EP4638010A1 - Methods and materials for performing electrochemical assays

Info

Publication number: EP4638010A1
Application number: EP23908562.4A
Authority: EP
Inventors: Alexander Revzin; Alan M. Gonzalez Suarez; Seonhwa LEE; Josue AMADOR-HERNANDEZ
Original assignee: Mayo Foundation for Medical Education and Research; Mayo Clinic in Florida
Current assignee: Mayo Foundation for Medical Education and Research; Mayo Clinic in Florida
Priority date: 2022-12-21
Filing date: 2023-12-21
Publication date: 2025-10-29
Also published as: WO2024138016A1

Abstract

This document provides devices having a fluidic module and an electrical module. Methods of using such devices also are provided herein.

Description

METHODS AND MATERIALS FOR PERFORMING ELECTROCHEMICAL ASSAYS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Patent Application Serial No. 63/434,351 , filed on December 21, 2022. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

TECHNICAL FIELD

This document relates to methods and materials for performing electrochemical assays. For example, this document provides systems and devices having a fluidic module and an electrical module as well as methods for using such systems and devices.

BACKGROUND INFORMATION

Microfluidic devices can be useful tools that enable rapid, low-cost, and automated biological or chemical assays.

SUM ARY

This document provides methods and materials for performing electrochemical assays. For example, this document provides systems and devices having a fluidic module and an electrical module. In some cases, a device described herein can be configured to conduct an electrochemical assay. In some cases, such an assay can include detecting one or more extracellular vesicles (EVs), in which the EV may be the target analyte. In some cases, such an assay can include detecting one or more target analytes from one or more EVs (e.g., any analyte that may be present in a lysate or other sample obtained from EVs). Methods of using such systems and devices also are described herein.

Accordingly, in a first aspect, provided herein is a deydce (e.g., for conducting an electrochemical assay) including: a fluidic module configured to provide a chamber (e.g., a first chamber), a reservoir, and a releasable valve; and an electrical module configured to provide a working electrode within said chamber and to provide a reference electrode within said reservoir.

In some embodiments, said releasable valve is configured to minimize fluidic communication between said chamber, or a portion thereof, and said reservoir.

In some embodiments, said working electrode is configured to be attached (e.g., directly or indirectly) to a capture agent for capturing a target analyte. In some embodiments, said device further includes a plurality of chambers. In some embodiments, a first chamber is one of said plurality of chambers. In some embodiments, said first chamber is configured to be in fluidic communication with said reservoir, and a second chamber of said plurality of chamber is configured to be in fluidic communication with said first chamber. In some embodiments, said second chamber is configured to lyse a sample to provide one or more target analytes and configured to deliver said one or more target analytes to said first chamber.

In some embodiments, each of said plurality of chambers is configured to be in fluidic communication with said reservoir. In some embodiments, each of said plurality of chambers includes an individually addressable working electrode.

In some embodiments, said electrical module further includes a counter electrode within said reservoir.

In some embodiments, said releasable valve includes one or more structures configured to provide fluidic communication between said chamber and said reservoir at a pressure from about 0.5 psi to about 2 psi. In some embodiments, said one or more structures include an inlet, an outlet, and a first constricted portion disposed between said inlet and said outlet. In some embodiments, the first constricted portion includes a change in dimension along one or more of x, y, or z axes.

In some embodiments, the device further includes an expanded portion disposed between said first constricted portion and said outlet. In some embodiments, the expanded constricted portion comprises a change in dimension along one or more of x, y, or z axes. In some embodiments, the expanded portion includes a liquid bypass region. In some embodiments, the device further includes a second restricted portion and an expanded portion, wherein said expanded portion is further disposed between said first and second restricted portions.

In some embodiments, said fluidic module includes a non-conductive material, polymer, elastomer, glass, or a combination thereof.

In some embodiments, said working electrode further includes an attached capture agent. In some embodiments, said capture agent includes one or more of a nanoparticle, a microparticle, a dye, a detectable agent, a protein, an antibody, a nucleic acid, an aptamer, a small molecule, or a combination thereof. In some embodiments, the device further includes a linker disposed between a surface of said working electrode and said attached capture agent. In some embodiments, said linker includes a covalent bond, an optionally substituted alkylene, or an optionally substituted heteroalkylene. In some embodiments, said electrical module further includes a bond pad and a connector configured to electrically connect said working electrode to said bond pad.

In some embodiments, said device is a monolithic device.

In some embodiments, said fluidic module and said electrical module are configured to be separated.

In some embodiments, an adhesive layer (e.g., including any adhesive described herein) disposed between said fluidic module and said electrical module.

In a second aspect, provided herein is a system including: a device described herein; an optional lysing plate configured to provide one or more target analytes for said device; a platform configured to electrically connect said device to a controller configured to control said electrical module; and a user interface configured to receive and transmit information between said device and said controller.

In some embodiments, said lysing plate includes a plurality of wells, wherein a surface of the plurality of wells includes a capture agent configured to capture at least one of the one or more target analytes.

In some embodiments, said controller includes a multiplexer, a potentiostat, and/or a printed circuit board for making one or more electrical contacts.

In a third aspect, provided herein is a method for conducting an electrochemical assay. In some embodiments, said method includes:

(a) providing any device herein or a system herein;

(b) delivering a sample to said chamber of said device;

(c) providing one or more reagents for said electrochemical assay to said reservoir of said device;

(d) actuating said releasable valve, thereby providing electric communication or fluidic communication between said chamber and said reservoir; and

(e) detecting one or more electrical signals from said working electrode, thereby detecting the presence or absence of a target analyte in said sample.

In some embodiments, each of (b), (c), and (d) can be conducted at the same time or in any order.

In some embodiments, said sample includes one or more extracellular vesicles, analytes from one or more extracellular vesicle, lysates from one or more extracellular vesicles, particles, micelles, or a combination of any of these.

In some embodiments, said target analyte includes a protein disposed on a surface of at least one of said one or more extracellular vesicles. In some embodiments, said target analyte includes a protein, a nucleic acid, a lipid, or other analyte (e.g., any described herein) from at least one of said one or more extracellular vesicles.

In some embodiments, said one or more reagents include a nanoparticle, a metal, a dye, a fluorophore, a detectable reporter, an electroactive agent, or a combination thereof.

In some embodiments, (c) of said providing and (d) of said actuating are conducted at the same time.

In some embodiments, said one or more electrical signals are determined between said working electrode and said reference electrode.

In a fourth aspect, provided herein is a method for detecting an analyte of an extracellular vesicle. In some embodiments, said method includes:

(a) capturing one or more extracellular vesicles on said working electrode of any device herein or any system herein; and

(b) detecting one or more electrical signals from said working electrode, thereby detecting the presence or absence of a target analyte on a surface of at least one of said one or more extracellular vesicles.

In some embodiments, the method further includes (e.g., after or before said capturing): (al) providing one or more reagents for an electrochemical assay to said working electrode in said chamber.

In some embodiments, the method further includes (e.g., after or before said capturing): (al) providing one or more reagents for an electrochemical assay to said reservoir.

In some embodiments, the method further includes (e.g., after or before said capturing): (al) actuating said releasable valve, thereby providing electric communication or fluidic communication between said chamber and said reservoir.

In some embodiments, said actuating provides said electric communication and said fluidic communication between said chamber and said reservoir.

In a fifth aspect, provided herein is a method for detecting an analyte of an extracellular vesicle. In some embodiments, said method includes:

(a) capturing one or more target analytes from one or more extracellular vesicles on said working electrode of any device herein or any system herein; and

(b) detecting one or more electrical signals from said working electrode, thereby detecting the presence or absence of at least one of said one or more target analytes from said one or more extracellular vesicles (e.g., within a lysate obtained from at least one of said one or more extracellular vesicles).

In some embodiments, the method further includes (e.g., before said capturing): (aO-1) lysing said one or more extracellular vesicles to release said one or more target analytes; and (aO-2) providing said one or more target analytes to said working electrode.

Definitions

By “electrical communication,’’ as used herein, refers to any structure or space through which an electrical signal may be conducted. Such structures and spaces can include a conductive material itself, such as conductive liquid, gas, or solid, or can contain the conductive material. Electrical communication, in some instances, can include electrochemical communication, in which the conductive material can include one or more chemical agents.

By “fluidic communication,’’ as used herein, refers to any duct, channel, tube, pipe, chamber, or pathway through which a substance, such as a liquid, gas, or solid may pass substantially unrestricted when the pathway is open. When the pathway is closed, the substance is substantially restricted from passing through. Typically, limited diffusion of a substance through the material of a plate, base, and/or a substrate, which may or may not occur depending on the compositions of the substance and materials, does not constitute fluidic communication.

By “microfluidic” or “micro” is meant having at least one dimension that is less than 1 mm. For instance, a micro fluidic structure (e.g., any structure described herein) can have a length, width, height, cross-sectional dimension, circumference, radius (e.g., external or internal radius), and/or diameter that is less than 1 mm.

By "nano” is meant having at least one dimension that is less than 1 gm but equal to or larger than about 1 nm. For instance, a nanostructure (e.g., any structure described herein, such as a nanoparticle) can have a length, width, height, cross-sectional dimension, circumference, radius (e.g., external or internal radius), and/or diameter that is less than 1 pm but equal to or larger than 1 nm. In other instances, the nanostructure has a dimension that is of from about 1 nm to about 1 pm.

As used herein, the terms “top,” “bottom,” “upper,” “lower,” “above,” and “below” are used to provide a relative relationship between structures. The use of these terms does not indicate or require that a particular structure must be located at a particular location in the apparatus.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary⁷ skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of an exemplary device 100 having a fluidic module 110 and an electrical module 150, according to some embodiments.

FIG. 2A-2C shows exemplary releasable valves, according to some embodiments. Provided are schematics of (A) a plan view of chambers 212 within the fluidic module, (B) a magnified view of the valved region 215, and (C) non-limiting examples of geometries for valves.

FIG. 3 shows an exemplary strategy for extracellular vesicle (EV) detection, according to some embodiments. EVs were captured on a working electrode then labeled with redox active immunoprobes. EV capture and surface marker detection were carried out in a microtiter plate that contained 16 electrochemical cells. Square wave voltammetry (SWV) was used to assess EV surface marker expression.

FIG. 4 shows an exemplary schematic diagram describing integration of the MUA- functionalized electrode array with a microwell layer, according to some embodiments. Electrodes are micropattemed on glass and then functionalized with MUA. A microstructured poly(dimethylsiloxane) (PDMS) mask (or cover) is used to protect working electrodes while exposing counter and reference electrodes during oxygen plasma treatment. Plasma treatment removes MUA from counter and working electrodes, and it can also condition the glass substrate for bonding with the PDMS microwell layer. Antibodies for capture of EVs are then covalently attached to working electrodes presenting MUA moieties. RE, CE, and WE represent reference electrode, counter electrode, and working electrode, respectively. MUA is 11 -mercaptoundecanoic acid.

FIG. 5 shows operation of an exemplary electrochemical microtiter plate, according to some embodiments. (A) The plate included a PDMS layer containing wells, fluidic channels, and capillary valves; and a glass substrate with array of Au electrodes. There were 16 circular working electrodes (2.5 mm diameter, one electrode per well), as well as reference and counter electrodes. WE, RE, and CE denote working, reference and counter electrodes, respectively. (B) Close-up view of 4 wells with working electrodes located next to reference and counter electrode. Reference electrodes were fabricated by painting silver paste atop Au. Note that wells containing working electrodes are connected to counter and reference electrodes via fluidic channels molded in PDMS, how ever, the solution with blue dye is confined to the wells. This happens because wells also contain capillary valves (see close up view) that increase surface tension locally. (C) The use of capillary valves allow s us to operate the microtiter plate in two phases. Phase 1 - plate preparation where working electrodes are functionalized with antibodies (Abs). EVs are captured and labeled with immunoprobes. Counter and reference electrodes are generally minimally affected during these steps due to capillary valves. Phase 2 - plate reading, where capillary valves are actuated by exceeding the threshold pressure. The electrolyte solution fills the fluidic channels connecting working electrodes to counter and reference electrodes.

FIG. 6 shows electrochemical characterization of an exemplary microtiter plate, according to some embodiments. (A) SWV curves w ere obtained for each of the 16 w orking electrodes connected to on-chip counter and reference electrodes. (B) SWV curves w ere obtained for working electrodes connected to (a) on-chip reference and counter electrodes and (b) off-chip Ag/AgCl reference and Pt counter electrodes, respectively. 5 mM | Fe(CN)6|^{4 3} in 0.1 M KC1 was used for parts A and B.

FIG. 7 shows performance of an exemplary electrochemical microtiter plate, according to some embodiments. (A) Assessing whether position of the well affects performance of the electrochemical immunoassay. These results suggest that wells produce consistent electrochemical signals regardless of the location on the plate and distance to the counter/reference electrode. Top: the sample loading configuration with podocin concentrations and positions. Bottom: SWV signals for different concentrations of recombinant podocin followed by labeling with AuNPs/anti-podocin@Pb²⁺. (B, C) Calibration curves obtained after immobilizing different concentrations of recombinant podocin and nephrin in the wells of the microtiter plate. The linear plot of the normalized total charge (Q = Q - Qo) changes as a function of the logarithm of the concentration of recombinant podocin or nephrin (0-500 ng/mL).

FIG. 8 shows nanoparticle-enabled electrochemical immunoassay for detection of podocin and nephrin. according to some embodiments. Electrochemical (SWV) analysis of electrodes after incubation with different concentrations of nephrin and podocin, and labeling with appropriate immunoprobes (AuNPs/anti-podocin@Pb²⁺ for podocin and AuNPs/anti- nephrin@ Pb²⁺ for nephrin). SWV curves shown concentration dependence of the electrochemical signal.

FIG. 9 shows characterization of urinary EVs. (A) NTA analysis of concentration and dimension of urinary EVs, according to some embodiments. EV particles had a diameter of 152 ± 62 nm. EV concentration in undiluted urine sample was 1.54 x 10⁸ particles/mL. (B) Representative TEM images of EVs before (left) and after (right) incubation with immunoprobes. Scale bar. 100 nm.

FIG. 10 shows characterization of EV capture in an exemplary microtiter plate, according to some embodiments. (A) Steps for electrode functionalization, EV capture and labeling with immunoprobes. (B) EIS measurement for individual surface modification steps: (a) Au electrode (insert), (b) Au/MUA, (c) Au/MUA/EDC-NHS, (d) Au/MUA/EDC-NHS/anti-CD63, (e) Au/MUA/EDC-NHS/anti-CD63/BSA. (f) Au/MUA/EDC-NHS/anti-CD63/BSA/EVs, and (g) Au/MUA/EDC-NHS/anti- CD63/BSA/EVs/AuNPs-anti-CD63@Pb²⁺. (C) Experimental groups used for assessment of assay specificity in the microtiter plate. Each electrode was functionalized with anti-CD63 Abs for the capture of EVs. Immunoprobes targeting podocin and nephrin on urinary EVs were then used for labeling and electrochemical detection (SWV). Immunoprobes functionalized with anti-CD63 and anti-IgG were used as positive and negative controls, respectively. Another control experiment involved assessing non-specific interactions of immunoprobes in the absence of EVs (w/o EVs). In this experiment, electrodes were functionalized with anti-CD63 and then exposed to immunoprobes targeting CD63 without capturing EVs. (D) SWV curves associated with different experiment groups. (E) Total charge (Q) values were determined by calculating area under the curve for SWV peaks. Note minimal redox activity for negative control groups.

