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WO2018119401A2 - Dispositifs et procédés pour déterminer un ou plusieurs analytes dans des fluides - Google Patents

Dispositifs et procédés pour déterminer un ou plusieurs analytes dans des fluides Download PDF

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Publication number
WO2018119401A2
WO2018119401A2 PCT/US2017/068203 US2017068203W WO2018119401A2 WO 2018119401 A2 WO2018119401 A2 WO 2018119401A2 US 2017068203 W US2017068203 W US 2017068203W WO 2018119401 A2 WO2018119401 A2 WO 2018119401A2
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Prior art keywords
metal
analyte
previous
equal
region
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WO2018119401A3 (fr
Inventor
Marta SUAREZ FERNANDEZ
Lisa Marshall
Martina MEDKOVA
Aaron Oppenheimer
A. David Boccuti
Roberet ETHEREDGE
Eric EVJE
Frederick P. FLOYD
Mihail Lungu
Ionel Catalin POPESCU
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Daktari Diagnostics Inc
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Daktari Diagnostics Inc
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Publication of WO2018119401A2 publication Critical patent/WO2018119401A2/fr
Publication of WO2018119401A3 publication Critical patent/WO2018119401A3/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/0606—Investigating concentration of particle suspensions by collecting particles on a support
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/0656—Investigating concentration of particle suspensions using electric, e.g. electrostatic methods or magnetic methods
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/16—Reagents, handling or storing thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0645—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0677—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
    • B01L2400/0683—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers mechanically breaking a wall or membrane within a channel or chamber
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells

Definitions

  • the present invention generally relates to methods and devices for determining and/or quantifying one or more analytes in fluids.
  • the present invention generally relates to methods and devices for determining and/or quantifying one or more analytes in fluids.
  • a device comprises at least one microfluidic channel, a first reaction chamber in fluidic communication with the microfluidic channel, and a first reservoir containing a first stored reagent.
  • the first reservoir is sealed, and the first reservoir is configured to introduce the first stored reagent into the first reaction chamber after unsealing of the first reservoir.
  • the device includes a source of magnetic particles present in, or configured to be introduced into, the first reaction chamber.
  • the device also includes a magnetic element present in, or configured to be introduced into, the first reaction chamber.
  • the device further includes a detection region downstream of the first reaction chamber, and an electrode associated with the detection region.
  • the device comprises at least one microfluidic channel, a first reagent inlet, a sample introduction region positioned downstream of the first reagent inlet, a mixing region positioned downstream of the sample introduction region, a second reagent inlet positioned between the mixing region, a binding region positioned downstream of the lysis region, and a detection region positioned downstream of the binding region.
  • the device comprises at least one microfluidic channel, a first reagent inlet, and a first reservoir containing a first stored reagent.
  • the first reservoir is sealed and positioned adjacent the first reagent inlet, and the first reservoir is configured to introduce the first stored reagent into the first reagent inlet upon unsealing of the first reservoir.
  • the device includes a sample introduction region, a mixing region, a lysis region, a second reagent inlet, and a second reservoir containing a second stored reagent.
  • the second reservoir is sealed and positioned adjacent the second reagent inlet.
  • the second reservoir is configured to introduce the second stored reagent into the second reagent inlet upon unsealing of the second reservoir, a binding region, and a detection region.
  • the device comprises at least one microfluidic channel, a first reagent inlet, a sample introduction region positioned downstream of the first reagent inlet, and a waste region.
  • the device includes a first valve positioned between, and configured to control fluidic communication between, the first reagent inlet and the waste region.
  • the device includes a second reagent inlet, and a second valve positioned between, and configured to control fluidic communication between, the second reagent inlet and the waste region.
  • the device also includes a binding region and a detection region.
  • the device comprises an inlet, a first channel intersection, a primary channel extending from the first channel intersection, and a waste channel extending from the first channel intersection.
  • the waste channel is parallel to the primary channel (e.g., in the direction of fluid flow).
  • the device also includes a waste region positioned
  • the device also includes at least two of a sample introduction region, a mixing region, a lysis region, a binding region, and a detection region connected to the primary channel and positioned between the inlet and the waste region. At least one channel of the device is a microfluidic channel.
  • the device comprises at least one microfluidic channel, a sample introduction region, a mixing region, a lysis region, and a first electrode.
  • the first electrode is associated with the lysis region, a binding region, a detection region.
  • the device also includes a second electrode, wherein the second electrode is associated with the detection region.
  • the device comprises at least one microfluidic channel, a sample introduction region, a mixing region, a lysis region, and a first magnetic element.
  • the first magnetic element is associated with the lysis region, a binding region, and a detection region.
  • the device also includes a second magnetic element, wherein the second magnetic element is associated with the detection region.
  • a method comprises introducing a sample comprising a plurality of analyte-containing biological particles into a fluidic device comprising at least one microfluidic channel.
  • the method involves introducing, to the analyte, a plurality of metal-containing particles and a plurality of capture structures such that the analyte binds with both a capture structure and a metal-containing particle to form the bound complex.
  • the method involves separating any unbound metal-containing particles from the bound complex, and determining the amount of analyte present.
  • the method comprises introducing, to a first analyte and a second analyte different than the first analyte, a plurality of metal-containing particles comprising a first type of metal-containing particle and a second type of metal-containing particle, and a plurality of capture structures such that the first analyte binds with a capture structure and a first type of metal-containing particle to form a first bound complex, and such that the second analyte binds with a capture structure and a second type of metal-containing particle to form a second bound complex, separating any unbound metal-containing particles from the first bound complex and the second bound complex, and determining the amount of the first analyte and the second analyte present.
  • the method comprises: in a fluidic device comprising at least one microfluidic channel, performing the steps of: introducing, to an analyte, a plurality of metal-containing particles and a plurality of capture structures such that the analyte binds with both a capture structure and a metal-containing particle to form the bound complex.
  • the plurality of metal-containing particles comprise a metal.
  • the method involves separating any unbound metal-containing particles from the bound complex, exposing the bound complex to an electrolyte, applying an electric potential to oxidize at least a portion of the metal from the metal-containing particles, applying an electric potential to deposit at least a portion of the metal onto a working electrode, and measuring current by changing a voltage on the working electrode to determine the amount of analyte present.
  • the method comprises introducing a sample comprising a plurality of analyte-containing biological particles into a fluidic device comprising at least one microfluidic channel, attaching at least a portion of the analyte-containing biological particles to one or more capture substrates/capture structures, exposing an analyte from the analyte-containing biological particles such that the analyte is available to form a bound complex, introducing, to the analyte, a plurality of metal-containing particles and a plurality of capture structures such that the analyte binds with both a capture structure and a metal- containing particle to form the bound complex, separating any unbound metal-containing particles from the bound complex, and determining the amount of analyte present.
  • for quantifying an analyte in a fluid comprises: introducing or exposing a plurality of capture structures to a fluid comprising the analyte such that the analyte binds with a capture structure; introducing or exposing a plurality of metal-containing particles to the capture structures-analyte mixture such that the metal-containing particles bind to the capture structures that did not bind analyte in the first step, to form a bound complex, wherein the plurality of metal-containing particles comprise a metal, and wherein prior to forming the complex, the plurality of metal-containing particles have an average particle size of at least 100 nm; separating any unbound metal-containing particles from the bound complex; exposing the bound complex to an electrolyte; applying an electric potential to oxidize at least a portion of the metal from the metal-containing particles; applying an electric potential to deposit at least a portion of the metal onto a working electrode; and measuring current by changing a voltage on the working electrode to determine the amount of an
  • the device comprises a plasma separation region upstream of the first reaction chamber.
  • the plasma separation region comprises a membrane.
  • the first reaction chamber is configured to lyse an analyte. In some embodiments of the devices described above and herein, the first reaction chamber is configured to bind at least a portion of a plurality of magnetic particles with an analyte.
  • the device comprises a source of a washing agent connected to the first reaction chamber.
  • the device comprises a mixing region in fluidic communication with the first reaction chamber. In some embodiments of the devices described above and herein, the device comprises a source of a lysing solution present in, or configured to be introduced into, the first reaction chamber.
  • the device comprises a waste region in fluidic communication with the first reaction chamber. In some embodiments of the devices described above and herein, the device comprises a source of metal-containing particles present in, or configured to be introduced into, the first reaction chamber. In some embodiments of the devices described above and herein, the device comprises a source of capture substrates/capture structures present in, or configured to be introduced into, the first reaction chamber. In some embodiments of the devices described above and herein, the device comprises a magnetic element present in, or configured to be introduced into, the detection region. In some embodiments of the devices described above and herein, the device comprises a lysis region positioned downstream of the mixing region. In some embodiments of the devices described above and herein, the second reagent inlet is positioned between the mixing region and the lysis region.
  • the method comprises prior to exposing the analyte, introducing, to the analyte-containing biological particles, a plurality of magnetic particles such that the analyte-containing biological particles binds with the plurality of magnetic particles.
  • the method comprises prior to introducing, to the analyte, a plurality of metal-containing particles and a plurality of capture substrates/capture structures, exposing an analyte from the analyte- containing biological particles such that the analyte is available to form a bound complex.
  • determining the amount of analyte present comprising determining the number of bound complexes.
  • the method comprises exposing the bound complex to an electrolyte.
  • the method comprises applying an electric potential to oxidize at least a portion of the metal from the metal-containing particles. In some embodiments of the methods described above and herein, the method comprises applying an electric potential to deposit at least a portion of the metal onto a working electrode.
  • the method comprises measuring current by changing a voltage on the working electrode to determine the amount of analyte present.
  • the method comprises depositing at least a portion of the metal onto a working electrode comprising reducing the metal.
  • the method or device comprises exposing the analyte comprises introducing a lysis solution to the analyte-containing biological particles.
  • the method or device comprises capturing the one or more capture substrates/capture structures in a reaction chamber of the fluidic device.
  • the method or device comprises attaching at least a portion of the analyte-containing biological particles to one or more capture substrates/capture structures.
  • the at least one microfluidic channel has an average cross-sectional dimension of at least 200 microns and less than 2 mm.
  • the at least one microfluidic channel has a volume of between 1 microliter and 100 microliters.
  • the device comprises a polymeric material.
  • the device comprises a one or more valves.
  • the device comprises a reagent reservoir in fluidic communication with the first reaction chamber and/or the second reaction chamber.
  • the mixing region comprises a propeller.
  • the mixing region comprises a shear generating mechanism.
  • the mixing region comprises a vibration generating mechanism.
  • the mixing region comprises a magnetic element.
  • the mixing region comprises an electrode.
  • the mixing region comprises a serpentine channel.
  • the lysis region comprises a lysis buffer.
  • a reservoir containing a lysis buffer is in fluidic communication with the lysis region.
  • the binding region comprises a source of a metal-containing particle. In some embodiments of the methods or devices described above and herein, the binding region comprises a source of a plurality of capture substrates/capture structures. In some embodiments of the methods or devices described above and herein, the detection region comprises an ASV detector. In some embodiments of the methods or devices described above and herein, the detection region comprises an electrolyte contained therein.
  • the electrode comprises a carbon electrode.
  • the electrode comprises a first layer comprising carbon deposited on a second layer comprising silver.
  • the method comprises exposing the bound complex to an electrolyte. In some embodiments of the methods described above and herein, the method comprises applying an electric potential to oxidize at least a portion of the metal from the metal-containing particles. In some embodiments of the methods described above and herein, the method comprises applying an electric potential to deposit (reduce) at least a portion of the metal onto a working electrode. In some embodiments of the methods described above and herein, the method comprises measuring current by changing a voltage on the working electrode to determine the amount of analyte present.
  • the fluid comprises a blocking solution.
  • the blocking solution comprises BSA or casein.
  • the buffer solution comprises a chlorine- containing salt, metal acetate, and/or a salt selected from the group consisting of sodium acetate, zinc acetate, copper acetate, NaCl, LiCl, CsCl, and combinations thereof.
  • the buffer solution comprises a salt having a concentration of 1 mM to 5 M.
  • the buffer solution has a pH from 4 to 10.
  • the buffer solution comprises 50 mM sodium acetate, at least 1 mM and less than or equal to 5 mM zinc acetate, and at least 50 mM and less than or equal to 200 mM sodium chloride.
  • the lysis solution comprises a detergent, a denaturant, a reducing agent, or combinations thereof. In some embodiments of the methods or devices described above and herein, the lysis solution comprises a detergent selected from the group consisting of anionic surfactants, zwitterionic surfactants, nonionic surfactants, and cationic surfactants.
  • the plurality of metal-containing particles have an average particle size of at least 100 nm. In some embodiments of the methods or devices described above and herein, the plurality of metal-containing particles have an average particle size of less than or equal to 2 microns. In some embodiments of the methods or devices described above and herein, the plurality of metal-containing particles are conjugated with a first antibody that can bind to the analyte.
  • the metal- containing particles comprise silver, copper, cobalt, cadmium, lead, zinc, tin, nickel, bismuth, platinum, manganese, chromium, gold, alloys thereof, oxides thereof, sulfides thereof, or combinations thereof.
  • metal-containing particles comprise a metal layer deposited on a non-metallic particle.
  • the plurality of capture structures have a mean cross-sectional dimension of at least 100 nm and less than or equal to 5 microns. In some embodiments of the methods or devices described above and herein, the plurality of capture structures comprise a magnetic material. In some embodiments of the methods or devices described above and herein, the plurality of capture structures are conjugated with a second antibody that can bind to the analyte. In some embodiments of the methods or devices described above and herein, the plurality of capture structures are not electrochemically active.
  • an average particle size of the plurality of metal-containing particles is at least 0.2 times and less than or equal to 2.5 times an average particle size of the plurality of capture structures.
  • the concentration of the electrolyte is less than 1.0 M, less than 0.8 M, less than 0.6 M, less than 0.4 M, less than 0.2 M, or less than 0.1 M after adding the electrolyte to the fluid.
  • the electrolyte does not remove the metal-containing particle from the bound complex upon introduction of the electrolyte.
  • at least 90%, at least 95%, or at least 99% of the metal-containing particles in the bound complex are not removed from the bound complex upon introduction of the electrolyte.
  • applying the electric potential to oxidize at least a portion of the metal from the metal-containing particles directly oxidizes the plurality of metal-containing particles.
  • oxidation of at least a portion of the metal from the metal-containing particles comprises oxidizing Ag° to Ag + .
  • changing a voltage on the working electrode comprises increasing the electric potential to about 0 V.
  • increasing the electric potential comprises increasing the voltage at a rate of at least 10 mV/s and less than or equal to 2 mV/s.
  • the plurality of metal-containing particles are directly oxidized with the applied potential without the use of an oxidizing agent.
  • the electrolyte undergoes an intermediate redox reaction.
  • the capture substrate/structure non- specifically captures the analyte or analyte-containing biological particle.
  • the analyte-containing biological particle is a blood cell.
  • the capture substrate/structure is a plurality of beads.
  • the plurality of beads have an average diameters of between 100 nm and 5 microns.
  • the plurality of beads are magnetic.
  • the capture substrate/structure is uncharged. In some embodiments of the methods or devices described above and herein, the capture substrate/structure is charged.
  • the sample prior to removing any components not bound to the capture substrate/structure, is mixed with the buffer solution for at least 5 seconds and less than or equal to 5 minutes.
  • the method comprises washing the capture substrate/structure with a hypotonic solution.
  • the exposing step occurs prior to the step of introducing the plurality of capture structures and the plurality of metal-containing particles. In some embodiments of the methods described above and herein, the exposing step occurs after the step of introducing the plurality of capture structures and the plurality of metal-containing particles.
  • exposing the analyte from the analyte-containing biological particles comprises adding a lysing solution to release the analyte from the analyte-containing biological particles.
  • exposing the analyte from the analyte-containing biological particles comprises mechanical agitation or shearing.
  • removing any components not attached to the capture substrate/structure comprises magnetic separation and/or washing.
  • the sample is whole blood, serum, plasma, urine, sputum, sweat, and/or other biological fluids.
  • the fluid or sample is whole blood.
  • the fluid or sample is plasma.
  • the analyte-containing biological particle is a virion, a bacterium, a protein complex, an exosome, a cell, or fungi.
  • the analyte is an antigen, a protein, a lipid, a glycolipid, nucleic acid, an amino acid, membrane protein (e.g., from a bacterium), a hormone, a small molecule, a metabolite, or a drug.
  • the plurality of metal-containing particles comprises mutiple types of metals.
  • the plurality of metal-containing particles may comprise one or more of, or two or more of, a first type of metal-containing particle, a second type of metal-containing particle, and a third type of metal-containing particle.
  • Four or more (e.g., 5, 6, 7, 8, 9, 10) types of metal-containing particles are also possible.
  • the first type of metal-containing particle comprises silver, copper, cobalt, cadmium, lead, zinc, tin, nickel, bismuth, platinum, manganese, chromium, gold, alloys thereof, oxides thereof, sulfides thereof, or combinations thereof.
  • the second type of metal-containing particle comprises silver, copper, cobalt, cadmium, lead, zinc, tin, nickel, bismuth, platinum, manganese, chromium, gold, alloys thereof, oxides thereof, sulfides thereof, or combinations thereof.
  • the first type of metal- containing particle comprises silver.
  • the second type of metal-containing particle comprises gold or copper oxide.
  • the device comprises a source of metal-containing particles present in, or configured to be introduced into, the first reaction chamber, wherein the source of metal containing particles comprises one or more of a first type of metal-containing particle, a second type of metal-containing particle, and a third type of metal-containing particle.
  • the metal-containing particles comprise two or more of a first type of metal-containing particle, a second type of metal-containing particle, and a third type of metal-containing particle.
  • the method comprises forming one or more of a first bound complex comprising a capture structure and a first type of metal-containing particle, a second bound complex comprising a capture structure and a second type of metal-containing particle, and a third bound complex comprising a capture structure and a third type of metal-containing particle. In some embodiments of the methods described above and herein, the method comprises separating any unbound metal-containing particles from the first bound complex, the second bound complex, and/or the third bound complex. In some embodiments of the methods described above and herein, the method comprises determining the amount of the first analyte, the second analyte, and/or the third analyte.
  • the method applying the electric potential to oxidize at least a portion of the metal from the metal-containing particles directly oxidizes two or more of the first type of metal-containing particle, the second type of metal-containing particle, and the third type of metal-containing particle.
  • applying the electric potential to oxidize at least a portion of the metal from the first type of metal-containing particle does not oxidize the metal from the second type of metal-containing particle, or the metal from a third type of metal-containing particle.
  • the first, second, and/or third type of metal-containing particles are directly oxidized with the applied potential without the use of an oxidizing agent.
  • the first type of metal-containing particle comprises silver, copper, cobalt, cadmium, lead, zinc, tin, nickel, bismuth, platinum, manganese, chromium, gold, alloys thereof, oxides thereof, sulfides thereof, or combinations thereof.
  • the second type of metal-containing particle comprises silver, copper, cobalt, cadmium, lead, zinc, tin, nickel, bismuth, platinum, manganese, chromium, gold, alloys thereof, oxides thereof, sulfides thereof, or combinations thereof.
  • the first type of metal-containing particle comprises silver.
  • the second type of metal-containing particle comprises gold.
  • the method or device further comprises introducing, to the first analyte, the second analyte, and a third analyte different than the first and second analytes, a third type of metal-containing particle different from the first and second types of metal-containing particles.
  • the third type of metal-containing particle comprises silver, copper, cobalt, cadmium, lead, zinc, tin, nickel, bismuth, platinum, manganese, chromium, gold, alloys thereof, oxides thereof, sulfides thereof, or combinations thereof.
  • the third type of metal-containing particle comprises copper oxide.
  • FIG. 1 is a schematic illustration of a device for determining an analyte, according to one set of embodiments
  • FIG. 2 is a schematic illustration of a device for determining an analyte, according to one set of embodiments
  • FIG. 3 is a schematic illustration of a device for determining an analyte, according to one set of embodiments
  • FIG. 4 is a schematic illustration of a device for determining an analyte, according to one set of embodiments
  • FIG. 5 is a schematic illustration of a device for determining an analyte, according to one set of embodiments
  • FIG. 6 is a process flow diagram of a method for determining an analyte, according to one set of embodiments
  • FIG. 7 A is a schematic illustration of a device for determining an analyte, according to one set of embodiments
  • FIG. 7B is a schematic illustration of a device for determining an analyte, according to one set of embodiments.
  • FIG. 7C is a schematic illustration of an electrode, according to one set of
  • FIG. 8 is a schematic illustration of a bound complex comprising a capture structure, a metal-containing particle, and an analyte, according to one set of embodiments;
  • FIG. 9 is a schematic drawing of an exemplary method for determining and/or quantifying an analyte, according to some embodiments.
  • FIG. 10 is a schematic drawing of an exemplary method for determining and/or quantifying an analyte, according to some embodiments.
  • FIG. 11 is a schematic drawing of an exemplary method for determining and/or quantifying an analyte, according to some embodiments.
  • FIG. 12A is a plot demonstrating the efficiency of HCV virion capture from blood samples as measured by RT-PCR, according to one set of embodiments
  • FIG. 12B is a plot demonstrating the cycle threshold from RT-PCT versus the concentration of virions in the sample, according to one set of embodiments;
  • FIG. 12C is a plot of peak current area ⁇ C) versus concentration of HIV core antigen p24 (pM), according to one set of embodiments;
  • FIG. 12D is a plot of current versus voltage for various concentrations of HCV core antigen cAg, according to one set of embodiments.
