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WO2021214555A1 - Dispositif de dosage immunologique électrochimique - Google Patents

Dispositif de dosage immunologique électrochimique Download PDF

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Publication number
WO2021214555A1
WO2021214555A1 PCT/IB2021/050523 IB2021050523W WO2021214555A1 WO 2021214555 A1 WO2021214555 A1 WO 2021214555A1 IB 2021050523 W IB2021050523 W IB 2021050523W WO 2021214555 A1 WO2021214555 A1 WO 2021214555A1
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WIPO (PCT)
Prior art keywords
electrochemical
immunoassay device
electrochemical immunoassay
sensor
reference electrode
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2021/050523
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English (en)
Inventor
Vijaywanth MATHUR
Shweta DALVI
Ulhas DAFALE
Asha SIVADAS
Bhakti SARVAIYA
Rajani KADAM
Nikhil SANE
Ramesh MAMDAPURKAR
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Diasys Diagnostics India Pvt Ltd
Original Assignee
Diasys Diagnostics India Pvt Ltd
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Filing date
Publication date
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Publication of WO2021214555A1 publication Critical patent/WO2021214555A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • 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
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4737—C-reactive protein

Definitions

  • the present disclosure relates to field of electrochemical immunoassay. More particularly, the present disclosure relates to a label-free, wash-free electrochemical immunoassay device for effective and instant analysis of an analyte in a body fluid specimen.
  • the immunoassay-based tests including ELISA, western blot, and the like, contain either anti-bodies or enzyme conjugated antigen as a part of the sensing system, to detect a target antigen.
  • the conventional immunoassay uses electrochemical sensor for detection, wherein the technique requires an enzyme label such as HRP or alkaline Phosphatase Enzyme to generate the electrochemical signal and multiple steps of washing for achieving good sensitivity. This makes the usage of the technique not only laborious, but also expensive and time consuming.
  • a general object of the present disclosure is to provide an electrochemical immunoassay device for detecting an analyte in a biological fluid sample.
  • Another object of the present disclosure is to provide an electrochemical immunoassay device that can be used label-free and wash free.
  • Another object of the present disclosure is to provide an electrochemical immunoassay device that can be directly used at a remote location.
  • Another object of the present disclosure is to provide an electrochemical immunoassay device that can provide quick and accurate results.
  • Another object of the present disclosure is to provide an electrochemical immunoassay device that is economical.
  • the present disclosure relates to field of electrochemical immunoassay. More particularly, the present disclosure relates to a label-free, wash-free electrochemical immunoassay device for effective and instant analysis of an analyte in a body fluid specimen.
  • the present disclosure provides an electrochemical immunoassay device for detecting an analyte in a biological fluid specimen
  • the electrochemical immunoassay device includes: a substrate, which includes: a receiving stage adapted receive the biological fluid specimen containing the analyte; and an electrochemical sensor.
  • the electrochemical sensor includes: a reference electrode; a first layer including a primary receptor associated with the analyte and a first redox reagent, the first layer deposited over the reference electrode; and a working electrode.
  • the electrochemical immunoassay device includes: a first flow-through membrane adapted to fluidically couple the receiving stage with the electrochemical sensor, the first flow-through membrane provided such that the first flow through membrane is at least partially over the first layer of the electrochemical sensor.
  • the first layer is adapted to receive the biological fluid sample from the receiving stage, whereupon the analyte in the received biological fluid specimen binds with the primary receptor to form an insulating complex, wherein the insulating complex increases resistance to movement of ions of the redox reagent between the reference electrode and the working electrode, thereby resulting in a reduced current between the reference electrode and the working electrode. Measurement of the reduced current is indicative of a concentration of the analyte in the received biological fluid sample.
  • the measured reduced current is inversely proportional to the concentration of the analyte in the received biological fluid sample.
  • the electrochemical immunoassay device can be configured within a housing, and wherein the housing is provided with contacts electrically coupled to the reference electrode and the working electrode to enable measurement of current between the reference electrode and the working electrode.
  • the housing can be in the form of a cartridge.
  • the first layer can include a second receptor.
  • the first flow-through membrane can include a second redox reagent.
  • the reference electrode can be any noble metal selected from a group comprising gold, silver, platinum, and any combination thereof.
  • the reference electrode can be deposited on the substrate using deposition techniques selected from a group comprising sputtering, laser ablation deposition, vapour-based deposition, and any combination thereof.
  • the electrochemical immunoassay device can be used to detect C-reactive proteins in a sample of human blood.
  • the receiving stage can be a plasma separation membrane.
  • the first flow-through membrane can be made of nitrocellulose.
  • the present disclosure provides a method for manufacturing an electrochemical immunoassay device, the method including: depositing, on a substrate, a receiving stage; depositing, on the substrate, an electrochemical sensor, the deposition of the electrochemical sensor including: depositing a reference electrode; depositing, over the reference electrode, a first layer including a primary receptor associated with the analyte and a first redox reagent; and depositing a working electrode.
  • the method includes providing, on the substrate, a first flow-through membrane such that it fluidically couples the receiving stage with the electrochemical sensor, wherein the first flow-through membrane is provided at least partially over the first layer of the electrochemical sensor.