FIG. 11 shows the use of SPR to characterize EV capture and immunoprobe binding. (A) SPR sensogram showing Ab immobilization, EV capture, and immunoprobe labeling steps. (B) SPR binding signal of EV capture on electrodes functionalized with (a) anti-CD63 Abs and (b) isotype control Abs. (C) SPR signals for captured EVs labeled with (a) AuNPs/anti-CD63@Pb²⁺ and (b) AuNPs/IgGhPb² . respectively. RU: Resonance Units.

FIG. 12 shows representative SEM images of EVs captured on an electrode surface functionalized with anti-CD63 Abs. Images before (left) and after (right) incubation with EVs (1.54 x 108 particles/mL). Scale bar. 2 pm.

FIG. 13 shows use of hepatic EVs to assess specificity of an electrochemical immunoassay. (A) Capture of hepatic EVs on electrodes functionalized with anti-CD63 was characterized by EIS. (B,C) SWV signals and total charge (Q) values after labeling EVs with immunoprobes (AuNPs/Abs@Pb²⁺). Each data point represents three measurements carried out in different wells (n = 3).

FIG. 14 shows the assessment of podocin-to-nephrin ratios in EVs from clinical urine samples. (A) Electrochemical detection of EVs expressing podocin and nephrin in the microtiter plate. Urine samples from normotensive (NT, n=4) and preeclamptic pregnancies (PE, n=4) were analyzed. AuNPs/anti-podocin@Pb²⁺ and AuNPs/anti-nephrinV Pb² immunoprobes w ere used to label EVs and generate electrochemical redox signals. EVs were also labeled with immunoprobes carrying isotype control Abs to establish the background signal. The values of normalized total charge (Q = QEVS - Qisotype control Abs) were used to quantify the podocin/nephrin ratio. (B) Comparison of podocin/nephrin ratios in urinary EVs from normotensive (NT) and preeclamptic (PE) pregnant women. The ratio represents Q for podocin / Q for nephrin. The analysis was carried out in triplicate (three electrodes) for each clinical sample, p < 0.002. (C) An SPR sensogram for analysis of podocin and nephrin expression in clinical sample PE3. (D) Comparison of podocin/nephrin ratios obtained with an electrochemical immunoassay and SPR for clinical urine samples. FIG. 15 shows an SPR analysis of clinical urine samples. (A) SPR sensogram showing surface preparation steps followed by incubation with patient urine. (B) Description of podocin/nephrin ratio calculation. We used a 3-channel SPR instrument and captured EVs from a given patient in all channels. Then, individual channels were perfused with anti- podocin, anti-nephrin or rabbit IgG (isotype control) Abs. SPR response (RU, Resonance Units) was obtained from the baseline changes before and after injection of Abs. Signal for binding of these Ab types (RU) was used to calculate podocin-to-nephrin ratio. Isotype control signal was subtracted from podocin and nephrin signals. Then, the podocin/nephrin ratio was calculated by dividing normalized podocin (RU_Podocin) to normalized nephrin (Rtty/ep/irm)-

FIG. 16A-16C shows schematics of an exemplary multilayer device 1600. Provided are schematics of (A) a plan view of the device, (B) a magnified view of the valved region 1615 indicating flow direction 1660. and (C) a magnified view of the valved region 1615 indicating blocked flow 1662.

FIG. 17A-17C shows schematics of an exemplary fluidic module 1710. Provided are schematics of (A) a three-dimensional model of the fluidic module, (B) a magnified view" of the valve region, and (C) a magnified, inverted view' of the valve region.

FIG. 18A-18C shows schematics of an exemplary releasable valve, according to some embodiments. Provided are schematics of (A) a plan view of a one-level stop valve, (B) a cross-sectional side view' of a two-level stop valve, and (C) a side view of non-limiting adhesive layers for use as a pressure sensitive adhesive (PSA) layer.

FIG. 19 show s an exemplary schematic diagram describing use of a non-limiting fluidic module.

FIG. 20 shows exemplary images of a non-limiting fluidic module being filled with an aqueous solution.

FIG. 21 show s exemplary images of a non-limiting fluidic module being filled with an aqueous solution including surfactant ((4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid) (HEPES) buffer with Tween 20 (polyethylene glycol sorbitan monolaurate surfactant) at 0.05%).

FIG. 22A-22B shows exemplary' lysing plates, according to some embodiments. Provided are schematics of (A) a plan view and (B) a three-dimension view' of non-limiting lysing plates 2292. 2294, 2296. DETAILED DESCRIPTION

This document provides methods and materials for performing electrochemical assays. For example, this document provides devices having both a fluidic module (e.g.. a microfluidic module) and an electrical module (e.g., an electrochemical module). The devices provided herein can be used to perform an assay (e.g., an electrochemical assay). For instance, the fluidic module of a device described herein can be used to deliver samples and reagents for conducting an assay, and the electrical module of that device can be used to capture an extracellular vesicle (EV) and detect the presence of a target analyte associated with the EV (e.g., disposed on a surface of the EV and/or disposed within an EV). In another instance, the fluidic module of a device described herein can be used to deliver samples and reagents for conducting an assay, and the electrical module of that device can be used to capture a target analyte from an EV and detect the presence of a target analyte associated with the EV (e.g., disposed on a surface of the EV and/or disposed within an EV). The target analyte can be detected by using a label (e.g., a label described herein). In some cases, the label can be an electroactive label or electrically detectable label.

FIG. 1 shows an exemplary device 100 including a fluidic module 110 and an electrical module 150. As can be seen, the fluidic module can be configured to provide a chamber 112, a reservoir 120, and a valved region 115 including a releasable valve. The releasable valve can be configured to control fluidic communication between the chamber and the reservoir.

Chamber 112 can be in fluidic and electric communication with a working electrode 152, which in turn can be functionalized with capture agent 113. The capture agent (e.g., a capture agent described herein) can be provided in any useful manner. In some cases, the capture agent can be directly bound to a surface of a working electrode. In other cases, the capture agent can be indirectly bound (e.g., by way of a linker) to a surface of a working electrode. In use, a sample can be delivered to chamber 1 12 (e.g., by way of chamber inlet 114, which in turn can be in fluidic communication with a channel, a fluidic manifold, and the like, for transporting the sample to the fluidic module); and the capture agent can bind to a target analyte in the sample. The capture agent can be selected based on the desired target analyte to be captured or analyzed by the device. In some cases, the target analyte may be an EV itself (e.g., an intact EV or a portion of an outer portion of an EV), and the capture agent can bind a component of the EV (e.g., a component disposed on an outer surface of an EV). In some cases, the target analyte may be an analyte from an EV (e.g., intravesicular content from within an EV or any component present within an EV), and the capture agent can such an analyte. Electrical or electrochemical measurements can be determined by one or more electrical signals from the working electrode.

Reservoir 120 can be in fluidic and electric communication with a non- working electrode (e.g., areference electrode 158 or a counter electrode 156, 156’). In use, reservoir 120 can be used to deliver one or more reagents to chamber 112 by way of a channel 111. Reagent(s) can be delivered to reservoir 120 (e.g., by way of a reservoir inlet 124, which in turn can be in fluidic communication with a channel, a fluidic manifold, and the like, for transporting the reagent(s) to the fluidic module).

Channel 111 can be disposed between chamber 112 and reservoir 120, and control of flow within this channel 111 can be controlled by the presence of valved region 115. For instance, valved region 115 can be used to separate chamber 112 from reservoir 120, or to separate the working electrode (in the chamber) from other non-working electrodes (in the reservoir, e.g., such as a reference electrode or a counter electrode). Such separation may be used to functionalize the working electrode with a capture agent, to incubate the sample within a chamber having the working electrode, or to separate the sample in the chamber from reagents in the reservoir prior to conducting an assay.

Valved region 115 can include one or more releasable valves, which can be actuated (i.e., opened or closed) by a stimulus (e.g., pressure, force, temperature, electric field, and the like). In some cases, a releasable valve can be actuated from a closed state to an opened state by a change in pressure (e.g., an increase of pressure) past a threshold. Such changes in pressure can be determined across valved region 115.

A releasable valve can include any useful structure configured to be actuated, thereby providing electric and/or fluidic communication between chamber 112 and reservoir 120. FIG. 2A provides an exemplary configuration of a fluidic module configured to provide a chamber 212, a reservoir 220, and a valved region 215. A plurality of chambers (212, 212’, 212”, 212’”) may be arranged to be in fluidic communication with a reservoir 220, which in turn can include a reservoir inlet region 224 and an inlet channel 222.

Fluidic communication between chamber 212 and reservoir 220 can be provided byway of a channel 211, but flow through channel 211 can be controlled by one or more valves. In some cases, a plurality' of valved regions 215 can be disposed within a chamber 212, thereby minimizing flow between chamber 212 (or a portion thereof) and reservoir 220 when the valves are closed. As seen in FIG. 2A, the valved regions can be used to define a peripheral region 212b and a central region 212a of chamber 212. in which fluidic communication can be minimized between a portion of chamber 212 (e.g., a central region 212a) and reservoir 220.

Valved regions, as well as valves, may be present in any usefill position within the fluidic module, such as within the chamber, within the channel disposed betw een the chamber and the reservoir, within the reservoir, as well as any other structure that can be used to provide (or prevent) fluidic communication between the chamber and the reservoir. Fluidic communication can include any magnitude of fluidic communication, which can include reducing, stopping, or increasing flow of fluids. Furthermore, fluidic communication can include control of flow within portions of structures, such as control of fluidic communication between a portion of the chamber and a portion of the reservoir.

The releasable valve can include one or more structures configured to provide fluidic communication between the chamber and the reservoir. Such structure(s) can respond to a threshold pressure (e.g., a pressure from about 0. 1 psi to about 5 psi, 0.1 psi to about 2 psi, 0.5 psi to about 5 psi, or about 0.5 psi to about 2 psi), in which applying pressure above the threshold to the reservoir or the chamber can result in actuating the valve. FIG. 2B provides exemplary structures of a valved region 215. As can be seen, the valve in the valved region can include an inlet 252 and an outlet 252, in which fluid can flow 255 therein. While flow is provided in one direction (from a peripheral region of the chamber 212b to a central region of the chamber 212a), flow may be provided in the opposing direction or in both directions. Both unidirectional and bidirectional valves can be used as described herein.

In some cases, the releasable valve can include constricted and expanded portions to create regions that are responsive to varying pressure. For example, a constricted portion 266 can be employed to provide a region of increased pressure w hen a fluid flows through that portion. In another example, an expanded portion 268 can be employed to provide a region of decreased pressure when a fluid flows through that portion. By using such structures, regions of desired pressure gradients can be designed and implemented in a releasable valve 260 to control flow⁷ through the valve. The constricted portion can be characterized by a region having a reduced dimension, such as a reduced length, weight, diameter, height, or the like (e.g.. as compared to a dimension of a chamber or a reservoir in fluidic communication with the releasable valve). In some cases, such a region may be channel. The expanded portion can be characterized by a region having an increased dimension, such as an increased length, weight, diameter, height, or the like (e.g., as compared to a dimension of the constricted portion in fluidic communication with the expanded portion or as compared to a dimension of a chamber or a reservoir in fluidic communication with the expanded portion). In some cases, such a region may be channel.

FIG. 2C provides exemplary schematics of valves having various inlets, outlets, constricted portions, and expanded portions. For example, valves for use as described herein can be designed to include (i) an inlet, an outlet, a first constricted portion disposed in proximity' to the inlet, a second constricted portion disposed in proximity to the outlet, and an expanded portion disposed between the first and second constricted portions; (ii) an expanded inlet, an outlet, and a constricted portion disposed between the inlet and outlet; (iii) an inlet, an expanded outlet, and a constricted portion disposed between the inlet and outlet; (iv) an inlet, an expanded outlet, and an expanding portion disposed between the inlet and outlet; (v) an arrangement as in (i) but having a smaller expanded portion; (vi) an inlet, an expanded outlet, and an expanding portion disposed between the inlet and outlet; (vii) an inlet, an outlet, a first constricted portion disposed in proximity to the inlet, a second constricted portion disposed in proximity to the outlet, and another expanded portion disposed between the first and second constricted portions; and/or (viii) an expanded inlet, an outlet, and a constricted portion disposed between the inlet and outlet.

Any of these constricted and/or expanded portions can have any useful geometry. The extent of change (e.g., extent or degree of changes in expanding or constricting a crosssection within the structure) can be designed to provide any useful changes in pressure, volume, or flow. Such changes can be characterized by flow induced by changes in pressure, surface tension, capillary force, and/or other forces. For example and without limitation, such constricted and/or expanded portions may includes changes in one or more dimensions to effect any useful changes in pressure, volume, or flow. Such changes in one or more dimensions can be along one or more of x, y, or z axes (e.g., as provided in FIG. 1. FIG. 2B, or FIG. 17A).

A plurality of valves may be arrayed, arranged, or otherwise configured in any manner to provide control of fluidic communication between a chamber (or a portion thereof) and a reservoir (or a portion thereof) as described herein.

Turning again to FIG. 1, device 100 can include an electrical module 150 having one or more electrodes. Electrical module 150 can include a substrate 151 having an electrode, such as a working electrode 152. Electrical module 150 can be configured to provide a working electrode to be in fluidic communication with a chamber. Such configurations can be implemented by spatially arranging the working electrode to be in fluidic communication within a chamber upon aligning the fluidic module with the electrical module, patterning the working electrode to be within the chamber, or in any other useful manner.

One or more non-working electrodes (e.g.. a counter electrode 156. 156’ and/or a reference electrode 158) can be disposed on a substrate 151 . Any useful electrode configuration can be used (e.g., a two- or three-electrode configuration). In some cases, the electrode configuration can be designed to allow for on-chip electrochemical measurements. In some cases, the electrode configuration can be designed to allow for each chamber to function as an independent electrochemical cell.

The electrical module can be configured to provide a counter electrode and/or a reference electrode to be in fluidic communication with a reservoir. Such configurations can be implemented by spatially arranging the counter/reference electrode to be in fluidic communication within a reservoir upon aligning the fluidic module with the electrical module, patterning the counter/reference electrode to be within the reservoir, or in any other useful manner.