  • FIGs. 12E-12F are plots of peak current area ⁇ C) versus HCV core antigen concentration (pM), according to one set of embodiments;
  • FIG. 12G is a plot of peak current area ⁇ C) for HCV clinical samples subjected to acid lysis, according to one set of embodiments;
  • FIG. 13 is a plot of current versus voltage for various concentrations of HIV core antigen p24, according to one set of embodiments
  • FIG. 14 is a plot of HIV core antigen p24 concentration (fM) versus peak current area ⁇ C), according to one set of embodiments;
  • FIG. 15 is a plot of HIV core antigen p24 concentration (fM) versus peak current area
  • FIG. 16 is a plot of current versus voltage for various concentrations of HIV core antigen p24, according to one set of embodiments.
  • FIG. 17 is a plot of particle size versus peak current area ( ⁇ ), according to one set of embodiments.
  • FIG. 18 is a plot of various HIV core antigen samples versus peak current area ⁇ C), according to one set of embodiments.
  • FIGs. 19-20 are plots of current versus voltage for (A) an NH 4 SCN electrolyte and (B) a NaCl electrolyte, according to some embodiments;
  • FIG. 21 is a plot of release of a metal-containing particle in a NH 4 SCN electrolyte and a NaCl electrolyte, according to one set of embodiments;
  • FIGs. 22A-22C are plots of electrolyte concentration versus peak current area ⁇ C), for various concentrations of bound complexes, according to one set of embodiments;
  • FIGs. 22D-22F are plots of current versus voltage for various concentrations of bound complexes, according to one set of embodiments;
  • FIG. 23 is a schematic illustration of a system for determining an analyte, according to one set of embodiments;
  • FIG. 24A is a plot of current versus voltage cycling for Ag/AgCl Only, Ag/AgCl Tip Over Carbon, Ag/AgCl Under Carbon, and Carbon Only reference electrodes, according to one set of embodiments;
  • FIG. 24B are plots of voltage versus time for carbon only and silver under carbon electrodes, according to one set of embodiments.
  • FIG. 24C is a plot of current versus voltage for detection of 500 fM of silver in solution, according to one set of embodiments.
  • FIGs. 25A-25D are plots of current versus voltage for ASV of solutions containing exemplary metal-containing particles, according to one set of embodiments.
  • Devices and methods for determining and/or quantifying one or more analytes in fluids are generally provided.
  • the devices described herein may be designed such that two or more operations (e.g., lysing, magnetic separation, resuspension, mixing, and/or reacting) are conducted within a single reaction chamber.
  • two or more operations e.g., lysing, magnetic separation, resuspension, mixing, and/or reacting
  • the device comprises two or more such reaction chambers.
  • the device may comprise two or more reaction chambers, each reaction chamber comprising various regions for mixing, lysing, binding, and/or detection.
  • the devices and methods described herein may be useful for determining and quantifying relatively low concentrations of one or more analytes present in a patient sample (e.g., a droplet of whole blood, a droplet of plasma, a droplet of serum (e.g., for troponin)).
  • the analyte(s) may be quantified, in some cases, by binding the analyte(s) to a plurality of metal-containing particles and measuring current by changing a voltage on a working electrode.
  • the device is a microfluidic device (e.g., comprising one or more microfluidic channels).
  • the analyte is an antigen for HIV, hepatitis C virus, or a protein (e.g., a cardiac marker such as Troponin I).
  • the reaction chamber may be configured for receiving, introducing, and/or exposing a plurality of capture structures (e.g., magnetic particles) and/or a plurality of metal-containing particles (e.g., silver particles, gold particles, copper oxide particles) to a fluid comprising the analyte(s) (and/or analyte-containing biological particle(s)).
  • a plurality of capture structures e.g., magnetic particles
  • metal-containing particles e.g., silver particles, gold particles, copper oxide particles
  • the analyte(s) or analyte-containing biological particle(s) may bind with both a capture structure and a metal-containing particle to form a bound complex which may then be subjected to conditions (e.g., electrochemical conditions) that allow quantification of the analyte (e.g., based on the amount of metal-containing particles present).
  • conditions e.g., electrochemical conditions
  • each of the analytes may bind with both a capture structure and a metal-containing particle of a different type (e.g., a first type of metal-containing particle, a second type of metal-containing particle, a third type of metal-containing particle) to form different bound complexes (e.g., a first bound complex, a second bound complex, a third bound complex).
  • a metal-containing particle of a different type e.g., a first type of metal-containing particle, a second type of metal-containing particle, a third type of metal-containing particle
  • bound complexes e.g., a first bound complex, a second bound complex, a third bound complex
  • the analyte(s) (and/or analyte-containing biological particle(s)) may be exposed (e.g., introduced to a lysing solution such that the analyte is lysed from the analyte- containing biological particle) such that, for example, one or more antigen(s) is released from the analyte-containing biological particle(s).
  • a magnetic element may be present in the reaction chamber.
  • the devices and methods described herein may, in some embodiments
  • embodiments permit the analysis of analytes from whole blood without additional filtering or separation steps, utilize materials and/or steps that do not require the release of the analyte from the bound complex, and/or have relatively high sensitivity as compared to certain existing analyte quantification methods.
  • one or more such steps may be performed in combination with other steps and/or with particular device configurations described herein.
  • all the operations e.g., reactions
  • all the operations necessary to isolate, detect, and quantify an analyte are present and/or conducted on/in the device.
  • more than one, or all, reactions and/or sample preparation steps performed before detection of the analyte are conducted within a single reaction chamber such that each reaction or step can be independently controlled (e.g., by controlling fluid flow into and out of the reaction chamber).
  • each reaction or step can be independently controlled (e.g., by controlling fluid flow into and out of the reaction chamber).
  • This configuration may advantageously result in less "loss" of material that may otherwise occur from transporting the materials into different chambers of the device.
  • each reaction is conducted in a different reaction chamber such that each reaction chamber can be independently addressed (e.g., by controlling fluid flow into and out of each region independently).
  • the device may comprise one or more microfluidic channels in fluidic communication with a reaction chamber (e.g., see FIGs. 1-5).
  • a device 100 comprises a microfluidic channel 110 in fluidic communication with a reaction chamber 120.
  • a reservoir 130 containing a stored reagent is in fluidic communication with a reaction chamber 120.
  • the reservoir may be configured to introduce the stored reagent into the reaction chamber.
  • the reservoir may be sealed (e.g., to prevent evaporation and/or contamination of any components), and the stored reagent may be released into the reaction chamber upon unsealing of the reservoir.
  • the device may comprise two or more reservoirs in fluidic communication with the reaction chamber, as described in more detail below.
  • device 100 further comprises a detection region 140 in fluid
  • reaction chamber e.g., for quantifying an analyte
  • device 102 comprises a magnetic element 125 present in, or adjacent (e.g., directly adjacent) reaction chamber 120.
  • the magnetic element may be configured to be introduced into the reaction chamber, e.g., during fabrication of the device and/or during use of the device.
  • the magnetic element may be activated (e.g., brought proximate the reaction chamber, turned on such that a magnetic field is present in the reaction chamber, or otherwise magnetized) such that any magnetic particles present in the reaction chamber are drawn towards the magnetic element.
  • the reaction chamber may contain an analyte bound to a magnetic particle such that, upon activation of the magnetic element, the analyte bound to a magnetic particle is drawn towards the magnetic element.
  • any particles not drawn towards the magnetic element may be removed from the reaction chamber (e.g., by flowing a fluid such as a wash agent into and/or through the reaction chamber).
  • a component when referred to as being "adjacent" another component, it can be directly adjacent to the component, or one or more intervening component also may be present. A component that is "directly adjacent" another component means that no intervening component is present.
  • the magnetic particle may be, in some cases, paramagnetic or superparamagnetic. That is to say, in some embodiments, the magnetic particles may not be magnetized except in the presence of a magnetic field.
  • the device comprises one or more sources of one or more agents and/or substrates in fluidic communication with the reaction chamber.
  • device 104 comprises a source 150 of capture substrates/capture structures (e.g., magnetic particles) and a source 160 of one or more agents (e.g., a lysing solution), each connected to and/or in fluidic communication with reaction chamber 120.
  • Source 150 may be configured, in some embodiments, to introduce a plurality of capture substrates into reaction chamber 120.
  • source 160 may be configured to introduce one or more agents into reaction chamber 120.
  • the source of capture substrates and/or source of one or more agents may be present or contained in the reaction chamber.
  • the reaction chamber may comprise one or more regions (e.g., within the reaction chamber) for housing the capture substrates and/or one or more agents which may be released from the reaction chamber (e.g., upon opening of a valve, upon application of pressure to the region, upon application of vacuum to the region).
  • each of sources 150 and 160 contain reagents that are used to interact with (e.g., react with, bind with) an analyte present in the reaction chamber. Additional sources may be connected to/in fluid communication with the reaction chamber (not shown). In some embodiments, the sources may be connected to the reaction chamber through one or more fluidic (e.g., microfluidic) channels.
  • FIG. 4 shows exemplary device 200 comprising a plurality of microfluidic channels (e.g., microfluidic channel 210), a reaction chamber 220, an inlet channel 230 (in fluidic communication with a first reservoir, not shown) and inlet channel 232 (in fluidic communication with a second reservoir, not shown) in fluidic communication with reaction chamber 220, source of capture substrates 250, source of metal particles 255 (e.g., a source of metal particles of a first type, a second type, and/or a third type), and detection region 240 downstream of reaction chamber 220.
  • exemplary device 200 comprises a waste channel 260 in fluidic communication with reaction chamber 220.
  • a waste region 265 may be in fluidic
  • exemplary device 200 comprises plasma separation region 270 upstream of reaction chamber 220.
  • the device may comprise one or more caps (e.g., cap 280) for reversibly sealing one or more regions of the device.
  • cap 280 may be used to reversibly seal plasma separation region 270 (e.g., to prevent contamination when the device is not in use).
  • Other configurations are also possible and are described in more detail herein.
  • the device(s) described herein may be used for determining and/or quantifying one or more analytes in fluids.
  • quantification and/or detection of an analyte in a device as described herein may comprise performing, within the reaction chamber of the device, one or more adding steps, a separation step, a resuspension step, an exposure step, and/or one or more application of electric potential steps, amongst others. For instance, referring now to FIG. 6, one or more steps of method 500 may be conducted in the reaction chamber of the device.
  • a plurality of metal-containing particles 510 may be added in step 515 and a plurality of capture structures 530 may be added in step 535 to a fluid comprising an analyte 520 present in the reaction chamber of the device.
  • a plurality of metal-containing particles 510 e.g., a first type of metal-containing particle, a second type of metal-containing particle, a third type of metal-containing particle
  • a plurality of capture structures 530 may be added in step 535 to a fluid comprising an analyte 520 present in the reaction chamber of the device.
  • adding step 515 and adding step 535 may take place substantially
  • adding step 515 and adding step 535 may occur at different times, as described above.
  • Adding step 515 and/or adding step 535 may comprise, in some embodiments, flowing a plurality of metal-containing particles and/or a plurality of capture substrates from a reservoir or source connected to and/or in fluidic communication with the reaction chamber, such that the plurality of metal-containing particles and/or the plurality of capture substrates are introduced into the reaction chamber, such that the analyte forms a mixture of bound complexes and unbound components 540.
  • a plurality of metal-containing particles may include more than one type of metal-containing particles in some embodiments.
  • a plurality of metal-containing particles comprises one or more of a first type of metal-containing particle, a second type of metal-containing particle, and a third type of metal-containing particle.
  • a plurality of metal-containing particles comprises two or more of a first type of metal- containing particle, a second type of metal-containing particle, and a third type of metal- containing particle.
  • a plurality of metal-containing particles comprises each of a first type of metal-containing particle, a second type of metal-containing particle, and a third type of metal-containing particle.
  • each type of metal-containing particle may be configured to bind with a different type of analyte (e.g., antigen) in a sample (or multiple samples).
  • analyte e.g., antigen
  • method 500 comprises separating, via separating step 545, any unbound components from the bound complex in the reaction chamber.
  • the methods described herein can be used to quantify analytes present in a fluid such as whole blood, serum, plasma, urine, sputum, sweat, and/or other biological fluids.
  • Whole blood for example, is generally challenging to analyze with traditional qualification methods without additional filtration, separation, and/or dilution steps, since such steps may, for example, inadvertently remove and/or damage the analyte.
  • the capture structure comprises a magnetic particle and a magnetic field and/or magnetic element positioned adjacent the reaction chamber, such that a magnetic field may be applied to the mixture of bound complexes and unbound components 540.
  • the bound complexes comprising the capture structure may be attracted to, and move towards, the magnet and/or magnetic field.
  • the unbound components may then be separated (e.g., via aspiration and/or removal of the supernatant comprising the unbound components) from the bound complexes.
  • the separating step comprises separating any unbound metal-containing particles from the bound complex.
  • the separating step produces a plurality of bound complexes 550 with substantially no unbound metal-containing particles (e.g., less than 5 wt%, less than 2 wt%, less than 1 wt%, less than 0.5 wt%, less than 0.1 wt% unbound metal-containing particles versus the total bound complex weight).
  • the plurality of bound complexes may comprise, in some embodiments, a first type of bound complex comprising a first analyte and a first type of metal-containing particle, a second type of bound complex comprising a second analyte and a second type of metal-containing particle, and/or a third type of bound complex comprising a third analyte and a third type of metal-containing particle.
  • the weight percent of unbound metal-containing particles may be determined, for example, by collecting the fluid not including the bound complex and performing dynamic light scattering to quantify the concentration of unbound metal-containing particles in the collected fluid.
  • a sample (e.g., a droplet of whole blood, a droplet of plasma) comprising a plurality of analyte-containing biological particles may be introduced into the device (e.g., into an inlet of the device).
  • at least a portion of the sample may enter (e.g., flow), via the inlet, into at least one microfluidic channel of the device.
  • at least a portion of the sample may be introduced into microchannel 110.
  • At least a portion of the sample may be subsequently flowed (e.g., by applying a pressure to the fluid contained within microfluidic channel 110) or drawn (e.g., by applying a vacuum to the chamber 120) into reaction chamber 120.
  • the plurality of analyte-containing biological particles are reacted (e.g., lysed) within the reaction chamber such that an analyte contained within the analyte-containing biological particles is exposed (e.g., available for further reaction/binding, or isolated from other portions of the biological particle).
  • a plurality of metal-containing particles and a plurality of capture substrates/capture structures may be introduced to the (exposed) analyte. In some such embodiments, at least a portion of the plurality of metal-containing particles and a portion of the plurality of capture
  • substrates/capture structures bind with the analyte such that the analyte forms a bound complex with the metal-containing particles and capture substrates/capture structures.
  • a sample suspected of containing the hepatitis C virion i.e., the analyte-containing biological particle
  • the hepatitis C virion may then be lysed (e.g., by introducing a lysis solution into the reaction chamber flowed from a reservoir or source in fluidic communication with and/or connected to the reaction chamber containing the lysis solution) such that cAg or other HCV antigen (i.e., the analyte) is exposed (e.g., available for binding).
  • the exposed analyte is capable of forming a bound complex with a capture structure and a metal- containing particle. Bound complexes are described in more detail, below.
  • a plurality of magnetic particles may be introduced to the analyte-containing biological particles (e.g., the sample comprising the analyte) such that the analyte-containing biological particles bind with the plurality of magnetic particles.
  • the analyte-containing biological particles e.g., the sample comprising the analyte
  • at least a portion of the plurality of magnetic particles may bind to the analyte-containing biological particles and do not bind to particles that do not contain the analyte (e.g., non-analyte containing particles).
  • any non-analyte containing particles and/or unbound metal-containing particles may be separated from the bound complex and/or may be removed from the reaction chamber.
  • the magnetic element e.g., magnetic element 125 in FIG. 2
  • the magnetic element may be activated such that the plurality of magnetic particles are attracted to the analyte-containing biological particles (e.g., via the activated magnetic field generated by the magnetic element) and the particles which do not bind to the plurality of magnetic particles may be removed (e.g., washed) from the reaction chamber.
  • analyte-containing biological particles bound to the plurality of magnetic particles may remain in the reaction chamber.
  • the plurality of analyte-containing biological particles are not first reacted, as described above, to expose an analyte contained within the analyte-containing biological particles.
  • a plurality of metal- containing particles and/or a plurality of capture substrates/capture structures may be introduced to the analyte-containing biological particles.
  • at least a portion of the plurality of metal-containing particles and/or at least a portion of the plurality of capture substrates/capture structures bind with the analyte- containing biological particles.
  • a sample suspected of containing the HIV virion i.e., the analyte-containing biological particle
  • a capture substrate e.g., magnetic particles
  • a plurality of capture substrates/capture structures bind with at least a portion of the analyte-containing biological particles such that particles not bound to the plurality of capture substrates/capture structures may be removed (e.g., washed) from the reaction chamber.
  • An analyte e.g., an analyte contained within the analyte-containing biological particle
  • Analyte-containing biological particle may then be exposed (e.g., isolated from other portions of the analyte- containing biological particle), in some cases, as described herein.
  • the analyte is present in the sample and is in a suitable form for binding and/or analysis (e.g., a protein circulating free in blood or in a complex in blood, such as cardiac troponin).
  • a suitable form for binding and/or analysis e.g., a protein circulating free in blood or in a complex in blood, such as cardiac troponin.
  • the analyte in the sample is not contained within an analyte-containing biological particle.
  • no lysing step is performed (e.g., a lysis solution is not introduced into the reaction chamber).
  • a sample suspected of containing the analyte e.g., an already exposed analyte
  • a plurality of metal-containing particles and/or a plurality of capture substrates/capture structures may be introduced to the analyte such that the analyte forms a bound complex with the metal-containing particles and capture substrates/capture structures.
  • particles not bound to the plurality of metal-containing particles and/or the plurality of capture substrate may be removed (e.g., washed) from the reaction chamber.
  • washing the analyte-containing biological particles and/or the exposed analyte comprises introducing a washing agent into the reaction chamber, e.g., where one or more of the processing steps (e.g., binding, reaction, separation, and/or exposing steps) described herein occurred.
  • the washing agent is contained within a source (e.g., source 160 in FIG. 3) connected to and/or in fluidic communication with the reaction chamber (e.g., reaction chamber 120 in FIG. 3).
  • the device comprises a waste channel connected to and/or in fluidic communication with the reaction chamber such that a wash buffer containing unbound particles may be flowed into the waste channel.
  • the device comprises a reaction chamber and/or detection region in which the bound complex may be introduced (e.g., exposed) to an electrolyte (e.g., an electrolyte that may facilitate detection of the analyte).
  • an electrolyte e.g., an electrolyte that may facilitate detection of the analyte.
  • the electrolyte is introduced in the reaction chamber (e.g., via a reservoir or source containing the electrolyte and in fluidic communication with the reaction chamber).
  • the electrolyte may be introduced into the detection region (e.g., via a reservoir or source containing the electrolyte and in fluidic communication with the detection region).
  • the method may be performed such that the step of introducing the electrolyte to the bound complex does not release the analyte from the metal-containing particle and/or the capture structure.
  • bound complex 550 may undergo introduction step 565 in the reaction chamber or in the detection region.
  • the introduction step comprises exposing bound complex 550 to electrolyte 560, e.g., by adding the electrolyte to a fluid containing the bound complexes.
  • exposing step 565 may occur simultaneously with adding step 515 and/or adding step 535. That is to say, in some embodiments, the electrolyte 560 may be added to, or initially present in, the fluid comprising the analyte 520, prior to separating step 545. In other embodiments, the electrolyte may be added to, or initially present in, the fluid comprising the metal-containing particles and/or comprising the capture structures.
  • electrolyte 560 may be added to the mixture comprising the bound complex and unbound components 540.
  • bound complex 550 may be exposed to electrolyte 560 after separating step 545.
  • method 500 comprises resuspending the bound complexes, via a resuspension step (not shown in FIG. 6) prior to separating step 545 and/or prior to electric potential application step 555, by introducing a fluid (e.g., a lysing solution, a washing buffer, an electrolyte) into the reaction chamber such that the bound complexes are resuspended in the fluid.
  • a fluid e.g., a lysing solution, a washing buffer, an electrolyte
  • the electrolyte may be designed to facilitate detection of the analyte.
  • the electrolyte is used to conduct ions across electrodes in an electrochemical detection method, as described in more detail below.
  • the electrolyte does not remove the metal-containing particle from the bound complex upon introduction of the electrolyte to the bound complex.
  • at least 90%, at least 95%, at 98%, at least 99%, or 100% of the metal-containing particles are not removed from the bound complex upon introduction of the electrolyte.
  • the device comprises a plasma separation region upstream of the reaction chamber.
  • device 106 comprises a plasma separation region 170 upstream, and connected to and/or in fluidic communication with, reaction chamber 120.
  • the plasma separation region comprises a membrane for separating plasma from the sample (e.g., comprising removing whole blood cells from the sample).
  • plasma separation of the sample is conducted prior to inserting the sample suspected of containing the analyte into the device.
  • suitable methods such as centrifugation, for separating plasma from a sample (e.g., a whole blood sample).
  • no plasma separation step is conducted (e.g., a whole blood sample is introduced into the device).
  • the plasma separation region comprises a membrane for separating red blood cells from plasma and analyte.
  • the membrane is a porous membrane.