  • the present disclosure provides electrochemical immunoassay device, which includes one or more additional electrochemical sensors, wherein the electrochemical device is configured for detecting one or more additional analytes present in the received biological fluid sample corresponding to corresponding additional primary receptors provided in the first layers of the additional one or more electrochemical sensors.
  • the electrochemical immunoassay device can include one or more additional first flow-through membranes fluidically coupling the receiving stage (108) with corresponding one or more additional electrochemical sensors, the one or more additional first flow-through membranes provided such that the one or more additional second flow-through membranes are provided at least partially over the first layer of corresponding one or more additional electrochemical sensors.
  • any one or more of the additional one or more electrochemical sensors can be configured as a reference sensor.
  • FIG. 1 illustrates an exemplary representation of an electrochemical immunoassay device (100) with a single electrochemical sensor (104), in accordance with an embodiment of the present disclosure.
  • FIG. 2A illustrates an exemplary representation of an electrochemical immunoassay device (200) including plurality of electrochemical sensors (204-1, 204-2) and a baseline sensor (202), arranged in T-shaped form, in accordance with an embodiment of the present disclosure.
  • FIG. 2B illustrates an exemplary representation of an exploded view (200A) depicting one of the electrochemical sensors (204-2) of FIG. 2A, showing an overlapping portion between a first layer (230-2) and reference electrode (212-2) comprising a primary receptor (220-2), in accordance with an embodiment of the present disclosure.
  • FIG. 3A illustrates an exemplary representation of an electrochemical immunoassay device (300) including plurality of electrochemical sensors (304-1, 304-2) and a baseline sensor (302), arranged in Y-shaped form, in accordance with an embodiment of the present disclosure.
  • FIG. 3B illustrates an exemplary representation of an exploded view (300A) depicting one of the electrochemical sensors (304-2) of FIG. 3A, showing an overlapping portion between a first layer (330-2) and reference electrode (312-2) comprising a primary receptor (320-2), in accordance with an exemplary embodiment of the present disclosure.
  • FIG. 4 illustrates an exemplary flow diagram for a method to manufacture an electrochemical immunoassay device (100), in accordance with an embodiment of the present disclosure.
  • FIG. 5 illustrates an exemplary plot of the graphical results of trial 1, in accordance with an exemplary embodiment of the present disclosure.
  • the present disclosure relates to field of electrochemical immunoassay. More particularly, the present disclosure relates to a label-free (no enzyme label), wash-free electrochemical immunoassay device for effective and instant analysis of a specimen for detection of type and/or concentration of one or more target analytes.
  • the electrochemical immunoassay device can include an electrochemical sensor and a receiving stage.
  • the electrochemical sensor can include a first layer that can be stacked over an area of a reference electrode coupled with a working electrode, wherein the area of the reference electrode can include a primary receptor immobilized thereon.
  • the electrochemical immunoassay device can further include a first flow-through membrane, which can be positioned to overlap partially with the receiving stage on one side, and with the electrochemical sensor on other side.
  • the first flow-through membrane can further include a second mediator and/or a secondary receptor.
  • the secondary mediator can be used for supporting continuous reduction and oxidation cycles of mediator, present in the first layer.
  • the receiving stage of the electrochemical immunoassay device can be configured to receive a specimen from a human body.
  • the specimen can include, but is not limited to, blood, saliva, urine, or nasal swab effluent.
  • the specimen can be applied to the receiving stage for the analysis of the specimen by the electrochemical immunoassay device.
  • the target analyte that can be detected by using electrochemical immunoassay device of the present disclosure can include, but is not limited to, an antigen, or a protein.
  • the protein can be a C-reactive protein.
  • the detection of type and/or concentration of target analyte in a specimen can provide useful information for detection of infectious diseases such as bacterial infections and viral infections.
  • the primary receptor can be any or a combination of an antibody, protein, or aptamers. Various other types of primary receptors can also be used such as Aptamers (synthetic DNA or RNA receptors).
  • the electrochemical immunoassay device of the present disclosure can be used to test specimen in significantly small amounts.
  • the electrochemical immunoassay device of the present disclosure can be used to test 10 to 20 pL of undiluted blood sample.
  • the electrochemical immunoassay device of the present disclosure can be used to perform instant testing of specimen.
  • the electrochemical immunoassay device of the present disclosure can be used to test specimen in a time period of 90 seconds to 600 seconds.
  • the electrochemical immunoassay device can include an electrochemical sensor and a receiving stage, partially overlapping with each other.
  • the analyte present in the specimen that needs to be detected can get separated by the receiving stage and further transported to the electrochemical sensor through capillary action, to bind with the primary receptor immobilized on the reference electrode area of the electrochemical sensor to form a first complex.
  • the first complex formation can form an insulator layer that can hinder access of the redox species (mediator and/or secondary mediator) to the electrode surface of the electrochemical sensor, thus limiting the rate of electron transfer.
  • the reduction in detection signal (or electric current) can be inversely proportional to the amount of target analyte bound to the electrode surface, which can be a representative of the presence and concentration of the target analyte in the specimen.