The electrical module can include one or more components to electrically connect the electrodes to a controller and/or a power source. As seen in FIG. 1, electrical module 150 can include a bond pad 153 and a connector 154, in which connector 154 forms an electrical connection between bond pad 153 and a working electrode 152. Other bond pads and connectors may be used in any useful number, configuration, or arrangement to provide electrical connections to the counter electrode(s) and reference electrode(s). Any of these electrical connections (e.g., bond pad, connector, electrodes, and the like) can be formed from any useful conductive material or ohmic metal (e.g., a transition metal, an alloy thereof, and the like).

FIG. 16A-16C shows another exemplary device 1600 including a fluidic module and an electrical module. As can be seen, the fluidic module includes a reagent inlet/outlet 1614 in fluidic communication with a chamber, an electrolyte inlet 1624 in fluidic communication with a reservoir, and an air purge 1626 in fluidic communication with a chamber. As can be seen in FIG. 16A, each chamber can include a separate reagent inlet/outlet. Alternatively, a reagent inlet/outlet can be in fluidic communication with a plurality of chambers. As can be seen in FIG. 16A, an inlet or outlet for air purge can be in fluidic communication with a plurality of chambers. Alternatively, each chamber can include a separate air purge port (e.g., air purge inlet/outlet).

As can be also seen in FIG. 16A, the electrical module includes working electrodes (WE) 1652A-D and a counter and reference electrodes (C, RE) 1650. The fluidic module can be configured to provide a chamber in fluidic communication with a working electrode, as well as a reservoir in fluidic communication with the counter and reference electrodes.

FIG. 16B-16C provides a magnified view of the valved regions 1615, in which the releasable valves in this region can be actuated to provide flow 1660 into a chamber and configured to block flow 1662. In some embodiments, the valved regions are provided in a multilayer device (e.g., provided as a microtiter plate being a fluidic module, which in turn can be used with an electrical module). The fluidic module can be formed from any material (e.g., any described herein). In some cases, the fluidic module is formed from a thermoplastic material (e.g., configured to provide one or more chambers, reservoirs, and valved region) with an adhesive layer (e.g., configured to attach to an electrical module and optionally further configured to enhance valving within the valved regions).

The valved regions can include one or more capillary valves having any useful dimensions. In some cases, a plurality of capillary valves is associated with each chamber (e.g., two. three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more capillary valves for each chamber). Any useful dimensions can be employed to provide desired pressure differences upon actuating the valve. In some cases, the cross-section of a valve can include a dimension from about 50 to 800 pm in width and from about 50 to 500 pm in height.

FIG. 17A show s an exemplary fluidic module 1710 including a plurality of chambers 1712A-D, valved regions 1715A-D, a reservoir 1720, and a reservoir inlet 1724. Here, the valves in the valved regions can be configured to provide liquid pinning that occurs in three dimensions. For example, one or more height differences (e.g., along the z-axis in FIG. 17A) can provide an additional constricted region. If a valve requires increased dimensions along the x- and/or y-axes (e.g., due to, for instance, manufacturing or processing conditions, material choice, electrochemical assay conditions, etc.), then controlling a dimension along the z-axis can further provide desired actuating conditions (e g., as determined by desired pressure differences, flow direction, and the like). For example and without limitation, changes in the height and geometry of the valves can be selected to minimize the capillary pressure and/or minimize the fluid flow in one direction, while maximizing the capillary pressure in the opposite direction. Such changes can be present when optimizing valves only in one direction (e.g., along the x- or y-axes, such as by modifying the width of a cross- sectional region within a 2D valve), and optimizing in other dimensions (e.g., along the x- or y-axes and along the z-axis, such as by modifying the width and height of a cross-sectional region within a 3D valve) can increase efficiency. FIG. 17B-17C provides three-dimensional models of valved regions, and FIG. 18A- 18B provides schematics of valved regions. In these figures, a capillary valve can include a one-level stop valve (labeled with 1 and 1 ’). The one-level stop valve includes a magnification angle (that in this case is |3 = 102°, but P can be any useful angle), which provides two anchor points (1 and 1 ’) to stop the liquid (FIG. 17B and FIG. 18A). In addition, the valve can include a liquid bypass region, which is disposed in a perpendicular direction (labels 2 and 2’ and black arrows in FIG. 17C) with respect to the flow coming from the microwell (gray arrow towards label 4 in FIG. 17C). This expanded region 1768 in proximity to the constricted region 17 6 provides a liquid bypass region, whereas the presence of the constricted region 1766 provides a liquid pinning region. This combination of the constricted and expanded regions allows for a liquid to actuate only when desired, in which the liquid can move sideways, preventing its continuous forward advance, which contributes to maintaining the valve in a closed position.

As seen in FIG. 18B, a valve can include a vertical elongation (e.g., of 90° or other useful angle) along the z-axis to provide a two-level stop valve. This three-dimensional arrangement (along the x-. y-, and z-axes) can provide an additional anchorage point for pinning the liquid within the valved region, in addition to the two anchors offered by the single-level stop valve. A valve may include the use of another layer (e.g., an adhesive layer) to provide changes in dimension along the z-axis. In some embodiments, the layer includes an adhesive layer disposed between a channel of the fluidic module and a surface of the electrical module. The adhesive layer can be provided in any useful format. For example and without limitation, FIG. 18C provides a non-limiting pressure sensitive adhesive (PSA) layer, which can include a thermoplastic carrier, a first adhesive layer disposed on a top surface of the thermoplastic carrier, and a second adhesive layer disposed on a bottom surface of the thermoplastic carrier. In use, the PSA layer can be aligned and then attached between fluidic module and electrical module. In some cases, the electrodes and capillary valves layer can be bonded with a PSA layer with a thickness of 50 pm. However, a different bonding layer (e.g., differing material and/or thickness) or strategy could be used to achieve similar results.

The devices described herein can include any useful configuration of chambers, reservoirs, inlets, and outlets to deliver fluid for functionalizing a surface of the fluidic module and/or conducting an assay. As seen in FIG. 19, additional ports (e.g., reagents inlets/outlets as in FIG. 16A) can be in fluidic communication with a chamber. Such ports could be used as an alternative way of filling or retrieving liquid from the chamber (e.g.. if a dimension, such as diameter, of the chamber were to be decreased). Without wishing to be limited by its use, the chambers could be washed without the risk of contaminating or damaging a surface of the working electrode disposed within the chamber. Also, the filling ports could be arranged so that, for example, an 8-channel pipette or other equipment could be used to wash, fill, or otherwise operate the fluidic module. FIG. 20 and FIG. 21 provide filling of devices with different aqueous solution.

The fluidic module can include enclosed, partially enclosed, or opened reservoirs, chambers, structures, etc. For example and without limitation, with the use of the inlet/outlet ports, the chamber having a working electrode could be enclosed from the top or could be opened, such as for an open well. For an enclosed chamber, a fluid can be injected into the chamber, and one or more air purge ports or vent ports (e.g., which can be valves or holes) can be configured to allow air that is confined inside the chamber to drain and also configured to perfuse the fluid towards the chamber. The location of such ports can be in any useful manner so as to provide fluidic communication with the chamber (e.g., see ports labeled a-d in panel 3 of FIG. 19). Both configurations, e.g., closed-chamber and open-well, could be used as alternatives depending on the application and sample volume.

The devices and systems herein can be adapted to facilitate capture of extracellular vesicles (EVs) or components from EVs. For example and without limitation, a separate chamber can be present in a fluidics module to capture EVs, and the device can be configured to subsequently move the liquid from such a chamber into another chamber including a working electrode. Such movement can include the use of one or more valves (e.g., any described herein).

In some cases, an independent chamber (e.g., closed-chamber, open-well, etc.) can be part of the electrochemical microtiter plate. For example and without limitation, a lysing chamber could be located in proximity to each chamber including the working electrode. In this sense, a monolithic device (e g., a monolithic microtiter plate) can be provided. In some cases, an independent chamber (e.g., closed-chamber, open-well, etc.) can be employed, and lysed sample volumes can be transferred to the fluidics module (e.g., transfer by way of a pipette or other equipment, such as syringes, tubing, pumps, etc.). FIG. 22A- 22B provides non-limiting examples of chambers for lysing sample(s). As can be seen, the chambers can be provided as a lysing plate, in which can be provided as part of a kit w ith the electrochemical microtiter plate (e.g., including the fluidics module and the electrical module). Optionally, the lysing plate can be pretreated with one or more capture agents (e.g.. antibodies, or others described herein, such as in an ELISA plate). The dimensions of the chambers could vary depending on application. For example, if 16 different samples were to be analyzed, 16 individual chambers could be provided. Other numbers of samples and numbers of chambers can be employed. If the same sample is being tested for different biomarkers, larger EV lysis chamber(s) could be provided to retrieve more sample. Thus, the lysis plate can include any useful number of chambers, in which each chamber can be any useful size (e.g., same or differing sizes). A multichannel pipette or other equipment would still be used to transfer sample volumes to and from the lysis plate. The devices described herein can include any useful configuration of chambers, reservoirs, ports, inlets, outlets, working electrodes, counter electrodes, and reference electrodes to provide individually addressable electrodes and/or electrochemically isolated chambers to conduct an assay. In some cases, a device described herein can include any useful combination of features, such as one or more chambers, reservoirs, and electrodes. In some cases, a plurality of chambers (e.g., 112, 112’) may be provided, in which each chamber is associated with an individual working electrode (e.g., 152, 152’). Optionally, each chamber can include an individual chamber inlet (114, 114').

In some cases, the individual chamber inlets can be connected to a single inlet (e.g., in a manifold to deliver the same sample to each chamber), and each working electrode (152, 152’) can be functionalized with differing capture agents. Such differences can include two different capture agents (e.g., to capture to different target analytes), as well as tw o different densities of the same capture agent (e.g., to capture the same target analyte but with differing surface concentrations of the capture agent on the surface of the electrode). Other different capturing strategies can be implemented and are encompassed by the present document.

In some cases, a device described herein can be designed to have a plurality of individual chambers that can be connected to a single reservoir, or a single chamber that can be connected to a single reservoir, or a single chamber that can be connected to a plurality of reservoirs. Such connections can be implemented to provide fluidic and/or electric communication by providing a channel, capillary, or other structure having a conductive material (e.g., including a fluid, an ion, a solution having ions, such as a buffer, and the like).

In some cases, a device described herein can be configured to provide a fluidic module and an electrical module in any useful manner. In some cases, a device described herein can be a monolithic device, in which a fluidic module and an electrical module are configured to provide a single structure. In some cases, a device described herein can include a fluidic module and an electrical module, each of which can include one or more layers (e.g., a plurality of layers). In some cases, a device described herein can be configured to provide detachable modules, in which a fluidic module and an electrical module are configured to be separated.

In some cases, a device described herein can be used with other useful components to allow for detection of desired targets or analytes. Examples of such components can include, without limitation, an on-board camera for visualization or imaging; a potentiostat for electrochemical or optical detection of results of biological assays; a heater to provide temperature cycling, and the like.

Fluidic module

The fluidic module can be employed with any useful fluids, compounds, reagents, materials, and the like. In some cases, the fluidic module can include at least one channel configured to deliver a test sample to a region (e.g., channels, reservoirs, chambers, structures, etc.) within the fluidic module. In some cases, the fluidic module can include one or more regions or features (e.g.. channels, reservoirs, chambers, structures, etc.) configured to provide one or more reagents for performing an assay. In some cases, the fluidic module can include one or more regions or features (e.g., channels, reservoirs, chambers, structures, etc.) configured to prepare a sample (e.g., by way of separating, mixing, metering, and the like). In some cases, the fluidic module can include one or more regions or features (e.g., channels, reservoirs, chambers, structures, etc.) that can be enclosed, partially enclosed, or completely open (e.g., including one or more openings to allow access to the channels, reservoirs, chambers, structures, etc., such as an open well plate having accessible reservoirs or chambers). Flow channels within the fluidic module can have any useful dimension (e.g., height, width, cross-sectional dimension, etc.) from about 0.01 pm to about 1000 pm, from about 0.05 pm to about 500 pm, from about 0.2 pm to about 250 pm, from about 1 pm to about 100 pm, from about 2 pm to about 50 pm, or from about 5 pm to about 40 pm. The flow channel can have any useful width:depth aspect ratio, e.g., between about 1 :1 and 50:1.

Many flow channel cross-sections, valve cross-sections, and membrane thickness profiles can be employed, e.g., including rectangular, trapezoidal, circular, ellipsoidal, parabolic, hyperbolic, and polygonal, as well as sections of the above shapes.

In some cases, the fluidic module can be a monolithic device, in which the structures are integrated into a single structure. In some cases, the fluidic module and the electrical module, together, can form a monolithic device (e.g., having flow channels, valves, as well as any inlets, outlets, or ports to provide access to flow channels and valves; as well as having electrodes, bond pads, electrical connectors, and the like) within a single structure. Such monolithic devices can be manufactured in any useful manner, such as by using any useful lithography, rapid prototyping, printing (e.g., 3D printing), deposition (e.g.. electrodeposition), sputtering, etching, molding processes, and the like.

The fluidic module can be formed from any useful material. Examples of such materials include, without limitation, non-conductive materials, elastomeric materials, rigid materials (e.g., glass, silicon, polymers, and the like), inert materials, biocompatible materials, biocompatible surfaces, substrates, polymers, and the like, as well as combinations thereof. In some cases, the material can be formed from an elastomeric material (e.g, an elastomeric polymer, such as poly(dimethylsiloxane), polyisoprene, polybutadiene, polychloroprene, polyisobutylene, poly(styrene-butadiene-styrene). polyurethane, silicone, and the like). In some cases, the polymer can include a synthetic polymer, a transparent polymer, a rigid polymer, a thermoplastic polymer, and the like. Yet other non-limiting examples of polymers include poly(methyl methacrylate) (PMMA), polyethylene terephthalate (PET, e.g., biaxially -oriented PET or bo-PET), an acrylic polymer, poly(dimethylsiloxane) (PDMS), polycarbonate (PC), cyclo-olefin copolymer (COC), polyethylene terephthalate glycol (PETG), polyethylene (PE, such as branched homopolymer PE), polyvinylchloride (PVC), polystyrene (PS), styrene copolymer, polyimide (PI), polypropylene (PP), polytetrafluoroethylene (PTFE), polynorbomene (PN), poly(4-methyl-l- pentene), silicone, and combinations or co-polymers thereof. Layers, channels, chambers, reservoirs, and devices described herein can be manufactured in any useful manner, such as by using any useful lithography (e.g., photolithography, electron beam lithography, etc.), rapid prototyping, printing (e.g., 3D printing), deposition, sputtering, etching, molding, milling (e.g., ion-milling or CNC milling), processes, and the like.