  • the membrane comprises a gradient in size of pores through the thickness of the membrane (e.g., with larger pores at a first side of the membrane and smaller pores on a second side of the membrane, opposite the first side).
  • the membrane has a relatively uniform pore size through the thickness of the membrane (e.g., having a mean pore size smaller than the size of blood cells such as human blood cells).
  • the plasma separation region comprises one or microfluidic and/or centrifugation configurations or components for separating plasma from whole blood (e.g., a narrow channel, such as a nanochannel).
  • the device may comprise a single reaction chamber (e.g., for conducting all reaction(s) and/or method steps involving the analyte prior to detection/quantification of the analyte in the detection region).
  • a single reaction chamber e.g., for conducting all reaction(s) and/or method steps involving the analyte prior to detection/quantification of the analyte in the detection region.
  • the reaction chamber comprises one or more regions for carrying out different parts of the processing of an analyte leading up to detection.
  • the reaction chamber comprises one or more of a sample introduction region, a mixing region, a lysis region, a washing region, a binding region, a waste region, and/or a detection region.
  • device 100 comprises a reaction chamber 120 comprising sample introduction region 180, mixing region 182, lysis region 184, washing region 186, and binding region 188.
  • Each region may overlap with another region in the reaction chamber.
  • the entire reaction chamber can be used for sample introduction, mixing, lysis, washing, and/or binding.
  • region may be located in a different position within the reaction chamber (i.e., such that the regions do not overlap).
  • the device may comprise two or more reaction chambers (e.g., a first reaction chamber, a second reaction chamber) for carrying out different parts of the processing of an analyte leading up to detection.
  • each reaction chamber is configured to perform one step of a method involved in determining and/or quantifying an analyte in a fluid suspected of containing the analyte. For example, as illustrated in FIG.
  • device 600 comprises at least one microfluidic channel (e.g., microfluidic channel 605) and reaction chambers 610, 620, 630, 640, and 650 each connected to and/or in fluidic communication with one another (e.g., a second reaction chamber 620 is downstream of a first reaction chamber 610 and connected to and/or in fluidic
  • microfluidic channel e.g., microfluidic channel 605
  • reaction chambers 610, 620, 630, 640, and 650 each connected to and/or in fluidic communication with one another (e.g., a second reaction chamber 620 is downstream of a first reaction chamber 610 and connected to and/or in fluidic
  • each reaction chamber (e.g., the first reaction chamber, the second reaction chamber, etc.) in the device comprises one or more of a sample introduction region, a mixing region, a lysis region, a washing region, a binding region, a waste region, and a detection region.
  • reaction chamber 610 comprises a sample introduction region
  • reaction chamber 620 comprises a mixing region
  • reaction chamber 630 comprises a lysis region
  • reaction chamber 640 comprises a binding region
  • reaction chamber 650 comprises a detection region.
  • each reaction chamber e.g., the first reaction chamber, the second reaction chamber, etc.
  • each reaction chamber may be connected to and/or in fluidic communication with an inlet channel (e.g., a reagent inlet).
  • inlet channel 612 may be in fluidic communication with first reaction chamber 610
  • inlet channel 622 may be in fluidic communication with second reaction chamber 620
  • inlet channel 632 may be in fluidic communication with third reaction chamber 630
  • inlet channel 642 may be in fluidic communication with fourth reaction chamber 640
  • inlet channel 652 may be in fluidic communication with fifth reaction chamber 650.
  • each inlet channel may be in fluidic communication with a reservoir or source comprising a stored reagent (e.g., a washing agent, a lysis buffer, a plurality of magnetic particles, a plurality of metal-containing particles, a plurality of capture substrates, an electrolyte).
  • a stored reagent e.g., a washing agent, a lysis buffer, a plurality of magnetic particles, a plurality of metal-containing particles, a plurality of capture substrates, an electrolyte.
  • the stored reagent may be stored within the reservoir or the source (e.g., sealed to prevent evaporation and/or contamination of the reagent).
  • the reservoir or source is positioned adjacent the inlet channel.
  • a valve, foil, or other obstruction may prevent the reagent inside the reservoir or source from being in fluid communication with other reagents during storage.
  • the reservoir or source may be configured to introduce the stored reagent into the inlet channel upon unsealing of the reservoir or source. Unsealing of the reservoir or source may comprise, in some
  • a source may be positioned within a reaction chamber (e.g., a reaction chamber comprises a source positioned within the reaction chamber containing a plurality of metal-containing particles).
  • the stored reagent may be in the form of a fluid (e.g., liquid), a dry reagent (e.g., a lyophilized reagent, an adsorbed reagent), a suspension (e.g., a suspension of particles), or any other suitable form.
  • the device comprises a waste channel (e.g., a waste channel in fluidic communication with a waste region.)
  • the waste channel may be in fluidic communication with one or more reaction chambers (e.g., in fluidic communication with two or more reaction chambers).
  • excess reagents and/or other fluids or reaction byproducts may be transferred from one or more reaction chambers to the waste region via the waste channel.
  • FIG. 7B shows device 600 comprising waste channel 618 in fluidic communication with first reaction chamber 610 via connecting channel 624.
  • waste channel 618 may be in fluidic communication with second reaction chamber 620 via connecting channel 634, in fluidic communication with third reaction chamber 630 via connecting channel 644, in fluidic communication with fourth reaction chamber 640 via connecting channel 654, and/or in fluidic communication with fifth reaction chamber 650 via connecting channel 664.
  • one or more connecting channels may intersect with one or more inlet channels.
  • inlet channel 622 may intersect with connecting channel 624
  • inlet channel 632 may intersect with connecting channel 634
  • inlet channel 642 may intersect with connecting channel 644, and/or inlet channel 652 may intersect with connecting channel 654.
  • a valve, foil, or other obstruction may prevent the fluid (or reagent) in a reaction chamber (e.g., the first reaction chamber) from entering a connecting channel in fluidic communication with the waste channel, until a desired time for transferring the fluid to the waste channel.
  • a reaction chamber may comprise a sample introduction region.
  • a sample may be introduced directly into the reaction chamber of the device, where it may be processed (e.g., subjected to exposure to metal-containing particles, magnetic particles, lysis, isolation, and/or other steps described herein) without substantially flowing into a channel or chamber downstream of the reaction chamber as described herein.
  • the sample introduction region may be configured to prepare the sample for further reaction steps and/or detection steps.
  • the sample introduction region comprises a source of an anticoagulant (e.g., for anticoagulation of a whole blood sample).
  • the sample introduction region may be configured to isolate the analyte-containing biological particles.
  • a plurality of capture substrates/capture structures may be introduced (e.g., in the presence of a capture buffer) such that the capture substrates/capture structures bind to the analyte or the analyte-containing biological particles.
  • any unbound particles not bound to the plurality of capture substrates/capture structures may be removed from the sample introduction region.
  • a magnetic element positioned proximate the sample introduction region may be activated such that a plurality of magnetic capture substrates/capture structures are isolated from the sample and the unbound particles may be washed out of the sample introduction region.
  • the sample introduction region may comprise a quality control component (e.g., quality control electrodes).
  • the quality control component detects the presence of a fluid in the sample introduction region (e.g., indicating that the device is working properly).
  • a quality control component includes at least two electrodes spaced apart from one another and the timing of detection of a fluid from each of the electrodes indicates whether the fluids are in the correct chambers at the right time.
  • the quality control elements may be present in other portions of the device in some embodiments (e.g., in one or more of a sample introduction region, a mixing region, a lysis region, a washing region, a binding region, a waste region, and/or a detection region).
  • the quality control element may also be positioned in a channel (e.g., a microchannel) to indicate that the sample or a reagent has reached a particular location in the microchannel (e.g., confirming that it is present at both the correct location and at the correct time in the progression of the assay).
  • optical devices e.g., a camera
  • a reaction chamber comprises a mixing region.
  • the reaction chamber comprising the mixing region is positioned downstream of the sample introduction region.
  • the mixing region may be used to facilitate, for example, attaching at least a portion of the analyte-containing biological particles to one or more capture substrates/capture structures.
  • the mixing region may be used for mixing a plurality of metal-containing particles and a plurality of capture
  • the mixing region may comprise any suitable method for mixing a fluid.
  • the mixing region comprises a propeller, a shear generating mechanism, a vibration generating mechanism (e.g., a vibrating actuator), a magnetic element, and/or a serpentine channel such that two or more reagents within the mixing region are mixed together.
  • a reaction chamber comprises a lysis region.
  • the lysis region may be positioned downstream of the mixing region.
  • a lysing solution may be present in a reservoir or source in fluidic communication with the lysis region such that, upon introduction of the sample into the lysis region, the analyte- containing biological particles present within the sample are reacted with the lysis solution and the analyte is exposed.
  • a reagent inlet may be positioned between the mixing region and the lysis region (e.g., for introduction of the lysis solution into the lysis region).
  • the analyte is exposed from the analyte-containing biological particles such that the analyte is available to form a bound complex (e.g., unhindered).
  • exposing the analyte from the analyte-containing biological particle comprises introducing to the lysis region a lysis solution and/or heating the lysis region (e.g., via an electrode located proximate the lysis region).
  • a valve, foil, or other obstruction may prevent the fluid(s) inside the reaction chamber from being in fluid communication with a downstream reaction chamber until a desired time at which the fluid(s) are to be fluidically transferred to the downstream reaction chamber (e.g., the valve, foil, or other obstruction may be opened and/or removed).
  • a reaction chamber comprises a binding region.
  • the binding region may be positioned downstream of the lysis region.
  • the binding region may be used, in some cases, for introducing, to the (exposed) analyte, a plurality of metal-containing particles and a plurality of capture structures such that the analyte binds with both a capture structure and a metal-containing particle to form the bound complex.
  • a plurality of metal-containing particles and a plurality of capture substrates/capture structures may be introduced into the binding region (e.g., via a reagent inlet in fluidic communication with the binding region) such that an analyte present in the sample introduction region forms a bound complex. Binding may take place in solution. However, in some embodiments, at least one of the components (e.g., a magnetic particle) is stationary during the binding step.
  • a reaction chamber comprises a detection region.
  • the detection region may be positioned downstream of the binding region. In alternative embodiments, however, the detection region is positioned downstream of the reaction chamber (e.g., as illustrated in FIG. 1).
  • the detection region and reaction chamber may be connected to one another via a channel (e.g., a microfluidic channel).
  • the detection region may be used for detecting and/or quantifying the analyte present in the sample.
  • the detection region may comprise an electrode (e.g., a working electrode) associated with the detection region.
  • detecting and/or quantifying the analyte comprises exposing the bound complex to an electrolyte (e.g., an electrolyte present in a reservoir or source sealed and positioned adjacent a reagent inlet in fluidic communication with the detection region), applying an electric potential to oxidize at least a portion of the metal from the metal-containing particles, applying an electric potential to deposit at least a portion of the metal onto a working electrode, and measuring current by changing a voltage on the working electrode to determine the amount of analyte present.
  • the detection region may comprise an anodic stripping voltammetry (ASV) detector.
  • ASV anodic stripping voltammetry
  • the bound complex may be flowed into a detection region (e.g., detection region 140 downstream of the reaction chamber 120 in FIG. 1) such that the analyte may be quantified.
  • the bound complex may be exposed to an electrolyte.
  • a reservoir containing the electrolyte may be in fluidic communication with the reaction chamber such that the electrolyte may be flowed into the reaction chamber and reacted with the bound complex.
  • the reservoir containing the electrolyte may be in fluidic communication with the detection such that the electrolyte may be flowed into the detection region and reacted with the bound complex.
  • Reaction chambers described herein may have any suitable shape (e.g., cross- sectional shape when viewed top-down).
  • suitable shapes for reaction chambers include triangular, square, rectangular, elliptical, circular, hexagonal, and polygonal. Other shapes are also possible.
  • the reaction chamber may have a particular internal volume (e.g., may contain a particular volume of fluid).
  • the reaction chamber has an internal volume of greater than or equal to 50 microliters, greater than or equal to 75 microliters, greater than or equal to 100 microliters, greater than or equal to 125 microliters, greater than or equal to 150 microliters, greater than or equal to 175 microliters, greater than or equal to 200 microliters, or greater than or equal to 225 microliters.
  • the reaction chamber has an internal volume of less than or equal to 250 microliters, less than or equal to 225 microliters, less than or equal to200 microliters, less than or equal to 175 microliters, less than or equal to 150 microliters, less than or equal to 125 microliters, less than or equal to 100 microliters, or less than or equal to 75 microliters. Combinations of the above-referenced ranges are possible (e.g., greater than or equal to 50 microliters and less than or equal to 250 microliters, greater than or equal to 50 microliters and less than or equal to 125 microliters). Other ranges are also possible.
  • the reaction chamber may have a particular width.
  • the reaction chamber has a width of greater than or equal to 6 mm, greater than or equal to 8 mm, greater than or equal to 10 mm, greater than or equal to 12 mm, greater than or equal to 14 mm, greater than or equal to 16 mm, or greater than or equal to 18 mm.
  • the reaction chamber has a width of less than or equal to 20 mm, less than or equal to 18 mm, less than or equal to 16 mm, less than or equal to 14 mm, less than or equal to 12 mm, less than or equal to 10 mm, or less than or equal to 8 mm.
  • the reaction chamber may have a particular length (e.g., measured orthogonal to the width). In some embodiments, the reaction chamber has a length of greater than or equal to 12 mm, greater than or equal to 15 mm, greater than or equal to 20 mm, greater than or equal to 23 mm, greater than or equal to 25 mm, greater than or equal to 30 mm, greater than or equal to 35 mm, greater than or equal to 40 mm, or greater than or equal to 45 mm.
  • the reaction chamber has a length of less than or equal to 50 mm, less than or equal to 45 mm, less than or equal to 40 mm, less than or equal to 35 mm, less than or equal to 30 mm, less than or equal to 25 mm, less than or equal to 20 mm, or less than or equal to 15 mm. Combinations of the above referenced ranges are possible (e.g., greater than or equal to 12 mm and less than or equal to 50 mm, greater than or equal to 12 mm and less than or equal to 23 mm). Other ranges are also possible.
  • the reaction chamber may have a particular depth.
  • the depth of the reaction chamber is greater than or equal to 0.15 mm, greater than or equal to 0.2 mm, greater than or equal to 0.25 mm, greater than or equal to 0.3 mm, greater than or equal to 0.4 mm, greater than or equal to 0.6 mm, greater than or equal to 0.8 mm, greater than or equal to 1 mm, greater than or equal to 1.2 mm, greater than or equal to 1.4 mm, greater than or equal to 1.6 mm, or greater than or equal to 1.8 mm.
  • the depth of the reaction chamber is less than or equal to 2 mm, less than or equal to 1.8 mm, less than or equal to 1.6 mm, less than or equal to 1.4 mm, less than or equal to 1.2 mm, less than or equal to 1 mm, less than or equal to 0.8 mm, less than or equal to 0.6 mm, less than or equal to 0.4 mm, less than or equal to 0.3 mm, less than or equal to 0.25 mm, or less than or equal to 0.2 mm. Combinations of the above-referenced ranges are possible (e.g., greater than or equal to 0.15 mm and less than or equal to 2 mm, greater than or equal to 0.25 mm and less than or equal to 1 mm). Other ranges are also possible.
  • the detection region may have any suitable shape (e.g., cross-sectional shape when viewed top- down).
  • suitable shapes for detection regions include triangular, square, rectangular, elliptical, circular, hexagonal, and polygonal. Other shapes are also possible.
  • the detection region may have a particular internal volume (e.g., may contain a particular volume of fluid).
  • the detection region has an internal volume of greater than or equal to 5 microliters, greater than or equal to 10 microliters, greater than or equal to 12 microliters, greater than or equal to 15 microliters, greater than or equal to 18 microliters, greater than or equal to 20 microliters, greater than or equal to 25 microliters, greater than or equal to 30 microliters, or greater than or equal to 35 microliters.
  • the detection region has an internal volume of less than or equal to 40 microliters, less than or equal to 35 microliters, less than or equal to 30 microliters, less than or equal to 25 microliters, less than or equal to 20 microliters, less than or equal to 18 microliters, less than or equal to 15 microliters, less than or equal to 12 microliters, or less than or equal to 10 microliters. Combinations of the above-referenced ranges are possible (e.g., greater than or equal to 5 microliters and less than or equal to 40 microliters, greater than or equal to 5 microliters and less than or equal to 20 microliters). Other ranges are also possible.
  • the detection region may have a particular width.
  • the reaction chamber has a length of greater than or equal to 1.5 mm, greater than or equal to 2 mm, greater than or equal to 2.5 mm, greater than or equal to 3 mm, greater than or equal to 3.5 mm, greater than or equal to 4 mm, greater than or equal to 4.5 mm, greater than or equal to 5 mm, or greater than or equal to 5.5 mm.
  • the reaction chamber has a length of less than or equal to 6 mm, less than or equal to 5.5 mm, less than or equal to 5 mm, less than or equal to 4.5 mm, less than or equal to 4 mm, less than or equal to 3.5 mm, less than or equal to 3 mm, less than or equal to 2.5 mm, or less than or equal to 2 mm. Combinations of the above-referenced ranges are possible (e.g., greater than or equal to 1.5 mm and less than or equal to 6 mm, greater than or equal to 1.5 mm and less than or equal to 3 mm). Other ranges are also possible.
  • the detection region may have a particular length (e.g., measured orthogonal to the width).
  • the reaction chamber has a width of greater than or equal to 8 mm, greater than or equal to 10 mm, greater than or equal to 12 mm, greater than or equal to 14 mm, greater than or equal to 15 mm, greater than or equal to 16 mm, greater than or equal to 18 mm, greater than or equal to 20 mm, greater than or equal to 22 mm, greater than or equal to 24 mm, greater than or equal to 26 mm, or greater than or equal to 28 mm.
  • the detection region has a length of less than or equal to 30 mm, less than or equal to 28 mm, less than or equal to 26 mm, less than or equal to 24 mm, less than or equal to 22 mm, less than or equal to 20 mm, less than or equal to 18 mm, less than or equal to 16 mm, less than or equal to 15 mm, less than or equal to 14 mm, less than or equal to 12 mm, or less than or equal to 10 mm. Combinations of the above- referenced ranges are possible (e.g., greater than or equal to 8 mm and less than or equal to 30 mm, greater than or equal to 8 mm and less than or equal to 15 mm). Other ranges are also possible.
  • the detection region may have a particular depth.
  • the depth of the reaction chamber is greater than or equal to 0.2 mm, greater than or equal to 0.25 mm, greater than or equal to 0.3 mm, greater than or equal to 0.4 mm, greater than or equal to 5 mm, greater than or equal to 0.6 mm, or greater than or equal to 0.8 mm.
  • the depth of the detection region is less than or equal to 1 mm, less than or equal to 0.8 mm, less than or equal to 0.6 mm, less than or equal to 0.5 mm, less than or equal to 0.4 mm, less than or equal to 0.3 mm, or less than or equal to 0.25 mm.
  • the device comprises one or more reservoirs or sources in fluidic communication with one or more reaction chambers.
  • the reservoir or source contains a stored reagent (e.g., a wash buffer, a lysis solution, a plurality of metal-containing particles, a plurality of capture substrates/capture structures, an electrolyte).
  • the reservoir or source is sealed and positioned adjacent an inlet (e.g., a reagent inlet) in fluidic communication with one or more reaction chambers.
  • the reservoir or source is configured to introduce the stored reagent into the reagent inlet upon unsealing of the reservoir or source.
  • the reservoir may have one or more characteristics described in commonly-owned U.S. Patent Application No. US 2015/0190802, entitled “Fluid delivery devices, systems, and methods", filed January 6, 2015, and U.S. Patent Application No. US 2015/0190805, entitled “Fluid delivery devices, systems, and methods", filed January 7, 2015, each of which is incorporated herein by reference in its entirety for all purposes.
  • the reservoir can include a sealed flexible material defining a cavity and a housing around the sealed flexible material.
  • the cavity can contain a reagent (e.g., a liquid reagent).
  • the sealed flexible material can include at least one breakable seal.
  • a controller can be adapted to press a top surface of the sealed flexible material to pressurize said reagent in said cavity to a pressure sufficient to deliver fluid though the breakable seal.
  • the reservoir may be deformable and includes at least one breakable seal.
  • one or more magnetic elements may be associated with one or more reaction chambers of the device.
  • a magnetic element may be associated with a reaction chamber comprising a lysis region.
  • a magnetic element may be associated with a reaction chamber comprising a detection region.
  • Such magnetic elements may be used, for example, for separating and/or removing particles not bound to a plurality of magnetic particles and/or metal-containing particles, as described herein.
  • one or more electrodes may be associated with one or more reaction chambers of the device. Such electrodes may be used, for example, for generating an electric field and/or a magnetic field within the reaction chamber, performing an electrochemical reaction (e.g., oxidation/reduction of a metal such as the metal in the metal- containing particle), and/or for heating the reaction chamber.
  • an electrode may be associated with a reaction chamber comprising a detection region for use in the quantification of the analyte.
  • an electrode may be associated with a reaction chamber comprising a lysis region.
  • the electrode may be used to heat at least a portion of the lysis region (and/or a fluid contained within the reaction chamber). In certain embodiments, heating at least a portion of the lysis region facilitates exposure of the analyte from the analyte-containing biological particle.
  • an electrode may be associated with (e.g., located adjacent, located directly adjacent, located within) a reaction chamber and/or detection region of the device such that it can be used to provide an electric potential to the reaction chamber and/or detection region of the device. In some embodiments, the electrode may be formed by methods described in U.S. Patent No.
  • Electrodes are also possible.
  • the electrode can be electrically connected to a power source.
  • the power source can selectively apply a potential to different deposits of the electrode to change a solid-fluid contact angle between the electrode and the fluid in the channel or reaction chamber of the device. This phenomenon, known as “electrowetting,” can be used to actively change the fluid flow dynamics in a channel.
  • a series of conductive transfer material deposits can selectively receive a pulse from a power source to create a pulsed flow of fluid.
  • the electrode e.g., electrode material
  • the electrode is a carbon-based electrode (e.g., a carbon electrode) and/or a silver-based electrode.
  • a carbon-based electrode may comprise, for example, conductive carbon such as carbon black and/or carbon ink.
  • a silver-based electrode may comprise, for example, silver and/or silver-chloride. Other electrodes and electrode materials are also possible.
  • the electrode (e.g., a reference electrode, a working electrode) comprises one layer (e.g., one active layer). In other embodiments, the electrode (e.g., a reference electrode, a working electrode) comprises two or more layers. For example, in some embodiments, the electrode comprises a first layer comprising silver and/or silver- chloride, and a second layer comprising carbon. In some embodiments, one or more layers of the electrode may be positioned on another layer of the electrode. To form an electrode, one or more layers of the electrode may be positioned on (e.g., screen printed onto) a portion of the device and/or onto an electrode foil.
  • the electrode comprises one or more carbon-based layers deposited or positioned on (e.g., screen printed on) a metal layer such as a silver-based layer (e.g., comprising silver and/or silver-chloride).
  • the metal layer may be formed, for example, from a metal-containing ink.
  • electrode 700 comprises a substrate 710 (e.g., an electrode foil), a silver-based layer 720 deposited on substrate 710, and carbon-based layer 730 deposited on substrate 710 and silver-based layer 720.
  • the carbon layer may be thinner than the metal-containing layer.
  • the electrode comprises a carbon trace (e.g., a thin layer of carbon) covering a metal-containing ink.
  • an electrode comprising a carbon-based layer deposited on a silver-based layer may prevent contamination of additional silver (e.g., silver ions) in an electrolyte when measuring silver content in the electrolyte (e.g., during application of a voltage to the electrode and/or during cycling of voltage across two or more electrodes).
  • the largest area (or width, or length) of the silver-based layer forming the electrode may be less than or equal to the largest area (or width, or length) of one or more of the carbon-based layer(s) forming the electrode.
  • the carbon-based layer substantially or entirely coats the silver-based layer, preventing or inhibiting silver ions from leaching into the electrolyte without substantially interfering with measurement of silver content in the electrolyte. Other configurations are also possible.
  • the device comprises at least one micro fluidic channel, a first reagent inlet, a sample introduction region, a mixing region, a lysis region, a second reagent inlet, a binding region, and a detection region.
  • the sample introduction region is positioned downstream of the first reagent inlet.
  • the mixing region is positioned downstream of the sample introduction region.
  • the lysis region is positioned downstream of the mixing region.
  • the second reagent inlet is positioned between the mixing region and the lysis region and in fluidic communication with the lysis region.
  • the binding region is positioned downstream of the lysis region.
  • the detection region is positioned downstream of the binding region.
  • one or more reservoirs each containing a stored reagent may be sealed and positioned adjacent the first reagent inlet and/or the second reagent inlet.
  • Other reaction chambers and/or inlets are also possible.
  • the device comprises at least one microfluidic channel, one reaction chamber, and a detection region in fluidic communication with the reaction chamber.
  • the various steps and/or components associated with the sample introduction region, mixing region, lysis region, and binding region described above in the context of multiple reaction chambers may be conducted in one reaction chamber.
  • the various steps and/or components associated with the detection region may be conduct downstream of the reaction chamber.
  • the device comprises a common waste channel.
  • fluid flow to one or more reaction chambers or other chambers described herein may be controlled through the use of valves that permit access to certain chambers or regions, but not others, and through the use of the common waste channel.
  • the device comprises a common waste channel in fluidic communication with one or more (e.g., two or more, three or more, four or more, or five or more) chambers or regions.
  • the common waste channel may facilitate the removal of waste (e.g., unbound particles, lysis solution, wash buffers, etc.) after any chamber or region of the device.
  • waste e.g., unbound particles, lysis solution, wash buffers, etc.
  • the waste channel is a microfluidic channel, although larger channel may also be used.
  • the waste channel may be connected to and/or in fluidic
  • microfluidic channels e.g., a microfluidic channel positioned between one or more chambers and/or one or more regions of the device.
  • the device comprises a waste region positioned downstream of the waste channel.
  • the waste region may be in fluidic communication with the waste channel such that any removed wastes (e.g., unbound particles, lysis solution, wash buffers, etc.) are transferred (e.g., flowed) to the waste region.
  • the waste may be removed from the device by removing the waste from the waste region.
  • the waste may remain in the waste region after completion of the analysis (e.g., the waste is disposed of with the disposal of the device itself).
  • the analyte binds with a metal-containing particle and a capture structure to form a bound complex comprising the metal-containing particle, the capture structure, and the analyte.
  • determining and/or quantifying an analyte in a fluid suspected of containing the analyte involves subjecting the fluid suspected of containing the analyte with capture structures (e.g., conjugated capture structures) and metal-containing particles (e.g., conjugated metal-containing particles) to form a bound complex.
  • capture structures e.g., conjugated capture structures
  • metal-containing particles e.g., conjugated metal-containing particles
  • bound complex 850 comprises a metal-containing particle 810 bound to analyte 820, and a capture structure 830 bound to the analyte.
  • a metal-containing particle 810 bound to analyte 820 bound to analyte 820
  • a capture structure 830 bound to the analyte Those skilled in the art would understand that while a single analyte is shown, more than one analyte may be bound to a capture structure and/or a metal-containing particle simultaneously.
  • adding plurality of capture structures 530 and metal-containing particles 510 to a fluid comprising an analyte 520 forms a mixture of bound complexes and unbound components 540.
  • the unbound components may comprise a portion of the capture structures not bound to the analyte, a portion of the metal-containing particles not bound to the analyte, and/ or other components present in the fluid comprising the analyte.
  • the analyte may be subjected to the capture structures and metal-containing particles in any suitable order.
  • the fluid suspected of containing the analyte-containing biological particle is first exposed to the capture structures to form an analyte-containing biological particle-capture structure complex in a reaction chamber (e.g., the reaction chamber comprising a binding region).
  • the fluid suspected of containing the analyte is first exposed to the capture structures to form an analyte-capture structure complex in a reaction chamber (e.g., the reaction chamber comprising a binding region).
  • the capture substrate may be used to isolate the analyte such that any non-analyte-containing materials may be removed (e.g., washed) from the reaction chamber and/or the device.
  • Such a complex may be then be exposed to the metal-containing particles to form a bound metal-containing particle-analyte-capture structure complex in the reaction chamber.
  • the fluid suspected of containing the analyte is first exposed to the metal-containing particles to form an analyte-metal-containing particle complex.
  • Such a complex may then be exposed to the capture structures to form a bound metal-containing particle-analyte-capture structure complex.
  • the fluid suspected of containing the analyte is exposed to a mixture of metal-containing particles and capture structures simultaneously.
  • the capture structures are stored with a reservoir or source in fluidic communication with a reaction chamber.
  • a plurality of capture structures may be added to the fluid in a reaction chamber such that the analyte binds to at least a portion of the plurality of capture structures.
  • the plurality of capture structures may be added by, for example, opening a valve or unsealing the reservoir or source containing the capture structures such that the capture structures are transferred (e.g., flow) into the reaction chamber.
  • the analyte may attach or bind to a capture structure in any suitable manner.
  • a single analyte attaches or binds to a single capture structure.
  • more than one analyte e.g., more than one type of analyte, or more than one number of analytes
  • the analyte may attach or bind with the capture structure via formation of a non-specific bond (e.g., non-specific adsorption).
  • the analyte may interact with a functional group present on the surface of the capture structure.
  • the analyte may bind with the capture structure and/or a functional group present on the surface of the capture structure via a bond such as an ionic bond, a covalent bond (e.g., carbon-carbon, carbon-oxygen, oxygen-silicon, sulfur- sulfur, phosphorus-nitrogen, carbon-nitrogen, metal-oxygen, or other covalent bonds), a hydrogen bond (e.g., between hydroxyl, amine, carboxyl, thiol, and/or similar functional groups), a dative bond (e.g., complexation or chelation between metal ions and monodentate or multidentate ligands), and/or by Van der Waals interactions.
  • a bond such as an ionic bond, a covalent bond (e.g., carbon-carbon, carbon-oxygen, oxygen-silicon, sulfur- sulfur, phosphorus-nitrogen, carbon-nitrogen, metal-oxygen, or other covalent bonds), a hydrogen bond (e.g., between hydroxyl, amine,
  • the plurality of capture structures comprise a plurality of particles.
  • the plurality of capture structures may have any suitable average particle size. Although other sizes are possible, in some cases the average particle size of the capture structures is relatively large (e.g., at least 100 nm, at least 200 nm) to facilitate manipulation of the particles by an external magnetic field.
  • Average particle size as used herein generally refers to the median (D50) diameter of the particles and is determined by dynamic light scattering, for example using a Malvern Particle Size Analyzer. Dynamic light scattering techniques will be generally known to those skilled in the art.
  • the plurality of capture structures (e.g., capture structure) have an average particle size of at least 50 nm, at least 100 nm, at least 200 nm, at least 250 nm, at least 300 nm, at least 400 nm, at least 500 nm, at least 600 nm, at least 700 nm, at least 800 nm, at least 900 nm, at least 1 micron, or at least 1.5 microns.
  • the plurality of capture structures have an average particle size of less than or equal to 5 microns, less than or equal to 2 microns, less than or equal to 1.5 microns, less than or equal to 1 micron, less than or equal to 900 nm, less than or equal to 800 nm, less than or equal to 700 nm, less than or equal to 600 nm, less than or equal to 500 nm, less than or equal to 400 nm, less than or equal to 300 nm, less than or equal to 250 nm, less than or equal to 200 nm, less than or equal to 100 nm, or less than or equal to 50 nm.
  • the average particle size of the capture structures may be measured prior to forming a complex with the analyte (e.g., a capture structure-analyte complex). In some embodiments, the average particle size of the capture structures may be measured prior to exposing the particles to the electrolyte used in a detection step.
  • the plurality of capture structures are a plurality of magnetic particles.
  • the plurality of magnetic particles may comprise any suitable magnetic (or magnetizable) material.
  • the magnetic material comprises a ferromagnetic material.
  • suitable magnetic materials include iron, nickel, cobalt, and alloys thereof and combinations thereof.
  • the plurality of capture structures are not electrochemically active (in the environment in which the particles are positioned).
  • the plurality of magnetic particles comprise a magnetic material which does not exchange electrons with conductive surfaces such as electrodes.
  • analytes bound to capture structures comprising a magnetic material may allow the use of a magnetic field to direct, separate, and/or isolate the analyte in the fluid (e.g., via use of a magnetic element located proximate to, or in direct contact with, the fluid or the reaction chamber containing the fluid).
  • a magnetic element e.g., magnetic element 125 in FIG. 2
  • the reaction chamber e.g., reaction chamber 120 in FIG. 2 and the magnetic element may be activated such that the analyte bound to capture structures is attracted to and moves towards (or away from) the magnetic element.
  • an external magnet may be placed on an external surface of the reaction chamber containing the fluid comprising the analyte bound to capture structure(s).
  • the plurality of capture substrates/capture structures may include a portion of a surface of the device, such as a surface of a microfluidic channel. In some embodiments, the plurality of capture substrates/capture structures are areas on a surface of a reaction chamber that have been functionalized with a capture entity. In certain embodiments, the plurality of capture structures comprise a plurality of microfluidic posts.
  • the device comprises a reaction chamber (or
  • microfluidic channel comprising a plurality of microfluidic posts (e.g., conjugated microfluidic posts), such that an analyte introduced into the device binds to the plurality of microfluidic posts.
  • a plurality of metal-containing particles may then be added to the reaction chamber such that the metal-containing particle, analyte, and microfluidic post forms a bound complex.
  • Suitable capture structures added to, or present in, the reaction chamber include , particles, and petri dish surfaces (e.g., at least a portion of a surface of a petri dish, multi-well plate, or the like). Other capture structures are also possible.
  • the plurality of capture structures are coated with one or more materials.
  • suitable materials may include polymers, silica, proteins (e.g., protein G conjugated, streptavidin conjugated, BSA conjugated), and materials with specific functional groups.
  • functional groups include hydroxyl, amino, carboxylate, carbonyl, ether, ester, sulfhydryl (thiol), silane, nitrile, carbamate, imidazole, pyrrolidone, carbonate, acrylate, alkenyl, and alkynyl.
  • Other functional groups are also possible and are known to those skilled in the art.
  • a plurality of metal-containing particles may be added to the fluid such that the analyte attaches or binds to at least a portion of the plurality of metal- containing particles.
  • the metal particles are stored with a reservoir or source in fluidic communication with a reaction chamber.
  • the plurality of metal particles may be added to a chamber (e.g., a reaction chamber) by, for example, opening a valve or unsealing the reservoir or source containing the metal particles such that the metal particles are transferred (e.g., flow) into the chamber.
  • the analyte may attach or bind to a metal- containing particle in any suitable manner.
  • a single analyte attaches or binds to a single metal-containing particle.
  • more than one analyte e.g., more than one type of analyte, or more than one number of analytes
  • the analyte may attach or bind with the metal-containing particle via formation of a non-specific bond (e.g., non-specific adsorption).
  • the analyte may interact with a functional group present on the surface of the metal-containing particle.
  • the analyte may bind with the metal-containing particle and/or a functional group present on the surface of the metal-containing particle via a bond such as an ionic bond, a covalent bond (e.g., carbon-carbon, carbon-oxygen, oxygen-silicon, sulfur- sulfur, phosphorus-nitrogen, carbon-nitrogen, metal-oxygen, or other covalent bonds), a hydrogen bond (e.g., between hydroxyl, amine, carboxyl, thiol, and/or similar functional groups), a dative bond (e.g., complexation or chelation between metal ions and monodentate or multidentate ligands), and/or by Van der Waals interactions.
  • a bond such as an ionic bond, a covalent bond (e.g., carbon-carbon, carbon-oxygen, oxygen-silicon, sulfur- sulfur, phosphorus-nitrogen, carbon-nitrogen, metal-oxygen, or other covalent bonds), a hydrogen bond (e.g., between hydroxyl
  • the plurality of metal-containing particles may have any suitable average particle size. Although other sizes are possible, in some cases the average particle size of the metal- containing particles is relatively large (e.g., at least 100 nm, at least 200 nm) to increase the signal-to-noise ratio when using certain detection methods, as described in more detail below.
  • the plurality of metal-containing particles have an average particle size of at least 70 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 250 nm, at least 300 nm, at least 400 nm, at least 500 nm, at least 600 nm, at least 700 nm, at least 800 nm, at least 900 nm, at least 1 micron, or at least 1.5 microns.
  • the plurality of metal-containing particles have an average particle size of less than or equal to 2 microns, less than or equal to 1.5 micron, less than or equal to 1 micron, less than or equal to 900 nm, less than or equal to 800 nm, less than or equal to 700 nm, less than or equal to 600 nm, less than or equal to 500 nm, less than or equal to 400 nm, less than or equal to 300 nm, less than or equal to 250 nm, less than or equal to 200 nm, less than or equal to 150 nm, or less than or equal to 100 nm.
  • the average particle size of the metal-containing particles may be measured prior to forming a complex with the analyte (e.g., a metal-containing particle-analyte complex). In some embodiments, the average particle size of the metal-containing particles may be measured prior to exposing the particles to the electrolyte used in a detection step.
  • the plurality of metal-containing particles have an average particle size that is within a particular range of the average particle size of the plurality of capture structures.
  • the plurality of metal-containing particles have an average particle size that is at least 0.05, at least 0.06, at least 0.07, at least 0.08, at least 0.1, at least 0.15, at least 0.2, at least 0.5, at least 0.8, at least 1, at least 2, at least 2.5, at least 3, at least 4, or at least 5 times the average particle size of the plurality of capture structures.
  • the plurality of metal-containing particles have an average particle size that is less than or equal to 6, less than or equal to 5, less than or equal to 4, less than or equal to 3, less than or equal to 2.5, less than or equal to 2, less than or equal to 1, less than or equal to 0.8, less than or equal to 0.5, less than or equal to 0.2, less than or equal to 0.15, less than or equal to 0.1, less than or equal to 0.08, or less than or equal to 0.07 times the average particle size of the plurality of capture structures.
  • the average particle sizes used may be those prior to forming a complex with the analyte (e.g., a capture structure-analyte-metal-containing particle complex). In some embodiments, the average particle sizes may be those prior to exposure of the particles to the electrolyte used in a detection step (e.g., in a detection region).
  • a plurality of capture substrates/capture structures that are larger (e.g., at least 2 times the average particle size) than a plurality of metal-containing particles may permit the separation of a bound complex from unbound complexes by size.
  • bound complexes may be separated from unbound complexes by size sorting techniques including, but not limited to, membrane separation, size exclusion chromatography, and centrifugation. Other size sorting and/or separation techniques are also possible.
  • the average particle size of the metal-containing particle and/or the average particle size of the capture structure may be chosen based on a balance of factors such as increasing the amplification of the signal during detection (e.g., quantification), improving the efficiency of forming bound complexes with the metal-containing particle, the capture structure, and the analyte, and/or more easily removing unbound particles from the fluid (e.g., when isolating bound complexes with a magnet, separating the bound complexes via size exclusion chromatography, centrifugation, or membrane separation).
  • factors such as increasing the amplification of the signal during detection (e.g., quantification), improving the efficiency of forming bound complexes with the metal-containing particle, the capture structure, and the analyte, and/or more easily removing unbound particles from the fluid (e.g., when isolating bound complexes with a magnet, separating the bound complexes via size exclusion chromatography, centrifugation, or membrane separation).
  • larger metal-containing particles generally lead to greater amplification of the signal during quantification using certain detection methods described herein, whereas smaller metal-containing particles (and/or capture structures) generally lead to improved ability of binding between the analyte and the metal-containing particle and the capture structure. For instance, smaller particles generally lead to a reduced amount of steric hindrance during binding with the analyte. Additionally, larger capture structures generally have a higher magnetic moment and thus are more easily moved by a magnetic element in the fluid (e.g., to remove unbound components).
  • relatively large metal-containing particles may be used.
  • relatively larger metal-containing particles may result in an increased amplification of the signal during detection, which may increase the sensitivity of the methods described herein as compared to traditional quantification methods which utilize relatively smaller (e.g., less than 100 nm) metal-containing particles.
  • the metal-containing particles may comprise any suitable metal that may be useful for detecting an analyte present in the fluid.
  • the metal is one that can be oxidized and/or reduced in the presence of one or more electrodes.
  • suitable metals include silver, copper, cobalt, cadmium, lead, zinc, tin, nickel, bismuth, platinum, manganese, chromium, gold, alloys thereof, oxides thereof, sulfides thereof, and combinations thereof.
  • the metal-containing particles may comprise CuO, Au, Ag, ZnSe, ZnO, Bi, CdS, CdSe, Co, CoO, PbS, PbSe, PbO, Ni, Pt, and/or Mn.
  • the metal-containing particles comprise silver.
  • the metal-containing particles comprise copper oxide.
  • the metal-containing particles comprise gold.
  • the metal is present as a layer on a nonmetallic particle.
  • at least a portion of a surface of a nonmetallic particle is coated with the metal.
  • suitable nonmetallic particles include polymers, silica, or the like.
  • the core of the metal-containing particle may be formed of the metal. In such embodiments, the surface of the metal-containing particle may be modified to tailor the surface chemistry of the particle as described herein.
  • the plurality of metal-containing particles comprises two or more types of metals (e.g., three or more, four or more, five or more, six or more, or seven or more types of metals).
  • the plurality of metal-containing particles comprises a first type of metal-containing particle and a second type of metal- containing particle, different than the first type of metal-containing particle.
  • a third type of metal-containing particle may present, different from the first or second metal types.
  • using a plurality of metal-containing particles comprising two or more types of metals may be useful for the detection of two or more different analytes (e.g., each analyte bound to a different type of metal).
  • devices and methods comprising a plurality of metal-containing particles comprising two or more types of metals may enable multiplex detection of two or more analytes.
  • the plurality of metal-containing particles comprises a first type of metal-containing particle comprising silver and a second type of metal-containing particle comprising copper oxide.
  • the plurality of metal-containing particles comprises a first type of metal-containing particle comprising silver and a second type of metal-containing particle comprising gold.
  • the plurality of metal-containing particles comprises a first type of metal- containing particle comprising silver, a second type of metal-containing particle comprising gold, and a third type of metal-containing particle comprising copper oxide.
  • two or more analytes may be introduced to a plurality of metal-containing particles comprising two or more types of metal-containing particles such that a first type of metal-containing particle binds to a first analyte, and a second type of metal-containing particle binds to a second analyte. Bound complexes with each analyte and the metal-containing particles may be formed, as described herein.
  • the two or more analytes may be determined and/or quantified by exposing each bound complex (e.g., a first bound complex comprising the first analyte, a second bound complex comprising the second analyte) to an electrolyte and applying a first electric potential and/or measuring current by changing a first voltage on the working electrode to determine the amount of the first analyte present.