The fluidic module can include an adhesive, which can be provided as a layer, a film, a substrate, an interlayer (e.g., disposed between two layers), or an interface (e.g., disposed on a surface of a layer). In some cases, the adhesive can include any useful material, such as a pressure sensitive adhesive (e.g., an acrylic, silicon, or acrylic-hybrid based adhesive optionally including a support layer), an acry lic adhesive, an acrylic-hybrid adhesive, a silicone adhesive, and/or an adhesion promoter (e.g., Dow Coming® 1200 primer, including light aliphatic petroleum solvent naphtha, xylene, tetrapropyl orthosilicate, tetrabutyl titanate, ethylene glycol methyl ether, tetra (2-methoxyethoxy) silane, and/or ethylbenzene). The adhesive may be provided as a planar layer. In some cases, the adhesive may include one or more channels, chambers, reservoirs, and the like. Other components can include ports in fluidic communication with flow channels in the fluidic module. Such ports can include, e.g., one or more test reagent ports, control reagent ports, vacuum ports, pressurization ports, inlet ports, outlet ports, and the like. Such ports can be used to introduce samples or reagents, as well as to control flow of fluids within the flow channels.

Electrical module

The electrical module can include one or more electrodes. Examples of electrodes that can be used as described herein include, without limitation, disc electrodes, planar electrodes, three-dimensional electrodes, porous electrodes, post electrodes, microelectrodes (e.g., having a longest dimension in the range of 1 pm to 1000 pm, such as a radium, width, or length from about 1 pm to 1000 pm), nanoelectrodes (e.g., having a longest dimension in the range of 1 nm to 100 nm, such as a radium, width, or length from about 1 nm to 100 nm), as well as arrays thereof.

Electrodes of a device described herein can be provided in any useful configuration, such as an array. In some cases, the electrodes of a device described herein can be individually addressable. In some cases, the electrodes of a device described herein can be provided in a two-electrode configuration (including reference and working electrodes) or a three-electrode configuration (including reference, working, and auxiliary electrodes).

The electrode of a device described herein can include any useful conductive material (e.g., gold, indium tin oxide, titanium, and/or carbon). Optionally, the working area is surface modified, e.g., with a linking agent and/or a capture agent described herein. These electrodes can include one or more other components that allow for detection, such as a ground electrode, a reference electrode, a counter electrode, a potentiostat, etc. The electrode can have any appropriate configuration, such as, e.g., a disk electrode, a spherical electrode, a plate electrode, a hemispherical electrode, a microelectrode, or a nanoelectrode; and can be formed from any appropriate material, such as gold, indium tin oxide, carbon, titanium, platinum, etc.

Any of the electrodes of a device described herein can be further functionalized with a conductive material, such as a conductive polymer, such as any described herein, including poly(bithiophene), polyaniline , or poly(pyrrole), such as dodecylbenzenesulfonate-doped polypyrrole; a metal, such as metal nanoparticles (e.g., gold, silver, platinum, and/or palladium nanoparticles), metal microparticles, a metal film (e g., palladium or platinum), etc.; a nanotube; etc. Other components

The device can include one or more components in addition to the fluidic module and the electrical module. For example and without limitation, a component can include an adhesive layer disposed between the fluidic module and the electrical module. For ease of use, the adhesive layer could optionally be integrated with the fluidic module, such that aligning and attaching the fluidic module to the electrical module could be simplified. In another embodiment, the adhesive layer could be integrated with the electrical module.

The device can include or be used with a component for lysing a sample. In some cases, the device can include a chamber configured to lyse a sample to provide one or more target analytes and to deliver the one or more target analytes to another chamber (e.g., a chamber configured to conduct an assay, such as a chamber including a working electrode).

In some cases, the device can include the use of a lysing plate configured to provide one or more target analytes for the device. The lysing plate may be integrated with the fluidic module. In some cases, the lysing plate may include wells that are then integrated into the fluidic module as chambers. In some cases, the lysing plate may be placed on a top surface of the fluidic module, such that a well of the lysing plate is in fluidic communication with a chamber of the fluidic module. In some cases, the lysing plate may be separate from the fluidic module, in which samples are lysed within wells of the lysing plate and then lysates are transferred to the fluidic module (e.g., transferred by way of pipettes, syringes, tubing, pumps, etc.).

A lysing plate can include any useful configuration. In some cases, the lysing plate can include a plurality of wells (e.g., two, three, four, five, six, seven, eight, or more wells). The wells may be arrayed in any useful manner. In some cases, a surface of the plurality of wells can include a capture agent configured to capture at least one of the one or more target analytes. Optionally, a linker can be disposed between the surface and the capture agent. Non-limiting examples of capture agents, linkers, and target analytes can include any described herein. FIG. 22A-22B provide non-limiting examples of lysing plates.

Linkers

Linkers can be present between two components (e.g.. an electrode and a capture agent; any surface and a capture agent; a first capture agent and a second capture agent; and the like). Linkers can include a bond (e.g., a covalent bond); an amino acid; a plurality of amino acids; a nucleotide; a plurality of nucleotides; an optionally substituted alkylene; an optionally substituted heteroalkylene (e.g., poly(ethylene glycol), such as -(OCFhCtLjn-. in which n is an integer of 1 to 100); an optionally substituted arylene; or an optionally substituted heteroarylene.

An alkylene can include a multivalent (e.g., bivalent, trivalent, tetravalent, etc.) form of an alkyl group. Exemplary alkylene groups include, without limitation methylene, ethylene, propylene, butylene, etc. In some cases, the alkylene group can be a C1-3, Ci-6, C1-12, C1-16. C1-18, Ci-20, Ci-24, C2-3, C2-6, C2-12, C2-16, C2-18, C2-20, or C2-24 alkylene group. The alkylene group can be branched or unbranched. The alkylene group can also be substituted or unsubstituted. For example, the alkylene group can be substituted with one or more substitution groups (e.g., halo, oxy, oxo, amino, and the like). A heteroalkylene can be an alky lene group containing one, two, three, or four non-carbon heteroatoms (e.g., independently selected from the group consisting of nitrogen, oxygen, phosphorous, sulfur, or halo).

In some cases, a linker of a device described herein can include one or more chemical signatures. In some cases, the chemical signature includes a click-chemistry signature, which arises from reacting a click-chemistry reaction pair (e.g., any described herein). Examples of click-chemistry signatures that can be used as described herein include, without limitation, a triazole, an unsaturated six-member ring, a covalent bond, and the like.

In some cases, the chemical signature of a linker of a device described herein can include a reaction signature, which arises from reacting a cross-linker reaction pair. Examples of cross-linker reaction pairs that can be used include, without limitation, those for forming a covalent bond between a carboxyl group (e.g., -CO2H) and an amino group (e.g., - NH2); or between an imido group (e.g., maleimido or succinimido) and a thiol group (e.g., - SH); or between an epoxide group and a thiol group (e.g., -SH); or between an epoxide group and an amino group (e.g., -NH2); or between an ester group (e.g.. -CO2R, in which R is an organic moiety, such as optionally substituted alkyl, aryl, etc.) and an amino group (e.g.. - NH2); or between an carbamide group (e g., -NHC(O)Het, where Het is a N-containing heterocyclyl) and an amino group (e.g., -NH2); or between a phospho group (e.g., - P(O)(OH)2) and an amino group (e.g., -NH2), such as l-ethyl-3 -(3 -dimethylaminopropyl) carbodiimide (EDC) and dicyclohexylcarbodiimide (DCC), optionally used with N- hydroxysuccinimide (NHS) and/or N-hydroxysulfosuccinimide (sulfo-NHS). Other examples of cross-linkers that can be used as described herein include, without limitation, those for forming a covalent bond between an amino group (e.g., -NH2) and a thymine moiety, such as succinimidyl-[4-(psoralen-8-yloxy)]-butyrate (SPB); a hydroxyl group (e.g., -OH) and a sulfur-containing group (e.g., free thiol. -SH. sulfhydryl, cysteine moiety, or mercapto group), such as p-maleimidophenyl isocyanate (PMPI); between an amino group (e.g., -NH2) and a sulfur-containing group (e.g.. free thiol, -SH, sulfhydryl, cysteine moiety, or mercapto group), such as succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB) and/or succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate (SMCC); between a sulfur- containing group (e.g., free thiol, -SH, sulfhydryl, cysteine moiety, or mercapto group) and a carbonyl group (e.g., an aldehyde group, such as for an oxidized glycoprotein carbohydrate), such as N-beta-maleimidopropionic acid hydrazide-trifluoroacetic acid salt (BMPH). 3-(2- pyridyldithio) propionyl hydrazide (PDPH), and/or a 3-(2-pyridyldithio)propionyl group (PDP); and between a maleimide-containing group and a sulfur-containing group (e.g., free thiol, -SH, sulfhydryl, cysteine moiety, or mercapto group). Yet other examples of crosslinkers that can be used as described herein include, without limitation, those for forming a covalent bond between two or more unsaturated hydrocarbon bonds, e.g., mediated by radical polymerization, such as a reaction of forming a covalent bond between a first alkene group and a second alkene group (e.g., a reaction between acrylate-derived monomers to form a polyacrylate, polyacrylamide, etc.).

A linker used in a device described herein can include one or more reaction pairs. In some cases, the reaction pair can be one of a click- chemistry reaction pair, which can include a first click-chemistry group and a second click-chemistry group that reacts with that first click-chemistry group. Examples of click-chemistry groups that can be used include, without limitation, a click-chemistry group, one of a click-chemistry reaction pair selected from the group consisting of a Huisgen 1,3-dipolar cycloaddition reaction between an alkynyl group and an azido group to form a triazole-containing linker; a Diels-Alder reaction between a diene having a 4n electron system (e.g., an optionally substituted 1,3 -unsaturated compound, such as optionally substituted 1,3 -butadiene, l-methoxy-3-trimethylsilyloxy-l,3- butadiene, cyclopentadiene, cyclohexadiene, or furan) and a dienophile or heterodienophile having a 2TT electron system (e g., an optionally substituted alkenyl group or an optionally substituted alkynyl group); a ring opening reaction with a nucleophile and a strained heterocyclyl electrophile; and a splint ligation reaction with a phosphorothioate group and an iodo group; and a reductive amination reaction with an aldehyde group and an amino group.

A linker used in a device described herein can include one or more reactive groups. Examples of reactive groups that can be used as described herein include, without limitation, an amino (e.g., -NH2), a thio (e.g., a thioalkoxy group or a thiol group), a hydroxyl, an ester (e.g., an acrylate), a carboxyl (e.g., -CO2H or a deprotonated form thereof), an imido (e.g.. a maleimido or a succinimido), an epoxide, an isocyanate, an isothiocyanate, an anhydride, an amido, a carbamido (e.g., a urea derivative), an azide, an optionally substituted alkynyl, or an optionally substituted alkenyl.

In some cases, a linker used in a device described herein can include a binding reaction signature, which arises from reacting a binding reaction pair. Examples of binding groups and binding reaction pairs that can be used as described herein include, without limitation, those for forming a covalent bond between biotin and avidin, biotin and streptavidin, biotin and neutravidin, desthiobiotin and avidin (or a derivative thereof, such as streptavidin or neutravidin), hapten and an antibody, an antigen and an antibody, a primary antibody and a secondary antibody, and lectin and a glycoprotein.

Capture agents

A device described herein can be configured to provide one or more capture agents. In use, the capture agent can include a group that binds to or otherwise modifies a target. Examples of capture agents that can be used as described herein include, without limitation, one or more of a nanoparticle, a microparticle, a dye, a detectable agent, a protein, an antibody, a nucleic acid, an aptamer, a small molecule, or a combination thereof.

A capture agent that can be used in a device as described herein can directly or indirectly bind the target analyte. In some cases, a capture agent can include one or more labels that can be used to directly or indirectly detect a target analyte. For direct detection, a label can be conjugated to a capture agent that binds to the target. For instance, a capture agent can be an antibody that binds the target, and the label for direct detection can be a nanoparticle attached to the capture agent. For indirect detection, a label can be conjugated to a second capture agent that further binds to a first capture agent. Any appropriate methods can be used to arrange combinations of labels, capture agents, and linking agents to detect a target of interest.

In some cases, a capture agent of a device described herein can be a protein, such as an antibody or a fragment thereof. In some cases, a capture agent of a device described herein can be a protein, enzyme, saccharide, DNA, RNA, peptide, or whole cell. In some cases, a capture agent of a device described herein can include, for example, an amine, carboxyl, boronic acid, maleimide, thiol, biotin, or avidin group.

In some cases, multiple capture agents can be configured into a device described herein and used to bind the target and provide a detectable signal for such binding. For example, multiple capture agents can be configured into a device described herein and used for a sandwich assay, which can involve at least two capture agents and can optionally include a further capture agent that includes a label allowing for detection.

Examples of capture agents that can be used as described herein include, without limitation, one or more of the following: a protein that binds to or detects one or more target analytes (e.g., an antibody or an enzyme), a globulin protein (e.g., bovine serum albumin), a peptide, a nucleotide, a nanoparticle, a microparticle, a sandwich assay reagent, a catalyst (e.g.. that reacts with one or more targets), and/or an enzyme (e.g., that reacts with one or more targets, such as any described herein). The capture agent can optionally include one or more labels, e.g., a label described herein. In some cases, a device described herein can be designed to include more than one capture agent, optionally with one or more linking agents, can be used to detect a target analyte of interest. In some cases, a capture agent of a device described herein can be used in combination with a label (e.g., any described herein) to detect a maker. Examples of labels that can be used as described herein include, without limitation, one or more fluorescent labels, colorimetric labels, quantum dots, nanoparticles, microparticles, barcodes, radio labels (e.g., RF labels or barcodes), avidin, biotin, tags, dyes, an enzyme that can optionally include one or more linking agents and/or one or more dyes, as well as combinations thereof etc.

Systems

One or more devices described herein can be used in combination with a platform to form a system. The platform can be configured to electrically connect the device to a controller configured to control the electrical module. The controller can include a component to receive, transmit, combine, filter, or otherwise manipulate one or more electrical signals, such as, e.g., with a multiplexer, a potentiostat, a printed circuit board for making one or more electrical contacts, and the like.

Optionally, the system can further include a user interface configured to receive and transmit information between a device (e.g.. a device described herein) and the controller. The user interface can include a component to receive, transmit, store, or otherwise employ instructions, data, and the like, between the user interface and the device. In some cases, the component can receive, transmit, store, or otherwise employ instructions, data, and the like, between the user interface and the controller.

Systems described herein can include one or more other components. Examples of other components that can be used as described herein include, without limitation, one or more detectors (e.g., optical detectors, microscopes, photodetectors, etc.); imagers (e.g., configured for image acquisition) or optical readers; heaters; light emitting diodes; optical circuit elements, such as a filter, an objective, a lens, a mirror, a dichroic mirrors, fiber optics components, or a grating; a device manifold configured to interface a microfluidic device with one or more connections (e.g., tubing, fluidic connectors, ferrules, and the like) to pressure manifold(s); transducers; electronic signal filters; active/passive circuit elements (e.g., such as transistors, diodes, and resistors); amplifiers; feedback circuits; and the like.

Methods and uses thereof

One or more devices described herein can be used to ear ’ out any appropriate method. In some cases, a device described herein can be used to carry out an assay (e.g.. an immunoassay and/or an electrochemical assay).