  • a second electric potential different than the first electric potential may be applied, and/or current may be measured by changing a second voltage on the working electrode to determine the amount of the second analyte present.
  • a third electric potential different than the first and second electric potentials may be applied, and/or current may be measured by changing a third voltage on the working electrode to determine the amount of the third analyte present.
  • the detection region may comprise an ASV detector (e.g., for conducting ASV).
  • the two or more analytes may be determined and/or quantified substantially simultaneously (e.g., by measuring current by changing the voltage between a first voltage and a second voltage, and/or between a second voltage and a third voltage) on the working electrode to determine the amount of each analyte present.
  • the two or more analytes may be determined and/or quantified sequentially.
  • the first analyte bound to the first type of metal-containing particles may be introduced to the detection region and determined/quantified, and the second analyte bound to the second type of metal-containing particles (e.g., in a second bound complex) may then be introduced to the detection region and determined/quantified.
  • the third analyte and/or third type of metal-containing particle may be introduced to the detection region and determined/quantified,
  • the two or more analytes may be introduced into the detection region substantially simultaneously, but determined sequentially (e.g., by applying a first voltage, and then a second voltage different from the first voltage, to oxidize one or more of the metals in one or more of the pluralities of metal-containing particles). If a third analyte and/or third type of metal-containing particle is present, a third voltage different from the first and second voltages, may be applied to oxidize one or more of the metals in one or more of the pluralities of metal-containing particles).
  • an exemplary method comprises introducing, to a first analyte and a second analyte different than the first analyte, a plurality of metal-containing particles comprising a first type of metal-containing particle and a second type of metal-containing particle, and a plurality of capture structures such that the first analyte binds with a capture structure and a first type of metal-containing particle to form a first bound complex.
  • the second analyte binds with a capture structure and a second type of metal-containing particle to form a second bound complex.
  • the third analyte binds with a capture structure and a third type of metal-containing particle to form a third bound complex.
  • the method involves separating any unbound metal-containing particles from the first bound complex and the second bound complex (and third bound complex if present), and
  • the plurality of capture structures and the plurality of metal- containing particles may be simultaneously introduced to the reaction chamber containing a fluid (e.g., a sample) comprising the analyte (or analytes). In some cases, however, the plurality of capture structures may be added sequentially (e.g., prior to, or after) the introduction of the plurality of metal-containing particles to the reaction chamber.
  • a fluid e.g., a sample
  • the plurality of capture structures may be added sequentially (e.g., prior to, or after) the introduction of the plurality of metal-containing particles to the reaction chamber.
  • the plurality of capture structures may be added to the reaction chamber and incubated and/or mixed with the analyte(s) for any suitable amount of time such that at least a portion of the plurality of capture structures attach or bind to the analyte(s).
  • the plurality of capture structures and analyte may be incubated and/or mixed for at least 5 seconds, at least 10 seconds, at least 20 seconds, at least 30 seconds, 1 minute, at least 2 minutes, at least 3 minutes, or at least 4 minutes such that at least a portion of the plurality capture structures attach or bind to the analyte(s).
  • the plurality of capture structures and analyte may be incubated and/or mixed for less than or equal to 10 minutes, less than or equal to 5 minutes, less than or equal to 4 minutes, less than or equal to 3 minutes, less than or equal to 2 minutes, less than or equal to 1 minute, less than or equal to 30 seconds, less than or equal to 20 seconds, or less than or equal to 10 seconds such that at least a portion of the plurality of capture structures attach or bind the analyte. Combinations of the above referenced ranges are also possible (e.g. at least 5 seconds and less than or equal to 10 minutes, at least 1 minute and less than or equal to 5 minutes). Other ranges are also possible. In some other embodiments, the plurality of capture structures and analyte may be incubated for more than 5 minutes.
  • the plurality of metal-containing particles may be added to the reaction chamber and incubated and/or mixed for any suitable amount of time such that at least a portion of the plurality of metal-containing particles attach or bind to the analyte(s).
  • the plurality of metal-containing particles and analyte(s) may be incubated and/or mixed for at least 5 seconds, at least 10 seconds, at least 20 seconds, at least 30 seconds, 1 minute, at least 2 minutes, at least 3 minutes, or at least 4 minutes such that at least a portion of the plurality metal-containing particles attach or bind to the analyte(s).
  • the plurality of metal-containing particles and analyte(s) may be incubated and/or mixed for less than or equal to 10 minutes, less than or equal to 5 minutes, less than or equal to 4 minutes, less than or equal to 3 minutes, less than or equal to 2 minutes, less than or equal to 1 minute, less than or equal to 30 seconds, less than or equal to 20 seconds, or less than or equal to 10 seconds such that at least a portion of the plurality of metal-containing particles bind the analyte.
  • Combinations of the above referenced ranges are also possible (e.g. at least 5 seconds and less than or equal to 10 minutes, at least 1 minute and less than or equal to 5 minutes). Other ranges are also possible.
  • the plurality of metal-containing particles and analyte(s) may be incubated for more than 5 minutes. In some embodiments, the plurality of capture structures and the plurality of metal- containing particles are incubated and/or mixed with the analyte(s) substantially
  • the plurality of capture structures are incubated and/or mixed with the analyte(s) prior to incubating and/or mixing the plurality of metal- containing particles with the analyte(s).
  • the plurality of capture structures and/or the plurality of metal-containing particles are incubated and/or mixed with the analyte in a buffer solution or an electrolyte. That is to say, in certain embodiments, a buffer solution or an electrolyte may be added to the metal-containing particles, the capture structures, and/or the fluid comprising analyte.
  • the fluid may be mixed with the buffer solution or an electrolyte for any suitable amount of time (e.g., at least 1 minute and less than or equal to 5 minutes). Buffer solutions and electrolytes are described in more detail below.
  • the capture structures may be introduced to the reaction chamber containing the fluid comprising the analyte in a particular amount. In some embodiments, the capture structure may be introduced to the fluid in an amount of at least 5 ⁇ g / 100 ⁇ , at least 10 ⁇ g / 100 ⁇ , at least 25 ⁇ g / 100 ⁇ , at least 50 ⁇ g / 100 ⁇ , at least 100 ⁇ g / 100 ⁇ , or at least 250 ⁇ g / 100 ⁇ h.
  • the capture structure may be introduced to the fluid in an amount of less than or equal to 500 ⁇ g / 100 ⁇ , less than or equal to 250 ⁇ g / 100 ⁇ , less than or equal to 100 ⁇ g / 100 ⁇ , less than or equal to 50 ⁇ g / 100 ⁇ , less than or equal to 25 ⁇ g / 100 ⁇ , or less than or equal to 10 ⁇ g / 100 ⁇ h. Combinations of the above referenced ranges are also possible (e.g., at least 5 ⁇ g / 100 ⁇ ⁇ and less than or equal to 500 ⁇ g / 100 ⁇ ). Other ranges are also possible
  • the metal-containing particles may be introduced to the reaction chamber containing the fluid comprising the analyte in a particular amount. In some embodiments, the metal-containing particles may be introduced to the fluid in an amount of at least 5 ⁇ g / 100 ⁇ , at least 10 ⁇ g / 100 ⁇ , at least 25 ⁇ g / 100 ⁇ , at least 50 ⁇ g / 100 ⁇ , at least 100 ⁇ g / 100 ⁇ , or at least 250 ⁇ g / 100 ⁇ ⁇ .
  • the metal- containing particles may be introduced to the fluid in an amount of less than or equal to 500 ⁇ g / 100 ⁇ , less than or equal to 250 ⁇ g / 100 ⁇ , less than or equal to 100 ⁇ g / 100 ⁇ , less than or equal to 50 ⁇ g / 100 ⁇ , less than or equal to 25 ⁇ g / 100 ⁇ , or less than or equal to 10 ⁇ g / 100 ⁇ ⁇ . Combinations of the above referenced ranges are also possible (e.g., at least 5 ⁇ g / 100 ⁇ ⁇ and less than or equal to 500 ⁇ g / 100 ⁇ ).
  • the capture structure and/or metal-containing particle may each interact with an analyte via a binding event between pairs of biological molecules (e.g., a biological molecule present on the surface of the capture structure and/or metal-containing particle and the analyte), including proteins, nucleic acids, glycoproteins, carbohydrates, hormones, and the like.
  • biological molecules e.g., a biological molecule present on the surface of the capture structure and/or metal-containing particle and the analyte
  • proteins e.g., a biological molecule present on the surface of the capture structure and/or metal-containing particle and the analyte
  • proteins e.g., an antibody/peptide pair, an antibody/antigen pair, an antibody fragment/antigen pair, an antibody/antigen fragment pair, an antibody
  • fragment/antigen fragment pair an antibody/hapten pair, an enzyme/substrate pair, an enzyme/inhibitor pair, an enzyme/cofactor pair, a protein/substrate pair, a nucleic
  • a protein/nucleic acid pair a protein/nucleic acid pair, a peptide/peptide pair, a protein/protein pair, a small molecule/protein pair, a glutathione/GST pair, an anti-GFP/GFP fusion protein pair, a Myc/Max pair, a maltose/maltose binding protein pair, a carbohydrate/protein pair, a carbohydrate derivative/protein pair, a metal binding tag/metal/chelate, a peptide tag/metal ion-metal chelate pair, a peptide/NTA pair, a lectin/carbohydrate pair, a receptor/hormone pair, a receptor/effector pair, a complementary nucleic acid/nucleic acid pair, a ligand/cell surface receptor pair, a virus/ligand pair, a Protein A/antibody pair, a Protein G/antibody pair, a Protein L/antibody pair, an Fc receptor/antibody pair,
  • biotin/streptavidin pair a drug/target pair, a zinc finger/nucleic acid pair, a small
  • molecule/peptide pair a small molecule/protein pair, a small molecule/target pair, a carbohydrate/protein pair such as maltose/MBP (maltose binding protein), a small molecule/target pair, or a metal ion/chelating agent pair.
  • species include peptides, proteins, DNA, RNA, PNA.
  • the capture structure may interact with an analyte via a first type of binding event between a pair of biological molecules and the metal-containing particle may interact with the analyte via a second type of binding event between a pair of biological molecules, different than the first binding event.
  • the plurality of capture structures may be conjugated with a biological molecule or functional group, and/or the plurality of metal- containing particles may be conjugated with a biological molecule or functional group.
  • the plurality of capture structures and/or the plurality of metal-containing particles are conjugated with one or more antibodies.
  • the capture structure may interact with the analyte via a first antibody/antigen interaction and the metal- containing particle may interact with the analyte via a second antibody/antigen interaction, different than the first antibody/antigen interaction.
  • the capture structure may interact with the analyte at a first binding site on the analyte (e.g.
  • first epitope and the metal-containing particle may interact with the analyte at a second binding site on the analyte (e.g., a second epitope), different than the first binding site, on the analyte.
  • a second binding site on the analyte e.g., a second epitope
  • Other interactions and/or binding events between pairs of biological molecules are also possible.
  • the analyte is a protein, an indigent, a lipid, a glycolipid, nucleic acid, an amino acid, membrane protein (e.g., from a bacterium), a hormone, a small molecule, a metabolite, a drug, or the like.
  • the analyte is an antigen (e.g., for the hepatitis C virus (HCV), or an antigen for HIV).
  • HCV antigens such as El, E2, NS2, NS3, NS4 (e.g., NS4A, NS4B), and NS5 (e.g., NS5A, and NS5B).
  • the analyte is a core antigen.
  • core antigens which may be determined and/or quantified using methods described herein include HIV core antigen p24 and HCV core antigen (cAg).
  • a combination of HCV core antigen and one or more of the HCV antigens listed above may be determined.
  • the analyte is a protein (e.g., a cardiac marker such as
  • the protein is a protein found circulating free in blood or in a complex in blood.
  • the protein is a cardiac marker protein such as creatine kinase-MB (CK-MB), myoglobin, homocysteine, C-reactive protein (CRP), troponin T, or troponin I.
  • CK-MB creatine kinase-MB
  • CRP C-reactive protein
  • troponin T or troponin I.
  • they analyte is a hormone or small molecule used for diagnosis of endocrine disfunction, such as thyroid- stimulating hormone (TSH), triiodothyronine (T3), thyroxine (T4), or Vitamin D.
  • TSH thyroid- stimulating hormone
  • T3 triiodothyronine
  • T4 thyroxine
  • Vitamin D Vitamin D.
  • the devices and methods described herein may comprise multiplexing (e.g., the generation of a voltage/current signal that contains two or more peaks associated with two or more analytes present in a sample).
  • the devices and methods described herein may be useful for conducting diagnostic panels (e.g., an analyte panel) which may be helpful for diagnosing, assessing, or managing diseases or bodily conditions.
  • the devices and methods described herein may comprise two or more types of metal-containing particles for detecting and/or quantifying two more or analytes.
  • the analyte, or an entity capable of binding to an analyte is part of a panel.
  • a panel for diagnosing, assessing, or managing a particular disease or bodily condition may include in the detection region two or more binding entities capable of binding to two or more analytes of interest in a sample that would be indicative of the particular disease or bodily condition.
  • Non-limiting examples of panels include cardiac biomarker panels, metabolic panels, fertility panels, antigen/antibody combination panels, drug resistance panels, and genetic panels.
  • Non-limiting examples of analytes that may be detected in a cardiac biomarker panel include troponin, myoglobin, and CK-MB.
  • Non-limiting examples of analytes that may be detected in a metabolic panel include free T4, free T3, total T3, TSH, FSH, estradiol, and Vitamin D.
  • Non-limiting examples of analytes that may be detected in a fertility panel include estradiol, progesterone, LH, AMH, and beta-hCG.
  • Antigen/antibody combination panels may be used for the diagnosis of a disease including, but not limited to, Zika, Dengue, and Chikungunya virus infections. Other analytes and panels are also possible.
  • the plurality of capture structures and/or the plurality of metal-containing particles may interact and bind with one or more analytes (e.g., two or more, three or more, four or more, or five or more analytes), such as one or more antigens.
  • the plurality of capture structures and/or the plurality of metal-containing particles are conjugated with one or more antibodies and capable of binding to one or more analytes (e.g., one or more antigens).
  • the plurality of capture structures and/or the plurality of metal-containing particles may interact and bind with a core antigen and one or more additional antigens.
  • the capture structures and/or metal-containing particles may include more than one type of antibody that bind to different antigens.
  • the plurality of capture structures are conjugated with a first antibody and a second antibody
  • the plurality of metal-containing particles are conjugated with a third antibody and a fourth antibody, such that the first antibody and the third antibody form a bound complex with a first antigen (e.g., a core antigen), and the second antibody and the fourth antibody each bind with a second antigen (e.g., a non-core antigen).
  • a first antigen e.g., a core antigen
  • the second antibody and the fourth antibody each bind with a second antigen (e.g., a non-core antigen).
  • the first antibody and the third antibody form a bound complex with cAg and the second antibody and the fourth antibody each bind to at least one additional HCV antigen.
  • the second antibody and the fourth antibody form a second bound complex.
  • the plurality of capture structures and/or the plurality of metal-containing particles may be conjugated with a plurality of antibodies capable of binding to a plurality of analytes (e.g., antigens).
  • the antibody is an HIV-p24 antibody (e.g., commercial available from ZeptoMetrixTM such as anti-HIV-I p24, Clone: 39/5.4A).
  • the antibody is an HCV core antigen antibody (e.g., commercially available from Capricorn such as HCV-007-48489, HCV-007-48490, and HCV-007 -48491).
  • the devices and methods described herein may comprise a competitive assay format to determine the amount of analyte in a sample.
  • a plurality of capture structures may be added to the sample such that the capture structure may interact (e.g., bind) with an analyte via a binding events (e.g., a biological molecule present on the surface of the capture structure and the analyte).
  • some of the analyte present in the sample binds to at least a portion of the plurality of capture structures (e.g., the analyte may bind to a biological molecule present on the surface of the capture structure).
  • At least some of the analyte present in the sample does not bind to the capture structures (e.g., not all of the capture structures introduced to the sample bind to the analyte).
  • one or more metal-containing particles may interact (e.g., bind) with at least portion of the capture structures that did not bind with the analytes.
  • any unbound metal-containing particles may be removed.
  • the amount of metal-containing particles bound to capture structures that did not bind to the analyte is inversely proportional to the amount of analyte present in the sample.
  • a competitive assay comprises introducing a plurality of metal-containing particles to the sample such that the metal-containing particles may interact (e.g., bind) with an analyte via a binding events (e.g., a biological molecule present on the surface of the metal-containing particles and the analyte).
  • the analyte may bind to at least a portion of the plurality of metal-containing particles (e.g., the analyte may bind to a biological molecular present on the surface of the metal-containing particles). In some such embodiments, some of the analyte does not bind to some of the metal- containing particles.
  • a plurality of capture structures may be added to the sample and may interact (e.g., bind) with at least portion of the metal-containing particles that did not bind with the analyte.
  • any metal-containing particles not bound to the capture structure may be removed.
  • the amount of metal-containing particles bound to capture structures that did not bind to the analyte is inversely proportional to the amount of analyte present in the sample.
  • a determining step as described herein may be used to determine the amount of analyte present in the sample.
  • the determining step comprises voltammetry, including but not limited to, anodic stripping voltammetry, cathodic stripping voltammetry, adsorptive stripping voltammetry, square wave voltammetry, linear sweep voltammetry, staircase voltammetry, cyclic voltammetry, alternating current voltammetry, chronoamperometry, normal pulse voltammetry, differential-pulse
  • voltammetry or the like. These methods may be used to quantify the amount of metal- containing particles present, thereby quantifying (e.g., the amount of metal-containing particles being inversely proportional to) the amount of analyte present in the sample.
  • the determining step comprises exposing the plurality of metal-containing particles bound to capture structures (e.g., without analyte) to an electrolyte, applying an electric potential to oxidize at least a portion of the metal from the metal-containing particles, applying an electric potential to deposit at least a portion of the metal onto a working electrode, and measuring current by changing a voltage on the working electrode to determine the amount of analyte that was present in the sample (e.g., the amount of metal-containing particles being inversely proportional to the amount of analyte present in the sample).
  • the device comprises at least one microfluidic channel.
  • one or more inlets, inlet channels, or fluidic channels e.g., that connect one or more reaction chambers or other chambers of the device
  • at least one microfluidic channel of the device has a particular average cross-sectional dimension. The "cross-sectional dimension" (e.g., a diameter) of the channel is measured perpendicular to the direction of fluid flow.
  • the average cross-sectional dimension (e.g., mean cross sectional dimension) of the channel is less than or equal to about 2 mm, less than or equal to about 1 mm, less than or equal to about 800 microns, less than or equal to about 600 microns, less than or equal to about 500 microns, less than or equal to about 400 microns, or less than or equal to about 300 microns.
  • the average cross-sectional dimension of the channel is greater than or equal to about 250 microns, greater than or equal to about 300 microns, greater than or equal to about 400 microns, greater than or equal to about 500 microns, greater than or equal to about 600 microns, greater than or equal to about 800 microns, or greater than or equal to about 1 mm. Combinations of the above-referenced ranges are also possible (e.g., between about 250 microns and about 2 mm, between about 400 microns and about 1 mm, between about 300 microns and about 600 microns). Other ranges are also possible. In some cases, more than one channel or capillary may be used.
  • At least one microfluidic channel of the device can have any suitable cross-sectional shape (circular, oval, triangular, irregular, trapezoidal, square or rectangular, or the like) and can be covered or uncovered. In embodiments where it is completely covered, at least one portion of the channel can have a cross-section that is completely enclosed, or the entire channel may be completely enclosed along its entire length with the exception of its inlet(s) and outlet(s).
  • a channel may also have an aspect ratio (length to average cross sectional dimension) of at least 2: 1, more typically at least 3: 1, 5: 1, or 10: 1 or more.
  • An open channel generally will include characteristics that facilitate control over fluid transport, e.g., structural characteristics (an elongated indentation) and/or physical or chemical characteristics
  • hydrophilicity hydrophilicity or other characteristics that can exert a force (e.g., a containing force) on a fluid.
  • the fluid within the channel may partially or completely fill the channel.
  • the fluid may be held within the channel, for example, using surface tension (e.g., a concave or convex meniscus).
  • At least one microfluidic channel of the device can have any suitable volume.
  • the volume of the channel may be at least 0.1 microliters, at least 0.5 microliters, at least 1 microliter, at least 2 microliters, at least 5 microliters, at least 7 microliters, at least 10 microliters, at least 12 microliters, at least 15 microliters, at least 20 microliters, at least 30 microliters, or at least 50 microliters.
  • the volume of the channel may be less than or equal to 100 microliters, less than or equal to 70 microliters, less than or equal to 50 microliters, less than or equal to 25 microliters, less than or equal to 10 microliters, or less than or equal to 5
  • microliters are also possible (e.g., between 1 microliter and 100 microliters). Other ranges are also possible.
  • the device may have a particular overall thickness.
  • the device e.g., the device comprising at least one microfluidic channel
  • the device has an overall thickness of greater than or equal to 1 mm, greater than or equal to 1.2 mm, greater than or equal to 1.4 mm, greater than or equal to 1.6 mm, greater than or equal to 1.8 mm, greater than or equal to 2 mm, greater than or equal to 2.2 mm, or greater than or equal to 2.4 mm. Combinations of the above-referenced ranges are also possible (e.g., less than or equal to 2.5 mm and greater than or equal to 1 mm). Other ranges are also possible.