In some cases, a method described herein can include: delivering a sample to a chamber of a device or system described herein; and providing one or more reagents for an assay (e.g.. an electrochemical assay) to a reservoir of a device or system described herein.

The sample may be prepared in any useful manner. In some cases, the sample may include extracellular vesicles or lysates from extracellular vesicles. To prepare the sample, the method can further include: lysing one or more extracellular vesicles to release said one or more target analytes; and providing the one or more target analytes to the chamber of a device or system described herein. Lysing can include any useful process, such as the use of a lysing plate (e.g., any described herein).

To initiate the assay, the method can further include: actuating a releasable valve, thereby providing fluidic and/or electric communication between the chamber and the reservoir. In some cases, providing reagents within the reservoir can actuate the releasable valve. For example, a releasable valve can be disposed between a chamber and a reservoir. The presence of the reagents in the reservoir can provide a change in pressure within a channel or a chamber in fluidic communication with releasable valve, thereby inducing flow between the reservoir and the chamber. In these cases, providing a sufficient amount of reagent within the reservoir can actuate the releasable valve.

Any appropriate reagent can be used. Examples of reagents that can be used as described herein include, without limitation, nanoparticles, metals, dyes, fluorophores, detectable reporters, electroactive agents, or combinations thereof. Other reagents that can be used include buffers, salts, and combinations thereof.

Upon completing one or more reactions to conduct the assay, the method can include: detecting one or more electrical signals from a working electrode. In use, the electrical signal can be determined between the working electrode and a reference electrode. Optionally, a counter electrode can be used. The presence or absence of the electrical signal can indicate the presence or absence of a target analyte in the sample.

The methods described herein can be used to detect one or more extracellular vesicles. In some cases, a method described herein can include detecting an analyte of an extracellular vesicle. In some cases, a method described herein can include: capturing one or more extracellular vesicles in a device (e.g.. on a working electrode or a capture agent bound to the working electrode); and detecting one or more electrical signals from the device. The presence or absence of the electrical signal can indicate the presence or absence of a target analyte on a surface of an extracellular vesicle or a target analyte from an extracellular vesicle.

The methods described herein can be used to detect an analyte of an extracellular vesicle (e.g., any analyte associated with, derived from, or obtained from an extracellular vesicle). In some cases, a method described herein can include: capturing one or more target analytes from extracellular vesicles in a device (e.g., on a working electrode or a capture agent bound to the working electrode); and detecting one or more electrical signals from the device. The presence or absence of the electrical signal can indicate the presence or absence of a target analyte from an extracellular vesicle.

In use, a method described herein can include the use of any appropriate reagent (e.g., a reagent described herein). In some cases, a method described herein can include (e.g., after or before capturing the extracellular vesicle): providing one or more reagents for an electrochemical assay to the working electrode in the chamber. In some cases, a method described herein can include (e.g., after or before capturing the extracellular vesicle): providing one or more reagents for an electrochemical assay to the reservoir. In some cases, a method described herein can include (e.g.. after or before capturing the extracellular vesicle): actuating the releasable valve, thereby providing electric communication or fluidic communication between the chamber and the reservoir.

Any appropriate sample can be assessed (e.g., for the presence, absence, or amount of one or more analytes) using the methods and materials (e.g., devices and systems) described herein. In some cases, a sample can include one or more extracellular vesicles, lysates from one or more extracellular vesicles, particles, and/or micelles. In some cases, the target analyte can include a protein disposed on a surface of an extracellular vesicle. In some cases, a sample can include intravesicular content (e.g., of an extracellular vesicle) including proteins, peptides, and/or nucleic acids. In some cases, a sample can be a biological sample. For example, a sample can contain whole cells, cellular fragments, DNA, RNA, carbohydrates, lipids, viruses, microorganisms, and/or proteins. Examples of samples that can be used in the methods, devices, and systems described herein include, without limitation, whole blood samples, serum samples, plasma samples, urine samples, saliva samples, mucus samples, sputum samples, bronchial lavage samples, fecal samples, buccal samples, nasal samples, amniotic fluid samples, cerebrospinal fluid samples, synovial fluid samples, pleural fluid samples, pericardial fluid samples, peritoneal fluid samples, urethral samples, cervical samples, genital sore samples, hair samples, and skin samples.

In some cases, a sample to be assessed (e.g., for the presence, absence, or amount of one or more analytes) using the methods and materials (e.g., devices and systems) described herein can be an environmental sample, a water sample, an agricultural sample, a soil sample, a food sample, a meat sample, a produce sample, a drink sample, a plant sample, a leaf sample, a root sample, a flower sample, a stem sample, a pollen sample, a seed sample, or an industrial sample (e.g., an air filter sample, sample collected from a work station, or a sample collected from a storage facility).

In some cases, the devices and systems described herein can retain the sample within the device for safe and clean disposal.

A sample to be assessed (e.g., for the presence, absence, or amount of one or more analytes) using the methods and materials (e.g., devices and systems) described herein can be obtained using any appropriate technique. For example, biological samples can be obtained using non-invasive (e.g., swab) techniques or invasive techniques (e.g., venipuncture, finger stick, or biopsy). In some cases, a whole blood sample can be obtained from a human using a glass capillary⁷ tube. For example, an environmental sample, an agricultural sample, and/or an industrial sample can be obtained using a surface swab technique. In some cases, a sample can be a liquid sample.

A liquid sample can be any appropriate volume. As described herein, very small volumes of a sample can be collected and accurately analyzed for the presence, absence, or amount of two or more analytes using the methods and materials described herein. For example, a liquid sample (e.g.. a whole blood sample) with a volume of about 1 pL to about 10 pL (e.g., from 1 pL to 10 pL, from 2 pL to 10 pL, from 3 pL to 10 pL, from 4 pL to 10 pL, from 1 pL to 9 pL, from 1 pL to 8 pL, from 1 pL to 7 pL, from 1 pL to 6 pL, from 2 pL to 8 pL, from 3 pL to 7 pL, or from 4 pL to 6 pL) can be obtained and analyzed for the presence, absence, or amount of two or more analytes using the methods and materials described herein. In some cases, a larger volume can be obtained from the source, and a small portion (e.g., a volume from 1 pL to 10 pL, from 2 pL to 10 pL, from 3 pL to 10 pL, from 4 pL to 10 pL, from 1 pL to 9 pL, from 1 pL to 8 pL, from 1 pL to 7 pL, from 1 pL to 6 pL. from 2 pL to 8 pL. from 3 pL to 7 pL. or from 4 pL to 6 pL) of that larger obtained volume can be used in the methods and materials (e g., device or system) described herein.

A sample to be assessed (e.g., for the presence, absence, or amount of one or more analytes) using the methods or materials (e.g., devices or systems) described herein can be obtained from any appropriate animal. In some cases, a sample to be assessed as described herein can be obtained from a mammal (e.g., a human such as a human neonate, human baby, human toddler, human child, or human adult). Examples of mammals that samples can be obtained from include, without limitation, primates (e.g., humans and monkeys), dogs, cats, horses, cows, pigs, sheep, rabbits, and rodents (e.g., mice and rats). Other examples of animals that samples can be obtained from include, without limitation, fish, avian species (e.g., chickens, turkeys, ostrich, emus, cranes, and falcons) and non-mammalian animals (e.g., mollusks, frogs, lizards, snakes, and insects).

In some cases, a sample to be inserted into a device or system described herein can be obtained from a source (e.g.. a human) and directly inserted into the device or system without being pre-processed. For example, a whole blood sample can be obtained from a mammal (e.g., a human) and directly inserted into a device or system provided herein without being pre-processed (e.g., without being treated or manipulated in any way).

In some cases, a sample to be inserted into a device or system described herein can be obtained from a source (e.g., a mammal or surface) and processed prior to being inserted into a device or system (e.g., can be pre-processed) described herein. Samples that are pre- processed can be pre-processed using one or more appropriate reagents (e.g., enzymes, acids, bases, buffers, detergents, anticoagulants, and/or aptamers) and/or techniques (e.g.. purification techniques, centrifugation techniques, amplification techniques, culturing techniques, and/or denaturing techniques). For example, a blood sample can be obtained from a mammal (e.g., a human) and treated with one or more anticoagulants. Examples of anticoagulants that can be used to pre-process a sample (e.g., a blood sample) include, without limitation, EDTA. citrate (trisodium citrate), heparinates (e.g., sodium, lithium, or ammonium salt of heparin or calcium-titrated heparin), and hirudin. In some cases, a sample (e.g., a sample suspected to contain a microorganism) to be inserted into a device or system described herein can be obtained from a source (e g., a food preparation surface) and pre- processed by culturing the sample with appropriate culture media for a period of time (e.g., 4 hours to 24 hours) prior to being inserted into the device or system. Examples of other pre- processing techniques that can be performed prior to inserting the sample into a device or system described herein include, without limitation, centrifugation to obtain cell-containing material, centrifugation to obtain cell-free material, filtration to remove cell containing material, cell lysis, nucleic acid purification, protein purification, nucleic acid amplification (e.g., polymerase chain reaction (PCR)), reverse transcription to obtain cDNA, reverse transcription PCR, nucleic acid denaturation, and isothermal amplification.

The systems, devices, and methods described herein can be used to process, analyze, or otherw ise manipulate a test sample. Examples of processes that can be used as described herein include, without limitation, preparing a sample (e.g., separating one or more components of a test sample), reacting a sample (e.g., performing one or more chemical, biological, or biochemical reactions, including binding reactions, enzymatic reactions, and the like), assaying a sample (e.g., performing one or more assays, such as any described herein), and the like. Multiple assays using one or more devices and/or systems described herein can be performed in parallel and/or serially.

Assays that can be performed using one or more devices and/or systems described herein include, without limitation, blood assays, panels. POC assays, and the like. Examples of assays that can be performed using one or more devices and/or systems described herein include, without limitation, multiple chemical and/or biochemical assay formats and/or platforms such as well-, micro well-, microfluidic-, gel-, magnetic particle-, solid chromatographic-based assay formats, for detecting and quantifying analytes of interest in a sample. Assay types may include, without limitation, sandwich, hybridization, competition, and other assays.

The systems, devices, and methods described herein can be used to detect the presence, absence, or amount of one or more analytes present within a small volume (e.g., less than 10 pL) of a sample (e.g., a blood sample) obtained from a mammal (e.g., a human). For example, this document provides methods and materials for using plasma separation and multiplex analyte detection to detect two or more analytes (e.g., proteins, carbohydrates, lipids, nucleic acids, intact cells, intact viruses, intact microorganisms, and/or chemicals) within a small volume of a blood sample.

Any appropriate type of analytes can be detected using the methods and materials described herein. For example, a device or system described herein can be configured to detect the presence, absence, or amount of a protein, carbohydrate, extracellular vesicle, lipid, nucleic acid, intact cell, intact virus, intact microorganism, and/or chemical. Examples of proteins that can be detected using the methods and materials described herein include. without limitation, enzymes such as lactate dehydrogenase (LDH), alanine transaminase (ALT), aspartate transaminase (AST), creatine phosphokinase (CPK), and metalloproteases (e.g.. ADAM12), receptors such as soluble chemokine receptors (e.g., CCRS and CXCR4), soluble growth factor receptors (e.g., EGFR), and soluble transferrin receptor, serum proteins such as albumin, transferrin, alpha- 1 anti-trypsin, and immunoglobulins, inflammatory' cytokines such as tumor necrosis factor alpha (TNF-a), interleukin 2 (IL-2), and interferon gamma (IFN-y), viral proteins such as HIV envelope protein gpl20 and E protein of SARS- CoV-2, bacterial proteins such as Mycobacterium tuberculosis surface protein Rv0227c and the MSCRAMM family of S. aureus, and fungal proteins such as Sspl and Sell. Examples of carbohydrates that can be detected using the methods and materials described herein include, without limitation, glucose, lactate, pyruvate, prostate-specific antigen (PSA), CA 125, and CA 19-9. Examples of lipids that can be detected using the methods and materials described herein include, without limitation, total cholesterol, triglycerides, high density lipoprotein (HDL), and low density lipoprotein (LDL). Examples of intact viruses that can be detected using the methods and materials described herein include, without limitation, human immunodeficiency viruses (e.g., HIV1 and HIV2), coronaviruses (e.g., COVID-19 virus), Zika viruses, influenza viruses A and B, adenoviruses, RSV viruses, parainfluenza viruses, human metapneumoviruses, rhinoviruses, enteroviruses, hepatitis A, B, C and E viruses, rotaviruses, human papillomaviruses, measles viruses, caliciviruses, astroviruses, West Nile viruses, Ebola viruses, Dengue fever viruses, African swine fever viruses, herpes simplex viruses (e.g., HSV-2), Norwalk and Norwalk-like viruses, enteric adenoviruses, yellow fever viruses, chikungunya viruses, Epstein-Barr viruses, parvoviruses, varicella zoster viruses, and Ross River viruses. Examples of intact microorganisms that can be detected using the methods and materials described herein include, without limitation, bacterial microorganisms such as Staphylococcus aureus (e.g.. MRSA and MSSA). Streptococcus pyogenes.

Streptococcus pneumoniae. Mycoplasma pneumoniae, Haemophilus influenzae. Chlamydia pneumoniae, Bordetella pertussis, Mycobacterium tuberculosis, E. coli (e.g., enterohaemorrhagic E. coli such as O157:H7 E. coli or enteropathogenic E. coli), Salmonella species (e.g.. Salmonella enterica), Listeria monocytogenes, Acinetobact er baumanni, Klebsiella oxytoca, Sarcoptes scabiei, Neisseria gonorrhoeae. Chlamydia trachomatis. Treponema pallidum, Campylobacter species (e.g., thermophilic strains of Campylobacter jejuni, C. lari, or C. coli), Bacillus cereus, Vibrio species, Yersinia enterocolitica, Shigella species, Enterococcus species (e.g., Enterococcus faecalis or E. faecium), Helicobacter pylori, and Clostridium species (e.g., Clostridium botulinum or Clostridium perfringens), fungal microorganisms such as Aspergillus species (e.g., A. flavus , A. fiimigatus , and A. niger). yeast (e.g., Candida norvegensis and C. albicans'). Penicillium species, Rhizopus species, and Alternaria species, and protozoan microorganisms such as Cryptosporidium parvum, Giardia lamblia, and Toxoplasma gondii. Examples of chemicals that can be detected using the methods and materials described herein include, without limitation, glucose, bilirubin, parathyroid hormone, bile acid, and urea.

Examples of other target analytes that can be detected using the methods and materials described herein include, without limitation, one or more markers indicative of a disease state or a health status (e.g., such as podocin and nephrin for preeclampsia, glycated hemoglobin for diabetes, or markers for stress or fatigue), a cardiovascular marker (e.g., CRP. D-dimer. troponin I or T), a blood marker (e.g., hematocrit, or hemoglobin), a cell (e.g., a leukocyte, neutrophil, B-cell, T-cell, lymphocyte, or erythrocyte), a viral marker (e.g., a marker for human immunodeficiency virus, hepatitis, influenza, or chlamydia), physiologically relevant markers (e.g., such as glucose, lactate, pH, and the like), a protein (e.g., myoglobin, troponin, insulin, or C-reactive protein), an enzyme (e.g., creatine kinase), a catecholamine (e.g., dopamine, epinephrine, or norepinephrine), a cytokine (e.g.. TNF-a or interleukins, such as IL-6, IL-12, or IL-1), an antibody (e.g., immunoglobulins, such as IgA), a biomolecule (e.g., cholesterol or glucose), a neurotransmitter (e.g., acetylcholine, glutamate, dopamine, epinephrine, neuropeptide Y, or norepinephrine), a signaling molecule (e.g., nitric oxide), an antigen (e.g., CD3, CD4, or CD8), an ion (e.g., a cation, such as K⁺, Na⁺, H~, or Ca²⁺, or an anion, such as CL or HCOs ), CO2, O2, H2O2, a cancer biomarker (e.g., human ferritin, carcinoembryonic antigen (CEA), prostate serum antigen, human chorionic gonadotropin (hCG), diphtheria antigen, or C-reactive protein (CRP)), a hormone (e.g., hCG, epinephrine, testosterone, human growth hormone, epinephrine (adrenaline), thyroid hormone (e.g., thyroid-stimulating hormone (TSH). thyroxine (TT4). triiodothyronine (TT3), free thyroxine (FT4), and free triiodothyronine (FT3)), adrenal hormone (e.g., adrenocorticotrophic hormone (ACTH), cortical hormone (F), and 24-hour urine- free cortisol (UFC)), a gonadal hormone (e.g., luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, estradiol (E2), and prolactin (PRL)), cortisol, leptin, or a peptide hormone, such as insulin), an inflammatory marker (e.g., CRP), a metabolite (e.g., glucose, cholesterol, triglyceride, creatinine, lactate, ammonia, ascorbic acid, peroxide, potassium, glutamine, or urea), a nucleic acid (e.g., DNA and/or RNA for detecting one or more alleles, pathogens, single nucleotide polymorphisms, mutations, etc.), an amino acid (e.g., glutamine), a drug (e.g., a diuretic, a steroid, a growth hormone, a stimulant, a narcotic, an opiate, etc.), etc. Other exemplary markers include one or more pathogens, such as Mycobacterium tuberculosis, Diphtheria antigen, Vibrio cholera, Streptococcus (e.g., group A), etc.

In some cases, the methods and materials described herein can be used in small animal research, neonatal blood analysis, analysis of blood for one or more preeclampsia biomarkers, analysis of blood for one or more cardiac biomarkers, point-of-care testing of infectious diseases (e.g., COVID-19, sexually transmitted diseases, or HIV), and/or point-of- care testing in an operating room to provide rapid turnaround results.

The invention wi 11 be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1: Using electrochemical immunoassay in a microtiter plate to detect surface markers of preeclampsia on urinary extracellular vesicles

Extracellular vesicles (EVs) are nanoscale particles (e.g., about 50 to 1000 nm in diameter) bound by a phospholipid membrane and secreted by cells. EVs are found in various bodily fluids such as serum, urine, and saliva, and are thought to mediate intercellular communication. ³ For example, EVs can contain biological information (e.g., protein and RNA cargo) reflective of parental cells and tissue of origin, making them a source of biomarkers for early disease diagnosis.⁴’⁶

Preeclampsia (PE) is a pregnancy-specific disorder characterized clinically by hypertension and the appearance of proteins such as albumin in patient urine. This disorder occurs in ~5% of pregnancies worldwide and is the second leading cause of matemal/fetal morbidity and mortality worldwide.⁷ PE can be associated with renal injury and disruption of renal barrier function. One manifestation of renal injury includes the appearance in urine of kidney cells called podocytes as well as podocyte-derived EVs.^8,9 Further analysis revealed that podocyte EVs carried podocin and nephrin, proteins expressed on podocytes and responsible for filtration function of the kidney glomeruli.⁵ While podocin levels remained constant, nephrin levels decreased in patients with PE causing the podocin-to-nephrin ratio to rise. Monitoring the ratio of two biomarkers is beneficial in that it reduces assay variability, which may be affected by many confounders inherent in urine analytes such as changes in pH. osmolality, and contamination. EV analysis can be carried out using digital flow cytometry - a complicated analytical technique.⁵ As described herein, an analytical/biosensing approach that would be more amenable to screening/testing in a clinical setting was implemented.

Electrochemical biosensors may be operated with inexpensive instrumentation.^10,11 Electrochemical detection of EVs^{6, 12-20} may benefit from a simple-to-use and sensitive means for detecting EV surface antigens. A nanoparticle (NP)-enabled electrochemical immunoassay can be employed to detect EV surface proteins. AuNPs were labeled with antibodies (Abs) to confer specificity and impregnated with metal ions to produce electrochemical signals. Such Ab-functionalized and metal ion-doped AuNPs (also referred to as non-limiting examples of immunoprobes) can be characterized by one or more of the following: 1) the presence of multivalent interactions to enhance affinity for the target, 2) the use of a large number of metal ions (~10⁴ ions per nanoparticle), which may be loaded produce electrochemical signals, and 3) different metal ions (e.g. Zn²⁺, Cd²⁺, Pb²⁺, and Cu²⁺) may be loaded into subsets of AuNPs to produce distinct redox peaks at different potentials and enable multiplexed detection of target analytes from the same electrode or well. ^13,21,22 Such NP-enabled electrochemical immunoassays can exhibit low limits of detection (i.e., 10⁵ particles/mL for EVs or low pg/mL for protein) and high specificity’ to the target of interest. ¹⁵ Accordingly, described herein are devices and methods that incorporate NP-enabled immunoassays into a microtiter plate format to enable clinical sample testing.

Electrochemical microtiter plates for high-throughput electrochemical analyses of biological analytes, including EVs, may be useful. ^18,23-27 In some instances, a microtiter plate format can be used to carry out electrochemical analysis of EV surface markers.^18,20 This strategy can include magnetic separation of EVs in one microtiter, followed by’ transferring EVs into a custom-made 96 well electrochemical microtiter plate. Each well of this plate can contain a 3 -electrode cell and be connected to its own potentiostat. EVs can be labeled with Abs carrying horseradish peroxidase (HRP). and expression of surface markers can be quantified based on enzymatic breakdown of electrochemically active substrates.

Described herein are devices for capturing EVs on an electrode surface, followed by labeling with redox active immunoprobes. Without wishing to be limited by any mechanism action, such an approach can allow for a more streamlined workflow (both EV capture and surface marker detection happen in the same device) and simplicity of hardware, where multiple electrochemical cells can share working/ counter electrodes and may’ be operated using a single potentiostat. Of course, if desired, multiple potentiostats can be used.

To facilitate immunoassays using an electrode surface to capture EVs, a method for functionalizing working electrodes with Abs and capturing EVs without affecting neighboring counter/reference electrodes was developed. In other words, such a method can be used to establish an electrical (e.g., or electrochemical) connection between electrodes. For example, a connection with counter/reference electrodes can be established after functionalization of the working electrode and capture of EVs from a biological sample.

In one non-limiting implementation of this method, a novel 16-electrode microtiter plate was fabricated with an on-board counter/reference electrode. Each well contained a working electrode and capillary valves to confine liquid during electrode functionalization and sample incubation steps (see, e.g., FIG. 3). After preparing the plate, capillary valves could be opened by exceeding their burst pressure (e.g., 0.72 psi) to establish an electrochemical connection between the working electrode and counter/reference electrodes. Once fabricated and characterized, this electrochemical microtiter plate was used to analyze urinary EVs from normotensive and preeclamptic pregnant women.

As described herein, the levels of podocin and nephrin expression on urinary EVs were used to diagnose renal injury' associated with preeclampsia. In some cases, a nanoparticle-enabled immunoassay can be integrated with an electrochemical plate for quantifying podocin and nephrin expression in urinary EVs. The strategy included capturing EVs on an electrode surface and then labeling EVs with gold nanoparticles that are both functionalized with antibodies for target specificity and impregnated with redox active metal ions for electrochemical detection. These immunoprobes produced an electrochemical redox signal proportional to the expression level of EV surface markers. Electrochemical immunoassays were carried out in a microtiter plate that contained 16 wells with working electrodes connected to on-board counter/reference electrodes via capillary valves. Upon validation with recombinant proteins, this microtiter plate was used for analysis of urinary EVs from healthy and preeclamptic pregnant women. This analysis revealed a higher podocin-to-nephrin ratio for preeclamptic women compared to healthy controls (4.31 vs. 1 .69) suggesting that this ratio may be used for disease diagnosis.

Example 2: Non-limiting materials and methods

Materials

4-Morpholineethanesulfonic acid (MES), 4-(2-hydroxyethyl)piperazine-l- ethanesulfonic acid (HEPES), NHS-activated AuNPs (20 run), Pb(NOa)2. 11- mercaptoundecanoic acid (MUA), l-ethyl-3-(3 dimethylaminopropyl carbodiimide (EDC), N-hydroxy-succinimide (NHS), and Tween-20 purchased from Sigma-Aldrich (St. Louis, MO). Total urine EV isolation kit was purchased from Invitrogen (Carlsbad, CA). Single donor human pregnancy urine was purchased from Innovative Research (Novi, MI). Dulbecco's Phosphate-buffered Saline (DPBS) was purchased from Coming (Coming, NY). Ethyl alcohol (EtOH) was purchased from Electron Microscopy Sciences (Hatfield, PA), while isopropyl alcohol (IP A) was purchased from Honeywell (Charlotte, NC). Mouse antihuman CD63 and mouse IgG isotype control were purchased from BD Biosciences (San Jose, CA). Rabbit anti-human podocin Ab was purchased from Bioss Antibodies (Woburn, MA). Rabbit anti-human nephrin Ab was purchased from biorbyt (St. Louis, MO). Rabbit IgG isotype control was purchased from Thermo Fisher Scientific (Waltham, MA). Rabbit antihuman CYP2E1 Ab was purchased from CYP450-GP (Vista, CA). Human recombinant nephrin protein was purchased from R&D Systems (Minneapolis, MN). Human recombinant podocin protein was purchased from BioVendor (Brno, Czech Republic).

Gold Etch type TFA was purchased from Transene Electronic Chemicals (Danvers, MA). CR-7s Chrome Etch was purchased from KMG Electronic Chemicals (Pueblo, CO). Ag/AgCl ink was purchased from CH Instruments (Bee Cave, TX). Silicon wafers were purchased from University Wafer (South Boston, MA, USA). Polydimethylsiloxane (PDMS) base and curing agent kit (Sylgard 184) was purchased from Ellsworth Adhesives (Minneapolis, MN, USA). SU-8 2050 photoresist and SU-8 developer were purchased from Kayaku Advanced Materials (Westborough, MA, USA). AZ 5214-E IR photoresist and AZ 300MIF developer were purchased from Integrated Micro Materials (Argyle, TX, USA).

Protocols for constructing AuNPs/ Abs@Pb²⁺¹⁵ were used to synthesize immunoprobes targeting several EV surface antigens including AuNPs/anti-podocin@Pb²⁺, AuNPs/anti-nephrin@Pb²⁺, AuNPs/anti-CD63@Pb²⁺. and AuNPs/anti-CYP2El@Pb²⁺. AuNPs/IgG@Pb²⁺ was also prepared and served as a negative (isotype) control.

Briefly, NHS-activated AuNPs (6.54xlOⁿ NPs/mL) were dispersed in 90 pL of 1 x PBS buffer containing 20 pg of antibody (Ab) solution and incubated for 2 hours at room temperature. Thereafter. 10 pL of quencher solution was added to eliminate the remaining unreacted NHS on AuNPs. To remove unbound Abs and any reagents, the resulting mixture was centrifuged at 6,000 g and 4°C for 30 minutes and washed three times with HEPES buffer (0.02 M, pH 7.0). Next, the as-synthesized AuNPs/ Abs conjugates were dispersed in 1 mL HEPES buffer (0.02 M, pH 7.0) with 0.025% Tween 20, followed by adding 20 pL 10 mM PblNCLh aqueous solution and kept stirring overnight. During this reaction, Pb²⁺ ions became complexed with amine group of Abs. Finally, the immunoprobes (AuNPs/Abs@Pb²⁺) were collected by centrifugation and washed thoroughly with DI water with 0.025% Tween 20 and redispersed in 1 mL HEPES buffer (0.02 M, pH 7.0) with 0.025% Tween 20 and stored at 4°C for further use.

Design and fabrication of the electrochemical microtiter plate

The plate included two layers: 1) gold electrodes micropattemed on glass and 2) wells, channels and capillary valves molded in PDMS. These layers were designed using CAD software (AutoCAD 2020, Autodesk Inc.) and were fabricated by photolithography and metal etching techniques.²⁸'³⁰

(1) Fabrication of electrode arrays. Glass slides (75 mm x 25 mm) were sequentially sputter-coated with a 10 nm Cr adhesion layer and 100 nm Au layer (Lance Goddard Associates, Santa Clara, CA). A 1 pm layer of photoresist (AZ5214-E IR) was deposited on a substrate by spin-coating at 5000 rpm. Sequentially, the substrate with photoresist was then soft baked on a hot plate at 110°C for 1 minute. The design containing the desired structures was exposed to the photoresist via a maskless aligner (pPG 101, Heidelberg Instruments. Germany). Unexposed photoresist was developed by substrate immersion in 300 MIF developer until all undesired photoresist was removed (~2 minutes). Then, patterned Au/Cr layers were etched to create 16 patterned circular-shaped working electrodes (2.5 mm in diameter), 4 reference electrodes, and 4 counter electrodes, respectively. The electrodes were connected via 20 pm leads to contact pads located on the edges of the glass substrate. Finally, the electrode patterned slide was sonicated in acetone to remove unexposed photoresist and then exposed to oxygen plasma for 2 min at 150 mW (YES-G500, Yield Engineering Systems, Freemont, CA). To construct Ag/AgCl reference electrode, 1 pL of Ag/AgCl ink was carefully applied on the Au surface and cured at 120°C for 20 minutes following the manufacturer’s instructions.

(2) Fabrication of a PDMS micro well layer. A master mold for the microwell layer was fabricated using polydimethylsiloxane (PDMS) soft lithography protocol.³⁰ Briefly, a 100 pm layer of photoresist (SU-8 2050) on a silicon wafer was patterned using the pPG 101. Exposed photoresist was developed using SU-8 developer and then was exposed to chlorotrimethylsilane vapor in a closed chamber to facilitate the PDMS detachment from the patterned wafer. For the PDMS microtiter plate, PDMS pre-polymers (10:1 = elastomer: curing agent) were poured onto the fabricated mold, degassed in a vacuum chamber, and baked at 80°C for 60 minutes. Afterward, the PDMS layer was peeled off from the mold, and holes for wells and electrolyte inlets were punched using a 5 mm punch and a catheter puncher (Accu-Punch MP10-UNV, Syneo, USA), repressively.