  • Fluids e.g., comprising the plurality of capture structures, the plurality of metal- containing particles, the capture substrates/capture structures, the electrolyte, the lysing solution, the buffer solution, and/or the analyte
  • a channel e.g., microfluidic channel
  • reaction chamber e.g., a pump, syringe, pressurized vessel, or any other source of pressure.
  • fluids can be pulled into the channel (e.g., microfluidic channel) or reaction chamber of the device by application of vacuum or reduced pressure on a downstream side of the
  • Vacuum may be provided by any source capable of providing a lower pressure condition than exists upstream of the channel or device.
  • sources may include vacuum pumps, Venturis, syringes and evacuated containers. It should be understood, however, that in certain embodiments, fluids described herein can be flowed with a changing pressure drop across an inlet and an outlet of the microfluidic channel and/or reaction chamber of the device by using capillary flow, the use of valves, or other external controls that vary pressure and/or flow rate.
  • the device comprises one or more valves to facilitate fluidic communication between a reaction chamber and the detection region, between two or more reaction chambers, between an inlet channel and one or more reaction chambers, and/or between a reservoir or source and one or more reaction chambers.
  • one or more valves may be positioned between them. For example, a fluid contained within a reservoir may be introduced and be flowed into a reaction chamber by opening a valve located between the reservoir and the reaction chamber.
  • valves facilitate flow between an inlet of the device and a fluidic channel (e.g., microfluidic channel) and/or reaction chamber.
  • a fluidic channel e.g., microfluidic channel
  • flowing a fluid into one or more microfluidic channels and/or reaction chamber can include opening an inlet valve and an outlet valve, closing all other valves in the device, and pumping or pulling the fluid into the microfluidic channel(s) and/or reaction chamber.
  • the valve may have one or more characteristics described in commonly- owned U.S. Patent Application No. US 2015/056717, filed August 20, 2014, and entitled "Microfluidic metering of fluids", which is incorporated herein by reference in its entirety for all purposes.
  • the electrolyte comprises a halide compound (e.g., NaCl, KC1, NaBr, KI).
  • the electrolyte comprises a thiocyanate (e.g., ammonium thiocyanate).
  • the electrolyte comprises an oligoelectrolyte or poly electrolyte.
  • Polyelectrolytes are known in the art and generally refer to polymers with a repeat unit comprising an electrolyte group.
  • Non-limiting examples of polyelectrolytes include poly(sodium styrene sulfonate), polypeptides, glycosaminoglycans, DNA, and polyacids (e.g., polyacrylic acid).
  • the electrolyte comprises a salt.
  • Simple screening tests can be employed to help select an electrolyte.
  • One simple screen test includes incubating the bound complex with the electrolyte for five minutes and then removing the electrolyte.
  • the amount of metal-containing particles bound in the bound complex can be measured using anodic stripping voltammetry and comparing the amount of metal-containing particles present in the removed electrolyte measured by anodic stripping voltammetry.
  • the bound complex is exposed to the electrolyte at a particular concentration.
  • the concentration of the electrolyte in the fluid containing the bound complex is less than 1.0 M, less than 0.8 M, less than 0.6 M, less than 0.4 M, less than 0.2 M, or less than 0.1 M.
  • the concentration electrolyte is greater than or equal to 0.01 M, greater than or equal to 0.05 M, greater than or equal to 0.1 M, greater than or equal to 0.2 M, greater than or equal to 0.4 M, greater than or equal to 0.6 M, or greater than or equal to 0.8 M. Combinations of the above referenced ranges are also possible (e.g., less than 1.0 M in greater than or equal to 0.01 M). Other ranges are also possible.
  • determining the amount of analyte present in the sample comprises quantifying the analyte in a detection region.
  • method 500 comprises determining step 590.
  • the determining step may comprise any suitable method for quantifying the amount of analyte present in the sample.
  • the determining step involves quantifying (e.g., directly or indirectly) the amount of metal- containing particles in the bound complex. Without wishing to be bound by theory, the amount of metal-containing particles in the bound complex is proportional to the amount of analyte present in the fluid.
  • the metal-containing particles are functionalized with an antibody that specifically binds to an analyte, such that the determining step determines the amount of analyte bound to the metal-containing particle.
  • the determining step comprises voltammetry, including but not limited to, anodic stripping voltammetry, cathodic stripping voltammetry, adsorptive stripping voltammetry, square wave voltammetry, linear sweep voltammetry, staircase voltammetry, cyclic voltammetry, alternating current voltammetry, chronoamperometry, normal pulse voltammetry, differential-pulse voltammetry, or the like. These methods may be used to quantify the amount of metal-containing particles present, thereby quantifying the amount of analyte present in the fluid as described in more detail below.
  • determining step 590 comprises the application of one or more electric potentials to bound complex 550.
  • an electrode located adjacent (e.g., directly adjacent) the detection region may provide one or more electric potentials to the detection region such that the analyte may be quantified.
  • method 500 comprises determining step 590, comprising applying one or more electric potentials (e.g., electric potential application step 555 and electric potential application step 575) to the fluid contained within the detection region (e.g., comprising the bound complex).
  • Those skilled in the art would be capable of selecting suitable methods for applying electric potential to the bound complexes including, but not limited to, providing a working electrode proximate to or in direct contact with at least a portion of the bound complex(es) and an auxiliary electrode, such that an electric potential can be applied to the bound complex(es).
  • electric potential application step 555 comprises applying an electric potential such that at least a portion of the metal from the metal-containing particles is oxidized, forming oxidized metal 570.
  • electric potential application step 575 comprises applying an electric potential (e.g., a first electric potential) to oxidized metal 570 such that at least a portion of the metal is deposited onto a working electrode (e.g., a working electrode in contact with at least a portion of the bound complex), forming deposited metal layer 580 on the working electrode (e.g., via a reduction step). The metal layer may then be stripped off of the working electrode.
  • a working electrode e.g., a working electrode in contact with at least a portion of the bound complex
  • an electric potential application step 555 comprises applying two or more electric potentials such that at least a portion of the metal from the first type of metal-containing particles is oxidized and at least a portion of the metal from the second type of metal-containing particles is oxidized.
  • electric potential application step 575 comprises applying two or more electric potentials (e.g., a first electric potential, a second electric potential) to the two or more oxidized metals such that at least a portion of the first metal and/or at least a portion of the second metal is deposited onto a working electrode (e.g., a working in contact with at least a portion of the bound complex(es)), forming one or more deposited metal layers on the working electrode (e.g., via a reduction step).
  • two or more electric potentials e.g., a first electric potential, a second electric potential
  • a third electric potential may be applied such that at least a portion of the metal from the third type of metal-containing particle is oxidized and/or such that at least a portion of the third metal is deposited onto a working electrode.
  • the methods described herein may involve multiple sequential oxidation-reduction- strip cycles depending on the number of types of metal-containing particles present in the sample. For example, a first metal from a first metal-containing particle may be oxidized, reduced (e.g., deposited onto a working electrode), and then stripped off of the working electrode during a first oxidation-reduction-strip cycle. Subsequently, a second metal from a second metal-containing particle may be oxidized, reduced (e.g., deposited onto a working electrode), and then stripped off of the working electrode during a second oxidation- reduction- strip cycle.
  • a first metal from a first metal-containing particle may be oxidized, reduced (e.g., deposited onto a working electrode), and then stripped off of the working electrode during a first oxidation-reduction-strip cycle.
  • a second metal from a second metal-containing particle may be oxidized, reduced (e.g., deposited onto a working electrode), and then stripped off of the working electrode
  • a third metal from a third metal-containing particle may be oxidized, reduced (e.g., deposited onto a working electrode), and then stripped off of the working electrode during a third oxidation-reduction-strip cycle. Further metals present in the sample may undergo this same process during further (e.g., fourth, fifth, sixth, etc.) oxidation-reduction- strip cycles.
  • the one or more electric potentials may be each, independently applied for any suitable amount of time.
  • the one or more electric potentials may each be applied for at least 1 second, at least 5 seconds, at least 10 seconds, at least 15 seconds, at least 30 seconds, at least 45 seconds, at least 60 seconds, at least 2 minutes, at least 4 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, or at least 30 minutes.
  • the one or more electric potentials may each be applied for less than or equal to 60 minutes, less than or equal to 30 minutes, less than or equal to 15 minutes, less than or equal to 10 minutes, less than or equal to 5 minutes, less than or equal to 4 minutes, less than or equal to 2 minutes, less than or equal to 60 seconds, less than or equal to 45 seconds, less than or equal to 30 seconds, less than or equal to 15 seconds, less than or equal to 10 seconds, or less than or equal to 5 seconds.
  • Combinations of the above referenced ranges are also possible (e.g., at least one second and less than or equal to 60 minutes, at least 15 seconds and less than or equal to 2 minutes, at least 30 seconds and less than or equal to 5 minutes, at least 1 minute and less than or equal to 10 minutes, at least 5 minutes and less than or equal to 30 minutes, at least 15 minutes and less than or equal to 60 minutes). Other ranges are also possible.
  • one or more positive electric potentials is applied such that at least a portion of the metal present in the metal- containing particles is oxidized.
  • the plurality of metal-containing particles are directly oxidized with the applied electric potential without the use of an oxidizing agent.
  • the metal-containing particles comprise silver and the positive electric potential may directly oxidize the silver from AgO to Ag+.
  • the first metal- containing particle comprises a first metal and the first electric potential may directly oxidize at least a portion of the first metal.
  • a second metal-containing particle comprises a second metal and the second electric potential may directly oxidize at least a portion of the second metal.
  • third type of metal-containing particle is used and a third electric potential may directly oxidize at least a portion of the third metal.
  • applying an electric potential to oxidize at least a portion of a metal from one type of metal-containing particle does not oxidize the metal from another type of metal-containing particles present in the sample or liquid.
  • applying the electric potential to oxidize at least a portion of the metal from the first type of metal- containing particle does not oxidize the metal from the second (or third or other) type(s) of metal-containing particles.
  • the first type of metal-containing particle can be quantified prior to the oxidation of the second type (or third type, or other types) of metal- containing particle present.
  • each positive electric potential may independently be at least 0 V, at least 0.1 V, at least 0.2 V, at least 0.5 V, at least 1 V, at least 1.2 V, at least 1.5 V, at least 1.7 V, at least 2 V, at least 2.5 V, at least 3 V, at least 3.5 V, at least 4 V, at least 4.5 V, at least 5 V, at least 5.5 V, at least 6 V, at least 7 V, at least 8 V, or at least 9 V.
  • the positive electric potential may be less than or equal to 10 V, less than or equal to 9 V, less than or equal to 8 V, less than or equal to 7 V, less than or equal to 6 V, less than or equal to 5.5 V, less than or equal to 5 V, less than or equal to 4.5 V, less than or equal to 4 V, less than or equal to 3.5 V, less than or equal to 3 V, less than or equal to 2.5 V, less than or equal to 2 V, less than or equal to 1.7 V, less than or equal to 1.5 V, less than or equal to 1.2 V, less than or equal to 1 V, less than or equal to 0.5 V, less than or equal to 0.2 V, or less than or equal to 0.1 V.
  • the above referenced ranges are also possible (e.g., at least 0V and less than or equal to 2V, at least 0.1 V and less than or equal to 2V, at least 0.1 V and less than or equal to 10 V). Other ranges are also possible.
  • the magnitude the positive electric potential may depend, at least in some part, on the electrode material (e.g., the reference electrode material, the working electrode material) and/or the metal-containing particle(s) material, and would be capable of selecting a suitable range of electric potentials based upon the teachings of this specification.
  • the reference electrode comprises a carbon material and the positive electric potential may be selected to be at least 0V and less than or equal to 2V.
  • each negative electric potential may independently be less than or equal to -0.1V, less than or equal to -0.2 V, less than or equal to -0.5 V, less than or equal to -0.7 V, less than or equal to -1 V, less than or equal to -1.2 V, less than or equal to -1.5 V, less than or equal to -1.7 V, less than or equal to -2 V, less than or equal to -2.5 V, less than or equal to -3 V, less than or equal to -3.5 V, less than or equal to -4 V, less than or equal to -4.5 V, less than or equal to -5 V, less than or equal to -5.5 V, less than or equal to -6 V, less than or equal to -7 V, less than or equal to -8 V,
  • the negative electric potential is greater than or equal to -10 V, greater than or equal to -9 V, greater than or equal to -8 V, greater than or equal to -7 V, greater than or equal to -6 V, greater than or equal to -5.5 V, greater than or equal to -5 V, greater than or equal to -4.5 V, greater than or equal to -4 V, greater than or equal to -3.5 V, greater than or equal to -3 V, greater than or equal to -2.5 V, greater than or equal to -2.0 V, greater than or equal to -1.7 V, greater than or equal to -1.5 V, greater than or equal to -1.2 V, greater than or equal to -1 V, greater than or equal to -0.7 V, greater than or equal to -0.5 V, or greater than or equal to -0.2 V.
  • the magnitude the negative electric potential may depend, at least in some part, on the electrode material (e.g., the reference electrode material, the working electrode material) and/or the metal-containing particle(s) material and would be capable of selecting a suitable range of electric potentials based upon the teachings of this specification.
  • the reference electrode comprises a carbon material and the negative electric potential may be selected to be less than or equal to -0.1 V and greater than or equal to - 2.0V.
  • the detection of two or more analytes may comprise applying a first series of electric potentials to determine the presence and/or amount of a first analyte (e.g., a first analyte bound to a first type of metal-containing particles), and applying a second series of electric potentials, different than the first series of electric potentials, to determine the presence and/or amount of a second analyte (e.g., a second analyte bound to a second type of metal-containing particles, different than the first type of metal-containing particles).
  • a first analyte e.g., a first analyte bound to a first type of metal-containing particles
  • a second series of electric potentials different than the first series of electric potentials
  • the detection may further comprise applying a third series of electric potentials to determine the presence and/or amount of the third analyte (e.g., a third analyte bound to a third type of metal-containing particle), where the third series of electric potentials is the same or different than the first series of electric potentials and/or the second series of electric potentials.
  • a third series of electric potentials to determine the presence and/or amount of the third analyte (e.g., a third analyte bound to a third type of metal-containing particle), where the third series of electric potentials is the same or different than the first series of electric potentials and/or the second series of electric potentials.
  • detection of two or more analytes comprises applying two or more electric potentials to generate a current versus voltage curve.
  • a peak on the current versus voltage curve may correspond to an oxidation state of a particular metal.
  • the first type of metal-containing particle may have a peak in current corresponding to a first voltage on a current versus applied voltage curve
  • the second type of metal-containing may have a peak in current corresponding to a second voltage on the current versus applied voltage curve.
  • the peak on the current versus applied voltage curve may correspond to voltages at which oxidation occurs and/or at which oxidized metal (e.g., metal from the metal containing particle) is deposited onto the working electrode.
  • oxidized metal e.g., metal from the metal containing particle
  • two or more metal-containing particles may be orthogonal - that is to say that the current versus voltage signal from one type of metal-containing particle does not influence the signal from a different metal-containing particle.
  • the first type of metal-containing particle may oxidize at a first voltage potential and the second type of metal-containing particle may oxidize at a second voltage potential, greater than the first voltage potential.
  • silver-containing particles will oxidize at a significantly lower voltage potential than gold-containing particles.
  • Silver could first be quantified by oxidizing the silver particles with a potential above the silver redox potential but below the gold redox potential and performing anodic stripping voltammetry. Then, the gold-containing particles may be oxidized at the higher potential and the anodic stripping voltammetry could be repeated at the potentials required for gold deposition and stripping. In such a two-step protocol, the electrolyte may be changed prior to oxidizing the gold-containing particles.
  • the third type of metal-containing particle may oxidize at a third voltage potential, greater than the first voltage potential and/or the second voltage potential.
  • the differences in voltage potential which oxidizes two or more of the metal-containing particles may permit detection of multiple analytes simultaneously (e.g., by applying an increasing voltage potential and observing peaks in the corresponding current).
  • An exemplary current versus voltage curve (for three types of metal-containing particles) is shown in FIG. 25D.
  • the metal in the metal-containing particle is already in an oxidized state and an initial electric potential could be chosen to reduce the metal in the metal-containing particle.
  • the difference between the electric potential at which a first metal in a first type of metal-containing particle oxidizes and the electric potential at which a second metal in a second type of metal-containing particle oxidizes may be greater than or equal to 0.1 V, greater than or equal to 0.2 V, greater than or equal to 0.3 V, greater than or equal to 0.4 V, greater than or equal to 0.5 V, greater than or equal to 0.6 V, greater than or equal to 0.7 V, greater than or equal to 0.8 V, greater than or equal to 0.9 V, greater than or equal to 1 V, greater than or equal to 1.2 V, greater than or equal to 1.4 V, greater than or equal to 1.6 V, greater than or equal to 1.8 V, greater than or equal to 2 V, greater than or equal to 2.5 V, greater than or equal to 3
  • the difference between the electric potential at which a first metal in a first type of metal-containing particle oxidizes and the electric potential at which a second metal in a second type of metal-containing particle oxidizes may be less than or equal to 10 V, less than or equal to 9 V, less than or equal to 8 V, less than or equal to 7 V, less than or equal to 6 V, less than or equal to 5 V, less than or equal to 4.5 V, less than or equal to 4 V, less than or equal to 3.5 V, less than or equal to 3 V, less than or equal to 2.5 V, less than or equal to 2 V, less than or equal to 1.8 V, less than or equal to 1.6 V, less than or equal to 1.4 V, less than or equal to 1.2 V, less than or equal to 1 V, less than or equal to
  • Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0.1 V and less than or equal to 10 V, greater than or equal to 0.1 V and less than or equal to 2 V). Other ranges are also possible.
  • the difference between electric potentials between two or more types of metal-containing particles may be measured using ASV.
  • the electrolyte undergoes an intermediate redox reaction.
  • the intermediate redox reaction oxidizes and/or promotes the deposition of the metal on the working electrode.
  • the potential from the working electrode directly oxidizes the metal from the metal containing particle.
  • the deposited metal layer may be removed from the working electrode by changing a voltage on the working electrode.
  • the current may be measured while changing the voltage to determine the amount of analyte present in the fluid.
  • current measuring step 585 comprises changing the voltage of the working electrode such that deposited metal 580 is removed from the working electrode, and quantifying the change in current on the working electrode.
  • an area under the curve of a plot of measuring current versus applied electric potential is generally proportional to the amount of metal deposited on the working electrode, which is also proportional to the amount of analyte originally present in the fluid. Accordingly, by measuring current by changing the voltage of the working electrode may determine the amount of analyte present in the fluid.
  • the current is measured as the electric potential is changed
  • the electric potential is changed at a particular rate. For example some embodiments, the electric potential is changed at a rate of at least 1 mV/s. In some embodiments, the electric potential is changed at a rate of at least 1 mV/s, at least 2 mV/s, at least 5 mV/s, at least 10 mV/s, at least 20 mV/s, at least 50 mV/s, at least 100 mV/s, at least 200 mV/s, at least 500 mV/s, at least 1 V/s, or at least 2 V/s.
  • the electric potential is changed at a rate of less than or equal to 5 V/s, less than or equal to 2 V/s, less than or equal to 1 V/s, less than or equal to 500 mV/s, less than or equal to 200 mV/s, less than or equal to 100 mV/s, less than or equal to 50 mV/s, less than or equal to 20 mV/s, less than or equal to 10 mV/s, less than or equal to 5 mV/s, or less than or equal to 2 mV/s.
  • the methods described herein may be useful for determining and/or quantifying the amount of one or more analytes present in the fluid or sample.
  • the fluid or sample is whole blood.
  • the fluid is a sample obtained from a patient such as whole blood, serum, plasma, urine, sputum, sweat, and/or other biological fluids. Methods for collecting such fluids are known in the art.
  • the fluid or sample is introduced into the device (e.g., via a microfluidic channel in fluidic communication with a reaction chamber).
  • the fluid or sample may be diluted (e.g., prior to determining and/or quantifying the amount of analyte present in the fluid or sample).
  • a buffer solution may be added to the reaction chamber containing the fluid or sample comprising the analyte (or analyte-containing biological particle) such that the fluid or sample is diluted.
  • the buffer solution is contained within a reservoir in fluidic
  • the fluid or sample may be diluted in a buffer solution prior to, or during, the introduction of the plurality of capture structures and/or the plurality of metal-containing particles to the reaction chamber.
  • an analyte in a fluid or sample is readily determinable without any subsequent process steps.
  • the analyte-containing sample is first processed to expose the analyte to allow it to be determinable by one or more methods described herein.
  • the analyte is present in (e.g., contained within or on) an analyte-containing biological particle, which is present in the fluid or sample.
  • the analyte-containing biological particle may be a virion, a bacterium, a protein complex, an exosome, a cell, or fungi.
  • the analyte may be exposed from the analyte-containing biological particle such that the analyte is available to form a bound complex (e.g., unhindered).
  • the analyte-containing biological particle is lysed (e.g., in the reaction chamber, in the lysis region) such that the analyte is released from the analyte-containing biological particle.
  • a lysing solution may be added to the reaction chamber containing the fluid comprising analyte such that the analyte is released from the analyte-containing biological particle. Lysing solutions are described in more detail below.
  • the analyte-containing biological particle is lysed via mechanical agitation, heating, washing, and/or shearing of the analyte-containing biological particle (e.g., via ultrasonic agitation).
  • the lysing step (e.g., comprising introducing the lysing solution to the reaction chamber containing the fluid comprising the analyte-containing biological particle) may open up HCV virions to release the core antigen, monomerize the core antigen, inactivate the host-derived antibodies against the core antigen, and/or dissociate the core antigen from the interactions with blood components other than the antibody against the core antigen.
  • exposing the analyte from the biological particle such that the analyte is available to form a bound complex comprises changing the pH, temperature, and/or ionic strength of the fluid comprising biological particle such that the analyte is available to form a bound complex.
  • an analyte-containing biological particle may be isolated (e.g., isolated from other components of the fluid sample).
  • a sample comprising a plurality of analyte-containing biological particles is added to a capture substrate, or the capture substrate is exposed to a fluid including the plurality of analyte-containing biological particles.
  • the capture substrate and a buffer solution are added to a sample comprising a plurality of analyte-containing biological particles. At least a portion of the analyte- containing biological particles may attach to the capture substrate. In certain embodiments, the analyte-containing biological particle may attach to the capture substrate via formation of a non-specific bond. In some cases, the analyte-containing biological particle may interact with a functional group present on the surface of the capture substrate.
  • the analyte-containing biological particle may attach to the capture substrate and/or a functional group present on the surface of the capture substrate via a bond such as an ionic bond, a covalent bond (e.g., carbon-carbon, carbon-oxygen, oxygen-silicon, sulfur- sulfur,
  • a bond such as an ionic bond, a covalent bond (e.g., carbon-carbon, carbon-oxygen, oxygen-silicon, sulfur- sulfur,
  • the capture substrate nonspecifically binds to the analyte-containing biological particle (e.g., a virion, a whole blood cell).
  • analyte-containing biological particle e.g., a virion, a whole blood cell
  • any components not attached to the capture substrate may be removed.
  • removing components not attached to the capture substrate involves a magnetic separation and/or a washing step (e.g., washing the capture substrate with a hypotonic solution).
  • the capture substrate comprises a plurality of magnetic particles.
  • a magnetic element located adjacent (e.g., directly adjacent) the reaction chamber may be activated such that a magnetic field is applied to the capture substrate such that the capture substrate (e.g., and any components bound to the capture substrate) is isolated within the reaction chamber, and any unbound components may be removed (e.g., washed) from the reaction chamber.
  • the components e.g., the analyte-containing biological particle, the analyte bound to the capture substrate may be resuspended by introducing a fluid (e.g., a lysing solution, a washing buffer, an electrolyte) into the reaction chamber.
  • a fluid e.g., a lysing solution, a washing buffer, an electrolyte
  • the components may be captured again to allow for one or more further washing steps to remove any unbound components. Accordingly, multiple capture and suspension steps may be performed.
  • suspending/resuspending the components may be stored in a reservoir or source in fluidic communication with the reaction chamber.
  • the analyte-containing biological particles may be suspending/resuspended in the electrolyte.
  • the analyte may be subjected to a process that exposes the analyte such that the analyte is available to form a bound complex, as described above, and the analyte may be quantified.
  • the capture substrate may comprise any suitable material capable of facilitating attachment of an analyte-containing biological particle.
  • capture substrate comprises a magnetic material.
  • the capture substrate may be charged.
  • the capture substrate may be uncharged.
  • the capture substrates/capture structures described herein may permit the nonspecific capture of analyte-containing biological particles, e.g., without the need for functionalizing the surface of the capture substrate with specific antibodies corresponding to the analyte and/or specific functional groups.
  • the capture substrate comprises a plurality of beads.
  • the plurality of beads may be magnetic.
  • the plurality of these may be substantially non-magnetic (e.g., polystyrene beads).
  • the plurality of beads may have any suitable size.
  • the plurality of beads have an average diameter of at least 100 nm, at least 200 nm, at least 500 nm, at least 1 micron, or at least 2 microns.
  • the plurality of beads have an average diameter of less than or equal to 5 microns, less than or equal to 2 microns, less than or equal to 1 ⁇ , less than or equal to 500 nm, or less than or equal to 200 nm. Combinations of the above referenced ranges are also possible (e.g., at least 100 nm and less than or equal to 5 microns). Other ranges are also possible.
  • suitable capture substrates/capture structures include non-magnetic resins, polymeric packing (e.g., polystyrene-divinylbenzene), and microfibers (e.g., electro spun microfibers).
  • polymeric packing e.g., polystyrene-divinylbenzene
  • microfibers e.g., electro spun microfibers
  • a buffer solution may be added to a sample and/or a fluid.
  • the buffer solution is stored in a reservoir or source connected to and/or in fluidic communication with the reaction chamber and/or another suitable chamber of the device.
  • the buffer solution comprises a salt.
  • the buffer solution comprises a chlorine-containing salt.
  • the salt is selected from the group consisting of sodium acetate, zinc acetate, NaCl, LiCl, CsCl, copper acetate, and combinations thereof.
  • the buffer solution may comprise metal acetate.
  • the buffer solution is selected for use with whole blood.
  • the buffer solution comprises a salt and has a salt
  • the buffer solution has a salt concentration of less than or equal to 5 M, less than or equal to 2 M, less than or equal to 1 M, less than or equal to 500 mM, less than or equal to 200 mM, less than or equal to 100 mM, less than or equal to 50 mM, less than or equal to 20 mM, less than or equal to 10 mM, less than or equal to 5 mM, or less than or equal to 2 mM. Combinations of the above-referenced ranges are also possible (e.g., at least 1 mM and less than or equal to 1 , at least 5 mM and less than or equal to 5 M). Other ranges are also possible.
  • the buffer solution comprises 50 mM sodium acetate, at least 1 mM and less than or equal to 5 mM zinc acetate, and at least 50 mM and less than or equal to 200 mM sodium chloride.
  • the buffer solution has a pH of at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9. In some embodiments, the buffer solution has a pH of less than or equal to 9, less than or equal to 8, less than or equal to 7, less than or equal to 6, or less than or equal to 5. Combinations of the above reference ranges are also possible (e.g., at least 4 and less than or equal to 10, at least 4 and less than or equal to 7). Other ranges are also possible.
  • a lysing solution may be added to a sample comprising a plurality of analyte-containing biological particles (e.g., cells) to expose the analyte from the analyte-containing biological particle such that the analyte is available to form a bound complex.
  • the lysing solution is stored in a reservoir or source connected to and/or in fluidic communication with the reaction chamber and/or another suitable chamber of the device.
  • the lysing solution comprises a detergent, a denaturant, a reducing agent, a strong acid (e.g., hydrochloric acid having, for example, a concentration of greater than or equal to 0.25 M and less than or equal to 1M), or combinations thereof.
  • a strong acid e.g., hydrochloric acid having, for example, a concentration of greater than or equal to 0.25 M and less than or equal to 1M
  • the detergent includes a surfactant.
  • the surfactant is an anionic surfactant (e.g., sodium dodecyl sulfate (SDS)), a cationic surfactant (e.g., alkyltrimethyl ammonium chloride, alkylmethyl ammonium bromide), a non- ionic surfactant (e.g., an alkyl poly(ethylene oxide) such as Triton X-100), or a zwitterionic surfactant (e.g., 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate, lauryl sulfobetaine, N-hexadecyl-N,N-dimethyl-3-ammonio-l-propanesulfonate).
  • SDS sodium dodecyl sulfate
  • a cationic surfactant e.g., alkyltrimethyl ammonium chloride, alkylmethyl am
  • the lysing solution comprises a denaturant such as quanidium hydrochloride, sodium thiocyanate, or urea.
  • the lysing solution comprises a reducing agent.
  • reducing agents include mercaptoethanol, DTT, glutathione, cysteine, tris(2- carboxyethyl)phosphine hydrochloride, cysteamine, dimethylamino ethanethiol,
  • diethylaminoethanethiol diethylaminoethanethiol, and diisopropylaminoethanethiol.
  • a blocking agent may be added to the fluid comprising the analyte.
  • the blocking agent may reduce any undesirable non-specific binding/adsorption.
  • the blocking agent is stored in a reservoir or source connected to and/or in fluidic communication with the reaction chamber and/or another suitable chamber of the device.
  • suitable blocking agents include bovine serum albumin (BSA), casein, fish gelatin, polyvinylpyrrolidone, pig gelatin, mouse serum, or the like.
  • devices and methods used to quantify the amount of analyte present in a fluid sample from a patient are provided.
  • the devices and methods described herein may be useful for detecting the presence of one or more antigens (e.g., a viral antigen such as cAg or p24) in a fluid sample from a patient.
  • a sample suspected of containing the hepatitis C virus may be added to a reaction chamber of a device, and a capture substrate may be introduced to the reaction chamber such that at least a portion of the viruses attach to the capture substrate. Any components from the sample not attached to the substrate may then be removed from the reaction chamber.
  • the virus may then be lysed (e.g., by introducing a lysis solution into the reaction chamber flowed from a reservoir or source in fluidic
  • analyte such as cAg or other HCV antigen is exposed and capable of forming a bound complex with a capture structure and a metal-containing particle.
  • a plurality of capture structures and a plurality of metal-containing particles may then be added to the reaction chamber to form the bound complex. Any unbound metal-containing particles may be separated from the bound complex and an electrolyte may be added to the bound complex. Voltammetry may be then performed to determine the amount of analyte present in a detection region downstream and in fluidic communication with the reaction chamber.
  • the analyte may be present in the sample in a manner suitable for forming a bound complex, and does not require using a capture substrate to isolate the analyte or lysing of a analyte-containing biological particle. While much of the description above relates to the use of a single reaction chamber, those skilled in the art would understand based upon the teachings of this specification that each reaction step may be conducted in an individual reaction chamber in fluidic
  • the presence of a particular antigen may indicate the patient carries a particular disease.
  • a patient may be diagnosed with a particular disease (e.g., hepatitis C) if the methods described herein determine that cAg (or another HCV antigen) is present in the fluid sample from the patient.
  • the devices and methods described herein may involve diagnosing a patient having (or suspected of having) hepatitis C by testing a sample (e.g., a whole blood sample) from the subject containing an HCV antigen and/or cAg using one or more methods described herein.
  • the methods may involve diagnosing the patient as not having hepatitis C, in embodiments in which the sample from the subject does not contain an HCV antigen and/or cAg.
  • a method involves identifying a patient, from two or more patients, as having or not having hepatitis C, by testing patient samples (e.g., whole blood samples) from the two or more patients according to one or more of the methods described herein. The method may involve determining the patient as having hepatitis C where the patient sample contains an HCV antigen and/or cAg, or determining that the patient does not have hepatitis C where the patient sample does not contain the HCV antigen and/or cAg.
  • a patient may be diagnosed with a particular disease or condition (e.g., HIV/AIDS) if the methods described herein determine that p24 (or another HIV antigen) is present in the fluid sample from the patient.
  • a method described herein may involve diagnosing a patient having (or suspected of having) HIV infection by testing a sample (e.g., a whole blood sample) from the subject containing an HIV antigen using one or more methods described herein. The method may involve diagnosing the patient as not having HIV infection, in embodiments in which the sample from the subject does not contain an HIV antigen.
  • a method involves identifying a patient, from two or more patients, as having or not having HIV infection, by testing patient samples (e.g., whole blood samples) from the two or more patients according to one or more of the methods described herein.
  • the method may involve determining the patient as having HIV infection where the patient sample contains an HIV antigen, or determining that the patient does not have HIV infection where the patient sample does not contain the HIV antigen.
  • a patient may be diagnosed with a particular disease or condition (e.g., heart disease and/or risk for myocardial infarction) if the methods described herein determine that Troponin I (or another suitable cardiac marker protein) is present in the fluid sample from the patient.
  • a method described herein may involve diagnosing a patient having (or suspected of having) heart disease by testing a sample (e.g., a whole blood sample) from the subject containing a cardiac marker protein such as Troponin I using one or more methods described herein. The method may involve diagnosing the patient as not having heart disease, in embodiments in which the sample from the subject does not contain, or contains a relatively low concentration of, a cardiac marker protein such as Troponin I.
  • a method involves identifying a patient, from two or more patients, as having or not having heart disease, by testing patient samples (e.g., whole blood samples) from the two or more patients according to one or more of the methods described herein.
  • the method may involve determining the patient as having heart disease where the patient sample contains the cardiac marker protein (or a relatively high concentration of the cardiac marker protein), or determining that the patient does not have heart disease where the patient sample does not contain the cardiac marker protein (or a relatively low concentration of the cardiac marker protein).
  • the device may be incorporated into a system (e.g., a cartridge) for performing a method described herein.
  • the system comprises one or more additional components (e.g., one or more additional components in fluidic communication with the device).
  • exemplary system 2300 comprises a device 2310 comprising at least one microfluidic channel (e.g., such as device 200 illustrated in FIG. 4).
  • one or more reservoirs 2320 for storing containing a stored reagent may be in fluidic communication device 2310, as described herein (e.g., one or more reservoirs may be in fluidic communication with an inlet of device 2310).
  • each reservoir may be sealed (e.g., to prevent evaporation and/or contamination of any components).
  • the one or more reservoirs may comprise a blister pack.
  • Blister packs are generally known in the art and may comprise any suitable material for containing and/or sealing a stored reagent therein.
  • the one or more reservoir e.g., the blister pack
  • the blister pack may comprise aluminum foil (e.g., aluminum foil laminate).
  • the blister pack may be unsealed (e.g., upon the application of pressure to the blister pack such that the blister pack bursts and/or breaks) such that the stored reagent is introduced into an inlet of the device.
  • the one or more reservoirs 2320 is positioned within a structural housing 2325.
  • the system comprises a blood and/or plasma separation module 2330.
  • the blood and/or plasma separation module may comprise the plasma separation region as described herein (e.g., the plasma separation region comprising a membrane for separating plasma from the sample).
  • the system comprises one or more absorbent regions.
  • system 2300 may comprise one or more absorbent regions 2340 associated with, for example, one or more reaction chambers.
  • the absorbent region comprises an absorbent pad.
  • suitable absorbent pads include fibrous pads and/or lateral flow assay conjugate pads.
  • the absorbent region comprises a plurality of lyophilized pellets.
  • the system comprises one or more adhesive layers comprising an adhesive.
  • system 2300 comprises an adhesive layer 2350 (e.g., positioned between device 2310 and blister pack 2320).
  • suitable adhesive include pressure sensitive adhesives and hot melt adhesives.
  • the device comprises one or more valves.
  • the system and/or valve(s) comprises a valve film.
  • system 2300 may comprise a valve film 2360 adjacent (e.g., directly adjacent) device 2310. The valve film may be used, in some cases, as a valve, foil, or other obstruction to control fluid flow.
  • valve film may be used to isolate the fluid(s) inside the reaction chamber or other suitable component of the device from being in fluid communication with a downstream channel and/or a downstream reaction chamber until a desired time at which the fluid(s) are to be fluidically transferred to the downstream channel and/or downstream reaction chamber.
  • the valve, foil, or other obstruction may be actuated by being opened and/or removed.
  • the valve film may comprise any suitable material (e.g., a polymer such as PET or TPE).
  • a polymer such as PET or TPE
  • the polymer is elastomeric.
  • the valve film may also have any suitable thickness.
  • the thickness of the valve film is greater than or equal to 0.1 mm, greater than or equal to 0.2 mm, greater than or equal to 0.3 mm, greater than or equal to 0.4 mm, greater than or equal to 0.5 mm, greater than or equal to 0.6 mm, greater than or equal to 0.7 mm, greater than or equal to 0.8 mm, or greater than or equal to 0.9 mm.
  • the thickness of the valve film is less than or equal to 1 mm, less than or equal to 0.9 mm, less than or equal to 0.8 mm, less than or equal to 0.7 mm, less than or equal to 0.6 mm, less than or equal to 0.5 mm, less than or equal to 0.4 mm, less than or equal to 0.3 mm, or less than or equal to 0.2 mm. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0.1 mm and less than or equal to 1 mm). Other ranges are also possible.
  • system 2300 comprises a cover 2370 and/or a cover 2375 adjacent (e.g., directly adjacent) a surface of device 2310.
  • the cover e.g., cover 2370
  • the cover is a microfluidic backbone cover (e.g., for enclosing one or more channels of device 2310 and/or for providing structural support for device 2310).
  • the cover is an electrode foil comprising one or more electrodes.
  • the electrodes may be printed (e.g., screen printed) on the cover (e.g., the electrode foil).
  • the cover e.g., the microfluidic backbone cover, the electrode foil
  • the cover may have any suitable thickness.
  • the thickness of the cover is greater than or equal to 0.1 mm, greater than or equal to 0.2 mm, greater than or equal to 0.3 mm, or greater than or equal to 0.4 mm.
  • the thickness of the cover is less than or equal to 0.5 mm, less than or equal to 0.4 mm, less than or equal to 0.3 mm, or less than or equal to 0.2 mm. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0.1 mm and less than or equal to 0.5 mm). Other ranges are also possible.
  • a device, system, or portions thereof used to perform a method described herein can be fabricated of any suitable material.
  • suitable material include polymers (e.g., polypropylene, polyethylene, polystyrene, polycarbonate, poly(acrylonitrile, butadiene, styrene),
  • the devices and methods described herein can be carried out or used in combination with the methods, components, systems, and/or devices (e.g., microfluidic devices) described in one or more of: U.S. Patent Number U.S. 8,852,875, issued October 7, 2014, and entitled “Methods for Counting Cells”; U.S. Patent Number 8,911,957, issued December 16, 2014, and entitled “Devices and methods for detecting cells and other analytes"; U.S. Publication No. 2015/190802, filed January 6, 2015, and entitled “Fluid delivery devices, systems, and methods”; U.S. Publication No. 2015/190805, filed January 7, 2015, and entitled “Fluid delivery devices, systems, and methods”; U.S.
  • a "subject” or a “patient” refers to any animal such as a mammal (e.g., a human), for example, a mammal that may be susceptible to a disease or bodily condition.
  • subjects or patients include a human, a non-human primate, a cow, a horse, a pig, a sheep, a goat, a dog, a cat or a rodent such as a mouse, a rat, a hamster, a bird, a fish, or a guinea pig.
  • the invention is directed toward use with humans.
  • a patient may be a subject diagnosed with a certain disease or bodily condition or otherwise known to have a disease or bodily condition.
  • a patient may be diagnosed as, or known to be, at risk of developing a disease or bodily condition. In other embodiments, a patient may be suspected of having or developing a disease or bodily condition, e.g., based on various clinical factors and/or other data.
  • small molecule refers to molecules, whether naturally occurring or artificially created (e.g. , via chemical synthesis) that have a relatively low molecular weight.
  • a small molecule is an organic compound (i.e., it contains carbon).
  • the small molecule may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyl, carbonyls, and heterocyclic rings, etc.).
  • the molecular weight of a small molecule is at most about 1,000 g/mol, at most about 900 g/mol, at most about 800 g/mol, at most about 700 g/mol, at most about 600 g/mol, at most about 500 g/mol, at most about 400 g/mol, at most about 300 g/mol, at most about 200 g/mol, or at most about 100 g/mol.
  • the molecular weight of a small molecule is at least about 100 g/mol, at least about 200 g/mol, at least about 300 g/mol, at least about 400 g/mol, at least about 500 g/mol, at least about 600 g/mol, at least about 700 g/mol, at least about 800 g/mol, or at least about 900 g/mol, or at least about 1,000 g/mol. Combinations of the above ranges (e.g., at least about 200 g/mol and at most about 500 g/mol) are also possible.
  • drug refers to an agent that is administered to a subject to treat a disease, disorder, or other clinically recognized condition, or for prophylactic purposes, and has a clinically significant effect on the body of the subject to treat and/or prevent the disease, disorder, or condition.
  • Drugs include, without limitation, agents listed in the United States Pharmacopeia (USP), Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Ed., McGraw Hill, 2001; Katzung, B. (ed.) Basic and Clinical
  • the drug is one that has already been deemed safe and effective for use in humans or animals by the appropriate governmental agency or regulatory body.
  • drugs approved for human use are listed by the FDA under 21 C.F.R.
  • the following example demonstrates a general method for determining and/or quantifying an analyte from a whole blood sample, for example in a microfluidic device, according to the methods described herein.
  • HCV virions were captured from plasma and blood samples after dilution of the whole blood samples 2-5 fold in a buffer containing sodium acetate, zinc acetate, and sodium chloride, and incubating with capture substrates (capture structures) while mixing for 5 minutes. Capture structures were then separated by placing the tubes into a magnetic separator and removing the unbound supernatant. The amount of captured virions in this process typically exceeded 90% (measured by RT-PCR) and is shown in FIGs. 12A-12B. After a brief wash step, particles were separated again and are ready for the next step - lysis.
  • Virions attached to the capture substrates were lysed in a lysis solution containing detergents, and/or denaturants and/or reducing agents to release the core antigen, which was subsequently detected.
  • the lysis step opened up HCV particles to release the core antigen, monomerized the core antigen, inactivated the host-derived antibodies against the core antigen, and dissociated the core antigen from the interactions with blood components other than the antibody against the core antigen.
  • Lysing can be performed, for example, by combining 100 ⁇ sample with 50 ⁇ treatment solution, of the following composition:
  • 100 ⁇ treated sample can be mixed, for example, with 100 ⁇ of reaction buffer, of the following composition:
  • ELISA was performed as a control.