The fabrication process was finished by placing a PDMS layer onto an electrode array to create 16 independent electrochemical cells with a maximum filling volume of 50 pL. Individual working electrodes were 2.5 mm in diameter, while PDMS wells were 5 mm in diameter and 2 mm in height.

Isolation and characterization of EVs

Three types of samples were used: (1) commercial pregnancy urine that was used for immunoassay characterization, (2) media conditioned by hepatic cells (Huh7) for selectivity experiments, and (3) patient urine samples for assessing clinical utility of our immunoassay.

(1) EVs from commercial urine samples were isolated using a total EV isolation kit (Invitrogen, Carlsbad, CA, USA).^13,31 Briefly, urine was mixed with EV isolation reagent and incubated for 1 h. After, EVs were collected by centrifugation at 10,000 g and dispersed in 1 X PBS.

(2) EVs from Huh7 cells were isolated by differential ultracentrifugation of conditioned media.³² EVs isolated at 100,000 g were resuspended l x PBS.

(3) Clinical urine samples were obtained from normotensive (NT) and preeclamptic (PE) pregnant women. Frozen urine samples (n=8) were thawed, filtered using 0.45 pm syringe filter to remove debris, and then used for testing clinical samples without additional processing.

Nanoparticle tracking analysis (NT A) was performed using a Nanosight NS300 (Malvern Panalytical, Malvern, UK) to evaluate the concentration and particle size of the isolated EVs. EV morphology and labeling EVs with immunoprobes were characterized by TEM using JEOL 1400 (JEOL USA Inc. Peabody, MA) at 80 kV. SEM was used to confirm the presence of EVs on the anti-CD63 functionalized electrodes using Hitachi S-4700 cold field emission SEM (Hitachi High Technologies America, Inc., Schaumburg, IL).

Functionalization of electrodes

The Au working electrodes were functionalized with anti-CD63 Abs for EV capture using the following steps. Au electrodes were immersed in 10 mM MUA in ethanol for 12 hours for self-assembly of this alkanethiol; then, electrodes were washed with ethanol and DI water and finally dried with nitrogen. Subsequently, working electrodes were blocked with pieces of PDMS and exposed to O2 plasma for 20 seconds. This process served to remove contamination from the reference and counter electrodes and to prime the glass substrate for bonding of PDMS. As the next step, the PDMS layer was aligned with and placed onto the glass substrate with an electrode array (see, e.g.. FIG. 4 for process description).

After that, Au electrodes in each well were treated with a 1 : 1 ratio of 200 mM of EDC and 100 mM of MES buffer (0.1 M, pH 5) for 1 hour to create amine-reactive groups. After the micro titer wells were rinsed with DI water, the electrodes were incubated with 50 pg/mL of anti-CD63 for 1.5 hours. After washing with l x PBS to remove excess Abs, working electrodes in wells were blocked with 1% BSA for 1 hour to avoid non-specific binding. After another washing step with 1 x PBS, a functionalized microtiter plate was stored at 4°C prior to use.

Electrochemical detection of EVs

For detection of podocin and nephrin on EVs, electrodes functionalized with anti- CD63 were incubated with the EV sample for 2 hours, followed by thorough washing with 1 x PBS. Next, wells of the microtiter plate were incubated with target-specific immunoprobes (i.e., AuNPs/anti-podocin@Pb²⁺ or AuNPs/anti-nephrin@Pb²⁺ at 8.95 x 10¹⁰ particles/mL) for 1 hour, washed thoroughly with DI water, and then analyzed using SWV. The time required for this assay was 3 hours. Wells of a microtiter plate were filled with 50 pL of sample and immunoprobe suspension. All EV detection experiments were performed in the manner described above.

Electrochemical detection of EVs was carried out by connecting working (Au), counter (Au), and reference electrodes (Ag/AgCl) to a multiplexer and a potentiostat (both from PalmSens). The SWVs were recorded in acetic acid/sodium acetate buffer (HAc/NaAc; 0.2 M, pH 4.5) in the -0.7 to 0.1 V range (versus Ag/AgCl) with 25 mV amplitude and 15 Hz frequency. Wells were filled with 50 pL of electrolyte for electrochemical detection. The electrodes were connected to a custom-designed 3D printed holder with pin-shaped connection to the circuit board and were addressed sequentially using a multiplexer and a potentiostat.

Electrochemical signals were recorded from three different working electrodes and were displayed as mean ± SD. To quantify electrochemical signals, SWV curves were converted to total charge (Q) by integrating the area under the redox curve. Then, Q values were normalized according to the formula (normalized Q, Q = QEVS- Qisoty e control Abs) and used to calculate the podocin/nephrin ratio by dividing Q of podocin to Q of nephrin. Surface plasmon resonance analysis ofEVs

A surface plasmon resonance (SPR) system (Biosensing Instruments, USA) was used to confirm surface functionalization steps and to benchmark clinical samples. Au SPR chips were prepared by self-assembly of MU A, activation of carboxylic groups with EDC-NHS, and immobilization of anti-CD63 Abs. BSA was used for blocking the surface to minimize non-specific bonding. Duration of the functionalization steps and concentration of reagents/ sample was the same as described herein for electrochemical detection. The flow rate used to inject reagent into the SPR instrument was 20 pL/minute and 10 pL/minute for EV capture. At completion of the EV capture step, a surface was washed with running buffer (1 x PBS), and the final baseline was recorded. SPR response (RU, Resonance Units) was obtained from the baseline changes before and after the sample injection.

Example 3: Characterization of a non-limiting electrochemical microtiter plate

Described herein are devices and methods that combine a NP-enabled electrochemical immunoassay for EV analysis ¹⁵ with an electrochemical microtiter plate. In some nonlimiting embodiments, such methods and devices can be used to enhance throughput of sample testing.

The immunoassay involved capture ofEVs on a working electrode. The plate was designed to selectively functionalize the working electrodes with Abs and incubate with a sample without affecting the counter and reference electrodes. The 16-well electrochemical plate also contained on-board counter/reference electrodes (see. e.g., FIG. 5A). A plate also included a micromolded PDMS layer having wells and gold electrodes patterned on glass. Each well contained one individually addressable working electrode and also had a fluidic connection to the counter/reference electrodes. This connection was checked by the capillary valves that rimmed each well and prevented fluidic communication with the counter/reference electrodes during functionalization and EV capture steps.

FIG. 5B shows that the solution (in this case blue dye) was confined to the wells and did not spill over into fluid channels leading to counter/reference electrodes. Two colors of food dye were used in FIG. 5C to illustrate the function of capillary valves during the surface functionalization and EV capture step (top, FIG. 5C), where the solution of blue dye is confined to wells, and the electrochemical measurement step, where a solution of yellow dye fills both the wells and fluidic channels communicating with counter/reference electrodes (bottom, FIG. 5C). The capillary valves were opened by introducing electrolyte through the inlet and exceeding their burst pressure (~0.7 Psi). Thus, fluidic/ionic connections between working electrodes and reference/counter electrodes were made at the time of electrochemical measurement.

Before using this microtiter for EV analysis, its performance was characterized using a soluble redox reporter (FeCN) (FIG. 6). SWV curves were obtained for each of the 16 working electrodes connected to on-board counter and reference electrodes. Working electrodes produced peaks of similar shape and amplitude (coefficient of variation. CV < 3.1%, n = 16). Next, working electrodes were connected to off-chip Ag/AgCl reference and Pt counter electrodes, and it was demonstrated that SWV curves for this setup were similar signals obtained with on-chip counter and reference electrodes (FIG. 6). This characterization confirmed that the microtiter plate enabled accurate electrochemical measurements.

Example 4: Assessing independence of electrochemical cells in the microtiter plate

To facilitate individual electrochemical measurements using this microtiter plate, the electrochemical cells were characterized to determine the extent of operating independently. Each working electrode had its own lead and contact, and therefore, was electrically independent. To simplify the design of the microtiter plate and reduce the number of electrical connections, one pair of reference and counter electrodes for addressing 16 w orking electrodes was used. Of course, a design including individual reference/counter electrodes (for each individual working electrode) can also be used.

While FIG. 5A shows four locations for micropattemed reference and counter electrodes, all of the electrode connections converged to a single contact pad that was connected to one potentiostat. A goal w as to confirm that immunoassay results did not vary depending on the location of the w orking electrode with respect to its counter/reference electrodes. To address this concern, a microtiter plate w as prepared where wells contained different concentrations of podocin (i.e.. 0.1, 5, and 100 ng/mL). In addition, the same solution of podocin was dispensed into three wells to examine working electrode position bias when making measurements (FIG. 7A, top). In these experiments, Au electrodes w ere functionalized with primary Abs using standard alkanethiol conjugation chemistry (described herein), then incubated w ith different concentrations of recombinant podocin and finally exposed with AuNPs/anti-podocin rt Pb² immunoprobe and analyzed using SWV. Here, the value of total charge (Q) w as estimated by integrating the area under the redox peaks and used to compare electrochemical results. As shown in FIG. 7A (bottom), the amplitude of redox peaks increased as the podocin concentration varied from 0. 1 to 100 ngfrnL. Furthermore, for a given podocin concentration, similar Q values (CV < 7.3%, n = 3) were observed regardless of the electrode location. The results confirmed that 16 working electrodes connected to counter/reference electrodes functioned as independent electrochemical cells.

Example 5: Detecting recombinant nephrin and podocin in the electrochemical microtiter plate

After assessing performance of the microtiter plate, the following was performed to characterize NP-enabled electrochemical immunoassays using known concentrations of recombinant proteins, podocin and nephrin. Such assessments can aid determination of figures of merit, limit of detection, and linear range for this immunoassay in the microtiter plate.

In this set of experiments, wells/working electrodes were functionalized with MUA and activated with EDC-NHS, incubated with a given concentration of podocin or nephrin and then labeled with appropriate immunoprobes (either AuNPs/anti-podocin@Pb²⁺ or AuNPs/ant-nephrin@Pb²⁺). As seen in FIG. 8, SWV analysis revealed linear correlation between the redox peaks at the formal potential of Pb ions (-0.32 V) and a protein concentration. SWV results were converted into calibration curves by plotting normalized charge Q (Q = Q - Qo) vs. concentration of podocin or nephrin and detection limits were determined using the formula: 3.3*o/slope of calibration curve, with o being the standard deviation of the y-intercept. As depicted in FIG. 7B-7C, calibration curves had a linear range of four logs from 0.05 to 500 ng/mL with a LOD of 10.6 pg/mL for podocin and 14.5 pg/mL for nephrin. These results were better than commercial ELIS As (e g., an ELISA kit from System Biosciences reports a LOD of 94 pg/mL and 190 pg/mL for podocin and nephrin, respectively). In addition, the figures of merit for immunoassays performed in the microtiter plate were similar to those in a standard electrochemical cell.¹⁵ Without wishing to be limited by any particular mechanism, the low- detection limits achieved with NP-enabled immunoassays may be attributed to a high concentration of metal ions doped into AuNPs (~10⁴ ions/particle), which amplify redox signals.

Example 6: Characterizing electrode functionalization, EV capture, and immunoprobe labeling in the microtiter plate

As the next step in assay development, the following was performed to characterize capture of EVs and detection of EV surface markers in the microtiter plate using electrochemical immunoassays. EV concentration and size distribution were routinely- assessed using NTA. As seen from FIG. 9A, the size distribution for EVs isolated from human pregnancy urine was 152 ± 62 nm. EVs were reconstituted in lx PBS at 10⁸ parti cles/mL for the characterization experiments described below. TEM analysis (see FIG. 9B) was carried out to confirm that immunoprobes bound to EVs.

A non-limiting process of modifying electrodes for capturing EVs is described in FIG. 10A. It included functionalization with a self-assembled monolayer comprised of MU A, followed by activation of terminal carboxyl groups with EDC-NHS and then immobilization of anti-CD63. Electrode surfaces were blocked by incubating with BSA to prevent nonspecific interactions. EVs express CD63 and were captured on electrode surfaces via Ab-Ag interactions.

Finally, wells/electrodes were incubated with target-specific immunoprobes, washed, and characterized electrochemically. The steps of this process were characterized byelectrical impedance spectroscopy (EIS). FIG. 10B shows the impedance spectra for the Au electrodes during the individual modification process, which were recorded in a solution containing 5 mM [Fe(CN)6]^{4 /3} in 0. 1 M KC1. As shown in FIG. 10B. the bare Au electrode presents a small semicircle at high frequencies with charge transfer resistance (R_ct) of 157 (curve a). After MUA-modified on the electrode (curve b), the value of Rct increased to 4654 Q, indicating that the negatively charged SAM on the electrode hinders the electron transfer of [Fe(CN)6]^{4 /3} .^33,34 After the activation of the SAM-modified electrode with EDC-NHS, the Rct decreased to 1487 (curve c) due to enhancement of the [Fe(CN)6]^{4 3} redox reaction rate at the electrode surface, indicating that the COO groups of SAM were successfully converted to NHS esters. After the electrodes were modified step-by-step with anti-CD63 Abs, BSA, and EVs, the Rct value increased gradually to 1699 Q, 1978 Q, and 2800 Q (curve d-f). These observations are logical given that capture of lipid nanoparticles (EVs) insulates the electrode and impedes electrode transfer. Incubation with immunoprobes (AuNPs/anti-CD63@Pb²⁺) (curve g) reduced Rct to 2434 Q which points to improved electron transfer. Thus, EIS pointed to successful capture of EVs on an electrode surface and to binding of immunoprobes to the EVs.

SPR analysis was also used to confirm Ab immobilization, EV capture, and immunoprobe labeling steps (see FIG. 11A). As described herein, SPR analysis was used for benchmarking electrochemical immunoassays when analyzing clinical samples. Example 7: Assessing specificity of the electrochemical immunoassay for detecting target surface markers on EVs

The following was performed to confirm that the immunoassay has high specificity for EV surface markers of interest. The first set of experiments focused on first capturing EVs and then assessing electrochemical signals when labeling with immunoprobes carrying anti-CD63 (positive control), anti-podocin and anti-nephrin (target markers), and rabbit IgG (isotype or negative control) (see FIG. IOC for anon-limiting experiment design). The same concentration of EVs (10⁸particles/mL) was used for all the conditions. A representative image of EVs captured on an electrode surface may be seen in FIG. 12. The electrochemical signals associated with the four types of immunoprobes are compiled in FIG. 10D.