  • lysis can also be performed, for example, by combining sample and treatment solution containing detergent and acid such as 0.25M HC1 and 7% Triton X-100, 3.5% dodecylethylmethacrylatedimethylammonium bromide (C12PS) and 7% dodecyltrimethylammonium chloride (C12TAC) and incubating for 5 minutes.
  • the treated sample may then be neutralized by adding a neutralization solution containing, for example, 0.25M Tris, pH 7.6.
  • the lysed components ("lysate") in the lysis may contain detergents at a
  • the first step of the detection module was to dilute the lysate so that the matrix was compatible with the detection assay.
  • the dilution factor is generally a function of the volume of the lysate, the concentration of the detergents, and/or the robustness of downstream components to the chosen detergents.
  • 3 ⁇ g of 200 nm HCV specific silver particles are added to the diluted lysate. Particles were incubated with the lysate at room temperature for 15 minutes. Prior to running the assay, the silver particles were coated with antibodies able to capture the analyte.
  • protocols for conjugating antibodies included EDC coupling of antibodies to carboxylic acids on lipoic acid molecules that have already been attached to the silver particles.
  • the size of the silver particle was chosen as a balance between amplification (larger silver particles generally lead to larger amplification during electrochemical quantification) and labeling efficiency (smaller silver particles generally lead to improved ability to label every antigen with a silver particle and a capture structure.)
  • the capture structures Prior to running the assay, the capture structures were coated with antibodies able to capture the analyte.
  • protocols for coating the particles with antibodies included EDC coupling to carboxylic acids present on the surface of the particles.
  • the size of the capture structure was chosen as a balance between steric hindrance (small particles are generally more likely to successfully bind to an analyte on a large silver particle) and magnetic moment (large particles generally have a higher magnetic moment and are more easily removed from solution with a magnet).
  • An exemplary schematic of the above procedure is shown in FIG. 9.
  • analyte cAg
  • silver particles cAg
  • capture structures including those with analyte and silver attached are gathered near the magnet.
  • Complexes containing a capture structure, an analyte molecule, and a silver particle are referred to as bound complex.
  • Wash buffer was then added and the capture structures, including all bound complexes, were resuspended by vortexing and pipetting up and down.
  • the resuspended bound complexes (mag) were then put back on the stage and the wash buffer was removed.
  • a schematic of the bound complexes is shown in FIG. 10.
  • the wash buffer was composed of, for example, 0.1% Casein and 0.05% tween-20 in
  • the capture structures and bound complexes were then resuspended in the electrolyte for the final electrochemical quantification of silver content.
  • the electrolyte was, for example, 0.1 M NH4SCN.
  • Other electrolytes such as NaCl, KC1, NaBr, and KI were also explored.
  • the remaining steps involve anodic stripping voltammetry (ASV) detection strategy and are schematized in FIG. 11.
  • ASV stripping voltammetry
  • the solution containing electrolyte, capture structures, and bound complexes was then transferred to a screen-printed electrode where the working, counter, and reference electrodes all included carbon ink. Under the working electrode was a magnet. After all capture structures migrated to the surface of the electrode, a positive potential was applied (30 seconds, +0.7V vs. carbon quasi-reference electrode). At this point the silver was oxidized from Ag(0) to Ag(I).
  • FIG. 12G shows the concentration of the HCV core antigen versus peak current area ⁇ C), comparing the analyte signal (50 fM) and the background signal (0 fM). Exemplary data obtained from HCV clinical samples is shown in FIG.12G.
  • HRP ELISA was performed in a 96 well plate with 100 p24 per well using the same antibodies as above. ELISA was detected via HRP quenched with TMB. The experiment was also performed with 50 ⁇ ⁇ of p24 per reaction according to Example 1 with 200 nm silver particles. Average of two duplicates was plotted for each technique.
  • FIGs. 13-16 show the limit of detection for various concentrations of p24 HIV antigen.
  • the following example demonstrates the effect of particle size on detection of p24 HIV antigen.
  • EDC coupling was used to conjugate antibody to a range of lipoic-acid coated silver particles from 100 nm to 600 nm.
  • Antibody loading was either kept constant at 60 ⁇ g/mg silver or adjusted according to surface area.
  • 3 ⁇ g of silver was combined with 50 ⁇ , of a 250 fM p24 solution in PBS, 0.1% casein, 0.05% tween-20.
  • signal increases with particle volume when silver particles are 100 nm, 200 nm or 325 nm.
  • the concentration of silver particles in the immunoassay for 100 nm, 200 nm, 325 nm, and 600 nm were approximately 18 pM, 2.3 pM, 540 fM, 85 fM respectively.
  • HIV+ plasma samples were purchased from SeraCare®. All samples except the healthy plasma were positive for HIV RNA but negative for HIV antibodies.
  • HIV virions were captured from plasma and blood samples after dilution of the whole blood samples 2-5 fold in a buffer containing sodium acetate, zinc acetate, and sodium chloride, and incubating with capture substrates (capture structures) while mixing for 5 minutes. Capture structures were then separated by placing the tubes into a magnetic separator and removing the unbound supernatant. After a brief wash step, particles were separated again and are ready for the next step - lysis.
  • 60 ⁇ of blood was mixed with 0.5 mg of capture structures and 240 ⁇ of capture buffer containing 62.5 mM sodium acetate, pH 5.6, 2.5 mM zinc acetate and 104 mM NaCl to achieve a final concentration of 50 mM sodium acetate, 2 mM zinc acetate and 83 mM sodium chloride. At least 2 mM zinc acetate was used for K3EDTA- treated blood, 4 mM for citrate-treated blood, and 1 mM for heparin-treated blood.
  • Capture structures with attached virion particles were separated from blood solution by placing sample on magnet stage. All capture structures, including those with virions, are gathered near the magnet. Wash buffer was then added and the capture structures were resuspended by vortexing and pipetting up and down. The resuspended capture structures were then put back on the stage and the wash buffer was removed.
  • the wash buffer was composed of, for example, 50 mM sodium acetate, pH 5.6.
  • Virions attached to the capture substrates were lysed in a lysis solution containing detergents, and/or denaturants and/or acidifying agents and/or reducing agents to release the p24, which was subsequently detected.
  • the lysis step opened up HIV particles to release the p24, monomerized p24, inactivated the host-derived antibodies against the p24, and dissociated the core antigen from the interactions with blood components other than the antibody against p24.
  • Lysing can be performed, for example, by adding 6ul treatment solution, containing 5%Triton X-100 (or 1% triton X-100 in PBS with 0.1% casein and 0.05% tween-20) to the capture structures containing attached virions and incubating at room temperature for 30 minutes. Capture structures were then removed from solution containing the lysed analyte by placing sample on magnet stage. Supernatant containing the virion-derived analytes was removed and placed to another tube. To inactivate the host-derived antibodies, 25 ⁇ of glycine-HCl pH 1.8 was added to the 6 ⁇ of solution containing lysed virions and sample was incubated at room temperature for 30 minutes. To neutralize the acid, 34 ⁇ of neutralization buffer containing 25 ⁇ of 1.5 M Tris, pH 11, 1% casein, 3 ⁇ 1% Tween-20.
  • 3 ⁇ g of 200 nm HIV specific silver particles are added to the neutralized lysate.
  • Particles were incubated with the lysate at room temperature for 15 minutes. Prior to running the assay, the silver particles were coated with antibodies able to capture the analyte.
  • protocols for conjugating antibodies included EDC coupling of antibodies to carboxylic acids on lipoic acid molecules that have already been attached to the silver particles.
  • the size of the silver particle was chosen as a balance between amplification (larger silver particles generally lead to larger amplification during electrochemical quantification) and labeling efficiency (smaller silver particles generally lead to improved ability to label every antigen with a silver particle and a capture structure.)
  • the capture structures Prior to running the assay, the capture structures were coated with antibodies able to capture the analyte.
  • protocols for coating the particles with antibodies included EDC coupling to carboxylic acids present on the surface of the particles.
  • the size of the capture structure was chosen as a balance between steric hindrance (small particles are generally more likely to successfully bind to an analyte on a large silver particle) and magnetic moment (large particles generally have a higher magnetic moment and are more easily removed from solution with a magnet).
  • An exemplary schematic of the above procedure is shown in FIG. 9.
  • the wash buffer was composed of, for example, 0.1% Casein and 0.05% tween-20 in
  • the capture structures and bound complexes were then resuspended in the electrolyte for the final electrochemical quantification of silver content.
  • the electrolyte was, for example, 0.1 M NH4SCN.
  • Other electrolytes such as NaCl, KC1, NaBr, and KI were also explored.
  • the remaining steps involve anodic stripping voltammetry (ASV) detection strategy and are schematized in FIG. 11.
  • ASV stripping voltammetry
  • the solution containing electrolyte, capture structures, and bound complexes was then transferred to a screen-printed electrode where the working, counter, and reference electrodes all included carbon ink. Under the working electrode was a magnet. After all capture structures migrated to the surface of the electrode, a positive potential was applied (30 seconds, +0.7V vs. carbon quasi-reference electrode). At this point the silver was oxidized from Ag(0) to Ag(I).
  • ASV Voltammetry
  • Silver was then stripped from the electrode, the second step for ASV.
  • the potential of the working electrode was ramped from reducing (-1.0 V) to oxidizing (0 V) at 10 mV/s.
  • the voltage was at the potential for oxidizing silver, the current peaked.
  • the area under the peak is directly related to the number of silver atoms oxidized. This number is then also directly related to the number of silver particles the analyte was able to successfully form bound complexes with.
  • Example 5 The following example demonstrates that the methods and electrolytes described herein do not release the silver particles from the antigen according to certain embodiments described herein.
  • silver particles were added to p24 HIV antigen and the supernatant was collected. As shown in FIGs. 19-21, substantially no silver particles were present in the supernatant, indicating that the electrolyte did not release the silver particles from the antigen when the bound complex was exposed to the electrolyte.
  • FIGs. 22A-22F show the effect of increasing the concentration of the electrolyte (NH 4 SCN), which resulted in changes in the peak area and shape.
  • 0.1M NH4SCN provided the largest peak area but distorted the shape of the peaks at high silver concentrations.
  • 0.5M NH4SCN, 1M NH4SCN, and 5 M NH4SCN were also tried and all of these decreased signal even farther.
  • Example 5 was performed with "defined bound complexes" where a known concentration of silver particles were introduced to the immunoassay with a high
  • the theoretical peak area for 1 pM of silver particles is approximately 1100 ⁇ C.
  • Plasma is first separated from blood components using a membrane based separator.
  • Analytes can include soluble protein markers found in plasma, such as Troponin I, or protein markers associated with or contained within pathogens such as bacteria or viruses, for example, core antigen of HCV.
  • soluble protein markers such as Troponin I
  • simple plasma dilution with a buffer containing, for example PBS, 0.1% casein, 0.05% Tween-20 and 0.5mg/ml mouse IgG can be required. Dilution can be performed, for example, by combining 25 ⁇ of plasma sample with 25 ⁇ buffer solution.
  • virions present in plasma sample may be lysed first in a lysis solution containing detergents, and/or acid, and/or denaturants and/or reducing agents to release the core antigen, which can be subsequently detected (for example, as described in Example 1).
  • the lysis step opens HCV particles to release the core antigen, monomerizes the core antigen, inactivates the host-derived antibodies against the core antigen, dissociates the core antigen from the interactions with blood components, endogenous antibodies against the core antigen, and dissociates the core antigen from the interactions with blood components other than the antibody against core antigen.
  • Lysing can be performed, for example, by combining 25 ⁇ of plasma sample with 25 ⁇ treatment solution, of the following composition:
  • 50 ⁇ treated sample can be mixed, for example, with 50 ⁇ of reaction buffer, of the following composition:
  • lysing can be performed, for example, by combining 25 ⁇ of plasma sample with 25 ⁇ of treatment solution, of the following composition:
  • the lysed components ("lysate") in the lysis may contain detergents at a concentration in which detection may not be accurately performed. Therefore, the first step of the detection module may include dilution of the lysate such that the matrix is compatible with the detection assay.
  • the dilution factor is generally a function of the volume of the lysate, the concentration of the detergents, and/or the robustness of downstream components to the chosen detergents.
  • the capture structures Prior to running the assay, can be, in some cases, coated with antibodies able to capture the analyte.
  • protocols for coating the particles with antibodies included EDC coupling to carboxylic acids present on the surface of the particles.
  • the size of the capture structure may be chosen as a balance between steric hindrance (e.g., smaller particles are generally more likely to successfully bind to an analyte on a large silver particle) and magnetic moment (e.g., larger particles generally have a higher magnetic moment and are more easily removed from solution with a magnet).
  • a reference electrode was fabricated by screen printing a layer of carbon ink over an
  • the Ag/AgCl reference electrode size was smaller in area (width, length) than the carbon reference electrode covering it. Silver content in the adjacent solution was measured via anodic stripping voltammetry.
  • FIG. 24A shows four different reference electrode configurations that were tested: Ag/AgCl Only, Ag/AgCl Tip, Ag/AgCl under Carbon, and Carbon Only using cyclic voltammetry.
  • the electrolyte used was 0.5M NaCl.
  • Ag/AgCl was not covered by carbon (Ag/AgCl Only and Ag/AgCl Tip)
  • silver ions were believed to have leached into the system, as evidenced by the growing background peaks near 0V (FIG. 24A).
  • the carbon- coated silver reference electrode (Ag/AgCl under Carbon) was also observed to generating a stable, reproducible reference potential (FIG. 24B).
  • Electrochemical measurement of silver content in solution via anodic stripping voltammetry was observed without interference from silver on the electrode, as shown in FIG. 24C for Ag/AgCl under Carbon reference electrode for solutions containing 500 fM silver (as compared to 0M Silver and NaCl only solutions).
  • the following example demonstrates the detection of two or more analytes in a sample (e.g., by using two or more types of metal-containing particles).
  • An electrode system with a stable reference potential was used - such as the system with carbon-coated Ag/AgCl described in Example 7.
  • Metal-containing particles (such as Ag and Au) were quantified by oxidizing a noble metal particle and then quantifying
  • Metal-containing particles where the metal has multiple possible redox states (such as CuO) were quantified by applying voltages to generate particular redox states or transitions. Conditions were found such that individual metal- containing particles are orthogonal - meaning that the signal from one type of metal- containing particle does not influence the signal from a different metal-containing particle.
  • silver nanoparticles will oxidize at a significantly lower potential than gold nanoparticles.
  • Silver could first be quantified by oxidizing the silver particles with a potential above the silver redox potential but below the gold redox potential and performing anodic stripping voltammetry. Then, the gold nanoparticles could be oxidized at the higher potential and the anodic stripping voltammetry could be repeated at the potentials required for gold deposition and stripping. In such a two-step protocol, the electrolyte may be changed prior to oxidizing the gold nanoparticles.
  • FIG. 25A shows a plot of current ( ⁇ ) versus voltage (V) for the simultaneous detection of three metal-containing particles (Ag, CuO, and Au). 0.5 ⁇ g of each metal- containing particle was dried on the working electrode of a screen-printed electrode.
  • FIG. 25B shows a plot of current ( ⁇ ) versus voltage (V) when the initial oxidation potential was set to 0.6V vs. Ag/AgCl for 30 seconds prior to depositing at -1.5V and stripping at 10 mV/s.
  • the gold nanoparticle did not appear to dissolve during the initial oxidation step, ultimately leading to a higher gold peak.
  • 0.5 ⁇ g of each metal-containing particle was dried on the working electrode of a screen-printed electrode. Particles were covered with ⁇ of 0.5M NaCl as an electrolyte.
  • FIG. 25C shows a plot of current ( ⁇ ) versus voltage (V) when different potentials were used for the deposition voltage, such that different metals were deposited. For example, at -1.3V, copper was not reduced to a form that can provide a peak during the stripping step. However, at -1.5V, copper deposited on the working electrode and was stripped during the final step of anodic stripping voltammetry.
  • FIG. 25D shows the identification of conditions such that individual metal-containing particles were orthogonal - meaning that the signal from one type of metal-containing particle did not influence the signal from a different metal-containing particle.
  • a reference to "A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase "at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.
  • At least one of A and B can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another
  • Examples of such terms related to shape, orientation, and/or geometric relationship include, but are not limited to terms descriptive of: shape - such as, round, square, circular/circle, rectangular/rectangle, triangular/triangle,
  • direction - such as, north, south, east, west, etc.
  • surface and/or bulk material properties and/or spatial/temporal resolution and/or distribution - such as, smooth, reflective, transparent, clear, opaque, rigid, impermeable, uniform(ly), inert, non-wettable, insoluble, steady, invariant, constant, homogeneous, etc.; as well as many others that would be apparent to those skilled in the relevant arts.
  • a fabricated article that would described herein as being " square” would not require such article to have faces or sides that are perfectly planar or linear and that intersect at angles of exactly 90 degrees (indeed, such an article can only exist as a mathematical abstraction), but rather, the shape of such article should be interpreted as approximating a " square,” as defined mathematically, to an extent typically achievable and achieved for the recited fabrication technique as would be understood by those skilled in the art or as specifically described.
  • two or more fabricated articles that would described herein as being " aligned” would not require such articles to have faces or sides that are perfectly aligned (indeed, such an article can only exist as a mathematical abstraction), but rather, the arrangement of such articles should be interpreted as approximating "aligned,” as defined mathematically, to an extent typically achievable and achieved for the recited fabrication technique as would be understood by those skilled in the art or as specifically described.

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Abstract

L'invention concerne en général des dispositifs et des procédés de détermination et/ou de quantification d'un ou de plusieurs analytes dans des fluides. Dans certains modes de réalisation, les dispositifs décrits ici peuvent être conçus de telle sorte qu'au moins deux opérations (par exemple, la lyse, la séparation magnétique, la remise en suspension, le mélange et/ou la mise à réagir) soient effectuées dans une ou plusieurs chambres de réaction. Les dispositifs et procédés décrits ici peuvent être utiles à la détermination et à la quantification de concentrations relativement faibles d'analytes présents dans un échantillon d'un patient (par ex., une gouttelette de sang total, une gouttelette de plasma, une gouttelette de fluide biologique). L'analyte peut être quantifié, dans certains cas, en liant l'analyte à une pluralité de particules contenant du métal et en mesurant le courant en changeant une tension sur une électrode active. Dans certains modes de réalisation, le dispositif est un dispositif microfluidique.
PCT/US2017/068203 2016-12-22 2017-12-22 Dispositifs et procédés pour déterminer un ou plusieurs analytes dans des fluides Ceased WO2018119401A2 (fr)

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CN110079828A (zh) * 2019-05-21 2019-08-02 西安汇创贵金属新材料研究院有限公司 一种金电解废液的处理装置及处理方法
WO2020091765A1 (fr) * 2018-10-31 2020-05-07 Xinova, LLC Détection de contaminants ou de particules comprenant la détection de la conductivité avec une buse de micro-injecteur
WO2020102429A1 (fr) * 2018-11-14 2020-05-22 The Board Of Trustees Of The Leland Stanford Junior University Dispositif microfluidique et procédés de diagnostic pour test d'allergie basé sur la détection de l'activation de basophiles
US20210208169A1 (en) * 2018-10-08 2021-07-08 Accure Health Inc. Automated assay processing methods and systems
CN114401591A (zh) * 2022-01-10 2022-04-26 奥特斯科技(重庆)有限公司 用于部件承载件制造、系统和方法中的含金属流体的再生器

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EP2345739B8 (fr) * 2004-05-03 2016-12-07 Handylab, Inc. Dispositif microfluidique permettant de traiter des échantillons contenant un polynucléotide
US7648844B2 (en) * 2005-05-02 2010-01-19 Bioscale, Inc. Method and apparatus for detection of analyte using an acoustic device
US7807454B2 (en) * 2006-10-18 2010-10-05 The Regents Of The University Of California Microfluidic magnetophoretic device and methods for using the same
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US20100075862A1 (en) * 2008-09-23 2010-03-25 Quanterix Corporation High sensitivity determination of the concentration of analyte molecules or particles in a fluid sample
EP2606154B1 (fr) * 2010-08-20 2019-09-25 Integenx Inc. Système d'analyse intégrée
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210208169A1 (en) * 2018-10-08 2021-07-08 Accure Health Inc. Automated assay processing methods and systems
US12449434B2 (en) * 2018-10-08 2025-10-21 Accure Health Inc. Automated assay processing methods and systems
WO2020091765A1 (fr) * 2018-10-31 2020-05-07 Xinova, LLC Détection de contaminants ou de particules comprenant la détection de la conductivité avec une buse de micro-injecteur
WO2020102429A1 (fr) * 2018-11-14 2020-05-22 The Board Of Trustees Of The Leland Stanford Junior University Dispositif microfluidique et procédés de diagnostic pour test d'allergie basé sur la détection de l'activation de basophiles
US12239982B2 (en) 2018-11-14 2025-03-04 The Board Of Trustees Of The Leland Stanford Junior University Microfluidic device and diagnostic methods for allergy testing based on detection of basophil activation
CN110079828A (zh) * 2019-05-21 2019-08-02 西安汇创贵金属新材料研究院有限公司 一种金电解废液的处理装置及处理方法
CN110079828B (zh) * 2019-05-21 2021-04-13 西安汇创贵金属新材料研究院有限公司 一种金电解废液的处理装置及处理方法
CN114401591A (zh) * 2022-01-10 2022-04-26 奥特斯科技(重庆)有限公司 用于部件承载件制造、系统和方法中的含金属流体的再生器

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