As seen by these SWV curves, immunoprobes targeting CD63 produced highest redox signal, consistent with high levels of expression for this surface marker. In addition, isotype control immunoprobes resulted in low levels of redox activity, suggesting minimal off-target binding for our immunoassay. Indeed, quantification of charge transfer presented in FIG. 10E, revealed 10-fold higher signals for immunoprobes targeting podocin and nephrin compared to isotype control. Another control experiment was designed to assess whether immunoprobes interacted with the electrode surface in the absence of EVs. In this experiment, electrodes were functionalized with anti-CD63 and then incubated with AuNPs/anti-CD63@Pb²' immunoprobes. As seen from FIG. 10D. signals for this condition were similar to redox signals for an isotype control group, suggesting minimal nonspecific interactions between immunoprobes and the electrode surface in the absence of EVs. FIG. 10E presents redox signals as total charge, an area under the curve of SWV signals, which makes it easier to compare voltammetry results from different experiments. Overall, FIG. 10D-10E confirm that electrochemical signals are specific to the target of interest and vary depending on the surface marker.

EV-specific and background signals were further characterized by SPR (FIG. 11B- 11C). When substrates w ere not configured for EV capture (i.e., functionalized with isotype control Abs). SPR signals were comparable to background (< I RU) upon incubation with EVs at 10⁸ particles/mL. With surfaces containing anti-CD63 Abs, the SPR signal was significantly higher than the background (29 RU) and increased further upon labeling with AuNPs/anti-CD63@Pb²⁺. In contrast, immunoprobes carrying isotype Abs produced minimal SPR signals after incubation with captured EVs. The contrast between the signals recorded for positive and negative control samples supports specificity of interactions between immunoprobes and EVs. Another way to test specificity of our immunoassay was to analyze EVs originating from a different organ. Urinary EVs are produced by kidneys and carry proteins responsible for renal barrier function such as nephrin and podocin. EVs from media conditioned by hepatic cells (Huh7), which are not expected to express podocin and nephrin, were tested. EVs were captured in an electrochemical microtiter plate and labeled EVs with immunoprobes targeting podocin and nephrin (kidney/podocyte markers), CD63 (ubiquitous EV marker), and CYP2E1 (hepatocyte-specific marker).^35,36 The concentration of hepatic EVs was adjusted to be comparable to the experiment with urinary EVs described in the preceding section. EIS characterization described in FIG. 13A revealed that EVs were successfully captured on the electrode surface. In fact, impedance signals for capture of hepatic EVs were similar to those observed for urinary EVs (2750 and 2800 for hepatic and urinary EV capture, respectively). This suggested similar surface density of EVs from both sources. After confirming successful capture of hepatic EVs, we proceeded to label EVs with immunoprobes. As shown in FIG. 11B-11C, labeling hepatic EVs with immunoprobes for podocin and nephrin produced negligible redox signals (< 1 pC/cm²), similar to the signal for isotype control immunoprobes (0.80 pC/cm²). Conversely, labeling with immunoprobes for CD63 and CYP2E1 resulted in prominent redox peaks (15.3 pC/cm² and 5.37 pC/cm² respectively, see FIG. 13C) reflective of positive signals. These results underscore once again that our immunoassay was specific to the target surface markers on EVs.

Example 8: Testing clinical samples in an electrochemical microtiter plate and benchmarking against SPR

The podocin to nephrin ratio is typically higher in PE pregnancies compared to normal (normotensive) pregnancies,⁵ as conducted using a relatively laborious technique of digital flow cytometry. The electrochemical immunoassay and microtiter plate was used to assess the podocin-to-nephrin ratio in urinary EVs of pregnant women. Urine samples from 4 normotensive (NT) as healthy controls and 4 preeclamptic (PE) pregnant women were collected and analyzed. An electrochemical microtiter plate was prepared as described previously by immobilizing anti-CD63 onto working electrodes and then blocking with BSA to minimize non-specific binding. Then, urine samples were passed through a 0.45 pm syringe filter to remove debris and loaded into wells for EV capture. Considering that particles in urine range in size from 10 to 30 pm,³⁷ 0.45 pm filter was expected to remove large particles while allowing EVs (mean 152 ± 62 nm) to pass. After filtering, urine samples were dispensed into a microtiter plate, incubated, and washed. Subsequently, micro wells were incubated with immunoprobes targeting podocin and nephrin (AuNPs/anti- podocin@Pb²⁺ and AuNPs/anti-nephrin@Pb²⁺), washed again and characterized with SWV. Additional wells were incubated with immunoprobes carrying isotype control rabbit IgG Abs to assess non-specific binding. Blocking with BSA and extensive washing after EV capture and immunoprobe incubation steps were used to minimize nonspecific interactions with the electrodes. The experiments were carried out in triplicate for each clinical sample. The value of normalized Q (Q = QEVS - Qisotype control Abs) was used to estimate marker expression level on EVs and to calculate the ratio of podocin to nephrin.

As displayed in FIG. 14A, the expression level of podocin increased in PE patients while the nephrin levels decreased. The podocin/ nephrin ratio was calculated by dividing Q of podocin to Q of nephrin in FIG. 14A and presented in FIG. 14B. The podocin-to-nephrin ratios in the urine of PE patients were higher than of NT healthy controls (p < 0.01). The average podocin-to-nephrin ratio was 4.31 for PE and 1.69 for NT samples, which is consistent with flow cytometry analysis of urinary EVs indicating an elevated podocin to nephrin EVs ratio in preeclampsia.⁵ These results confirm that podocin and nephrin on EVs in urine can be used as biomarkers of PE and also demonstrate clinical utility of our electrochemical immunoassay and microtiter plate. Furthermore, our detection method worked with minimally processed clinical samples and did not require EV isolation steps.

The method used herein was benchmarked against an established approach. SPR represents a useful benchmarking technology because of its similarity to the method described herein in both the substrate composition (Au-coated chips) and functionalization chemistry. Ab functionalization and specificity of EV captures steps were first confirmed by SPR (see FIG. 11). Then, SPR analysis of the same set of clinical urine samples that were filtered in the same manner as described above for electrochemical detection experiments were carried out. After characterizing surface functionalization and capture of urinary EV s (see FIG. 15A), the process proceeded to labeling with Abs. The SPR instrument had three channels that were used to label captured EVs with anti-podocin, anti-nephrin and isotype control Abs. FIG. 14C shows SPR binding signals of the PE3 sample that was analyzed electrochemically in FIG. 14A. The change of the SPR response at the endpoint (see FIG. 14C) was used to calculate the podocin to nephrin ratio which was 4.85 for this particular urine sample (i.e., PE3 sample) (see FIG. 15B for a detailed description of SPR-based analysis of the podocin/nephrin ratio). This is comparable to the ratio of 4.56 analyzed electrochemically in FIG. 14A. As shown in FIG. 14D, podocin/nephrin ratios of 8 clinical samples obtained by SPR and by electrochemical immunoassay showed good correlation (R² = 0.9) which suggests that the electrochemical immunoassay provides accurate information related to the levels of EV surface marker expression.

Accordingly, described herein are NP-enabled immunoassays integrated with an electrochemical microtiter plate for the detection of EV surface markers. The immunoassay relied on AuNPs functionalized with anti-podocin or anti-nephrin Abs and doped with Pb²⁺ to produce redox signals. Electrochemical immunoassays were carried out in a microtiter plate that contained capillary valves to allow functionalizing working electrodes with Abs and capturing urinary EVs without affecting reference and counter electrodes. The immunoassay performed in the electrochemical microtiter plate was found to be specific with negligible signals observed when using isotype control immunoprobes or when using correct immunoprobes in the absence of EVs. Furthermore, the NP-enabled immunoassay carried out in the electrochemical microtiter plate was used to analyze clinical samples and to demonstrate differences in podocin-to-nephrin ratios in urine of women with PE compared to healthy controls. It is also worth noting that SPR was rigorously characterized as a method for capturing EVs, assessing EV surface marker expression and benchmarking the electrochemical immunoassay method. In the future, the format can have an increased throughput from a 16-well to a 48- or 96-well format. In addition, more clinical sample testing can be performed to confirm the platform as a technology for screening and diagnosis of PE.

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Claims

WHAT IS CLAIMED IS:

1. A device comprising: a fluidic module configured to provide a first chamber, a reservoir, and a releasable valve, wherein said releasable valve is configured to minimize fluidic communication between said first chamber, or a portion thereof, and said reservoir; and an electrical module configured to provide a working electrode within said first chamber and to provide a reference electrode within said reservoir, wherein said working electrode is configured to be attached to a capture agent for capturing a target analyte.

2. The device of claim 1, wherein said device further comprises a plurality of chambers, and wherein said first chamber is one of said plurality of chambers.

3. The device of claim 2, wherein said first chamber is configured to be in fluidic communication with said reservoir, and wherein a second chamber of said plurality of chamber is configured to be in fluidic communication with said first chamber.

4. The device of claim 3, wherein said second chamber is configured to lyse a sample to provide one or more target analytes and configured to deliver said one or more target analytes to said first chamber.

5. The device of claim 2, wherein each of said plurality of chambers is configured to be in fluidic communication with said reservoir.

6. The device of claim 2, wherein each of said plurality of chambers comprises an individually addressable working electrode.

7. The device of any one of claims 1-6, wherein said electrical module further comprises a counter electrode within said reservoir.

8. The device of any one of claims 1-7, wherein said releasable valve comprises one or more structures configured to provide fluidic communication between said first chamber and said reservoir at a pressure from about 0.5 psi to about 2 psi.

9. The device of claim 8, wherein said one or more structures comprise an inlet, an outlet, and a first constricted portion disposed between said inlet and said outlet.

10. The device of claim 9, wherein the first constricted portion comprises a change in dimension along one or more of x, y, or z axes.

11. The device of claim 9, further comprising an expanded portion disposed between said first constricted portion and said outlet.

12. The device of claim 11, wherein the expanded portion comprises a liquid bypass region.

13. The device of claim 9, further comprising a second restricted portion and an expanded portion, wherein said expanded portion is further disposed between said first and second restricted portions.

14. The device of any one of claims 1-13, wherein said fluidic module comprises a non- conductive material, polymer, elastomer, glass, or a combination thereof.

15. The device of any one of claims 1-14, wherein said working electrode further comprises an attached capture agent.

16. The device of claim 15, wherein said capture agent comprises one or more of a nanoparticle, a microparticle, a dye, a detectable agent, a protein, an antibody, a nucleic acid, an aptamer, a small molecule, or a combination thereof

17. The device of claim 15, further comprising a linker disposed between a surface of said working electrode and said attached capture agent.

18. The device of claim 17, wherein said linker comprises a covalent bond, an optionally substituted alkylene, or an optionally substituted heteroalkylene.

19. The device of any one of claims 1-18, wherein said electrical module further comprises a bond pad and a connector configured to electrically connect said working electrode to said bond pad.

20. The device of any one of claims 1-19, wherein said device is a monolithic device.

21. The device of any one of claims 1-19, wherein said fluidic module and said electrical module are configured to be separated.

22. The device of any one of claims 1-21, further comprising an adhesive layer disposed between said fluidic module and said electrical module.

23. A system comprising: a device of any one of claims 1-22; an optional lysing plate configured to provide one or more target analytes for said device; a platform configured to electrically connect said device to a controller configured to control said electrical module; and a user interface configured to receive and transmit information between said device and said controller.

24. The system of claim 23, wherein said lysing plate comprises a plurality of wells, and wherein a surface of the plurality of wells comprises a capture agent configured to capture at least one of the one or more target analytes.

25. The system of claim 23, wherein said controller comprises a multiplexer, a potentiostat, and/or a printed circuit board for making one or more electrical contacts.

26. A method for conducting an electrochemical assay, wherein said method comprises:

(a) providing a device of any one of claims 1-22 or a system of any one of claims 23-25;

(b) delivering a sample to said first chamber of said device;

(c) providing one or more reagents for said electrochemical assay to said reservoir of said device; (d) actuating said releasable valve, thereby providing electric communication or fluidic communication between said first chamber and said reservoir; and

(e) detecting one or more electrical signals from said working electrode, thereby detecting the presence or absence of a target analyte in said sample, wherein each of (b), (c), and (d) can be conducted at the same time or in any order.

27. The method of claim 26, wherein said sample comprises one or more extracellular vesicles, analytes from one or more extracellular vesicle, lysates from one or more extracellular vesicles, particles, and/or micelles.

28. The method of claim 27, wherein said target analyte comprises a protein disposed on a surface of at least one of said one or more extracellular vesicles.

29. The method of claim 27, wherein said target analyte comprises a protein, a nucleic acid, or a lipid from at least one of said one or more extracellular vesicles.

30. The method of any one of claims 26-29, wherein said one or more reagents comprise a nanoparticle, a metal, a dye, a fluorophore, a detectable reporter, an electroactive agent, or a combination thereof.

31. The method of any one of claims 26-30, wherein (c) of said providing and (d) of said actuating are conducted at the same time.

32. The method of any one of claims 26-31, wherein said one or more electrical signals are determined between said working electrode and said reference electrode.

33. A method for detecting an analyte of an extracellular vesicle, wherein said method comprises:

(a) capturing one or more extracellular vesicles on said working electrode of a device of any one of claims 1-22 or a system of any one of claims 23-25; and

34. The method of claim 33, further comprising, after or before said capturing:

(al) providing one or more reagents for an electrochemical assay to said working electrode in said first chamber.

35. The method of claim 33, further comprising, after or before said capturing:

(al) providing one or more reagents for an electrochemical assay to said reservoir.

36. The method of any one of claims 34-35, wherein said one or more reagents comprise a nanoparticle, a metal, a dye, a fluorophore, a detectable reporter, an electroactive agent, or a combination thereof.

37. The method of any one of claims 33-36, further comprising, after or before said capturing:

(al) actuating said releasable valve, thereby providing electric communication or fluidic communication between said first chamber and said reservoir.

38. The method of claim 37, wherein said actuating provides said electric communication and said fluidic communication between said first chamber and said reservoir.

39. A method for detecting an analyte of an extracellular vesicle, wherein said method comprises:

(a) capturing one or more target analytes from one or more extracellular vesicles on said working electrode of a device of any one of claims 1-22 or a system of any one of claims 23-25; and

(b) detecting one or more electrical signals from said working electrode, thereby detecting the presence or absence of at least one of said one or more target analytes from said one or more extracellular vesicles.

40. The method of claim 39, further comprising, before said capturing:

(aO-1) lysing said one or more extracellular vesicles to release said one or more target analytes; and

(aO-2) providing said one or more target analytes to said working electrode.

41. The method of claim 39 or 40, further comprising, after or before said capturing:

42. The method of claim 39 or 40, further comprising, after or before said capturing: (al) providing one or more reagents for an electrochemical assay to said reservoir.

43. The method of any one of claims 41 or 42, wherein said one or more reagents comprise a nanoparticle, a metal, a dye, a fluorophore, a detectable reporter, an electroactive agent, or a combination thereof.

44. The method of any one of claims 39-43, further comprising, after or before said capturing:

(a2) actuating said releasable valve, thereby providing electric communication or fluidic communication between said first chamber and said reservoir.

45. The method of claim 39, wherein said actuating provides said electric communication and said fluidic communication between said first chamber and said reservoir.