  • the analyte present in the specimen that needs to be detected can bind to the secondary receptor on the first flow-through membrane, to obtain a complex which can get transported to the electrochemical sensor through capillary action.
  • the complex can further bind with the primary receptor immobilized on the electrochemical sensor to form a second complex.
  • the second complex formation can allow the subsequent attachment of the secondary receptor on the electrode surface of the electrochemical sensor, which can form an insulator layer that can hinder access of the redox species (mediator and/or secondary mediator) to the electrode surface of the electrochemical sensor, thus limiting the rate of electron transfer.
  • the reduction in detection signal (or electric current) can be inversely proportional to the amount of target analyte bound to the electrode surface, which can be a representative of the presence and concentration of the target analyte in the specimen.
  • the use of a secondary receptor can enable enhancement of signal amplitude, thereby requiring lesser sample.
  • the electrochemical immunoassay device can include a single electrochemical sensor or plurality of electrochemical sensors.
  • the electrochemical immunoassay device can comprise a single sensor for detection of a target analyte in a specimen.
  • the electrochemical immunoassay device can comprise a plurality of electrochemical sensors for detection of more than one type of target analyte or specimen.
  • the electrochemical sensors can comprise three electrochemical sensors, arranged in a T-shaped form or Y-shaped form.
  • the electrochemical immunoassay device can further include one or more baseline sensors, which can provide a baseline correction to detect signal of the electrochemical sensor.
  • the electrochemical immunoassay device can comprise a baseline sensor and one or more electrochemical sensors for detection of one or more target analytes in specimen.
  • the electrochemical immunoassay device can comprise a baseline sensor and two electrochemical sensors, arranged in a T-shaped form or Y-shaped form.
  • the electrochemical immunoassay device can include a housing, wherein the electrochemical immunoassay device can be configured in form of single use cartridge, which can be coupled with a holder and a computing device coupled to one or more analytical devices, for obtaining the signal by the continuous redox cycle in electrochemical sensor.
  • the electrochemical immunoassay device can be connected to one or more batteries.
  • the electrochemical characterizations can be carried out by cyclic voltammetry and the electrochemical detection was performed by differential pulse voltammetry technique using suitable mediators for signal enhancement.
  • the developed label-free, wash-free electrochemical immunoassay device can be a platform technology and can be adapted to any other immunoassays.
  • the analytical device can include, but not limited to, Differential Pulse Voltammetry (DPV).
  • DUV Differential Pulse Voltammetry
  • Various other analytical devices can also be included or coupled with the electrochemical immunoassay device.
  • the one or more electrochemical sensors and other components can be assembled in the cartridge form, by means of adhesives.
  • the electrochemical immunoassay device of the present disclosure can be used for performing measurements of antigen spiked in serum samples first and then spiked in whole blood samples, which can be detected from 0.5 mg/L to 150 mg/L with excellent specificity and reproducibility.
  • the present disclosure provides a system for effective and instant analysis of a specimen for detection of one or more target analytes.
  • the system can include an electrochemical immunoassay device, a holder, and a computing device.
  • the electrochemical immunoassay device can include an electrochemical sensor and a receiving stage.
  • the electrochemical sensor can include a first layer that can be stacked over an area of a reference electrode coupled with a working electrode, wherein the area of the reference electrode can include a primary receptor immobilized thereon.
  • the electrochemical immunoassay device can further include a first flow-through membrane, which can be positioned to overlap partially with the receiving stage on one side, and with electrochemical sensor on other side.
  • the holder can be configured to secure the device in a particular position and be electrically coupled to the device to aid the generation of an electrochemical signal.
  • the computing device can be configured to be coupled to the holder to process or display the generated electrochemical signal or detection signal.
  • the electrochemical immunoassay device of the present disclosure can include a single electrochemical sensor, as shown in FIG. 1.
  • FIG. 1 illustrates an exemplary representation of an electrochemical immunoassay device (100) with a single sensor, in accordance with an embodiment of the present disclosure.
  • the present disclosure provides electrochemical immunoassay device (100) for effective and instant analysis of a specimen for detection of type and/or concentration of one or more target analytes.
  • the electrochemical immunoassay device (100) can include an electrochemical sensor (104) and a receiving stage (108) that is provide on a substrate (150).
  • the electrochemical sensor (104) can include a first layer (130) that can be stacked over an area of a reference electrode (112) coupled with a working electrode (110), wherein the area of the reference electrode (112) can include a primary receptor (120) immobilized thereon.
  • the electrochemical immunoassay device (100) can further include a first flow-through membrane (106), which can be positioned to overlap partially with the receiving stage (108) on one side, and with electrochemical sensor (104) on other side.
  • the electrochemical sensor (104) can be a gold sensor that can be prepared by laser ablation of a sputtered gold sheet, to obtain screen printed portion in the form of the working electrode (110) and the reference electrode (112).
  • Various other material for the preparation of the electrochemical sensor (104) can also be used.
  • the primary receptor (120) can be immobilized on area of the reference electrode (112), which is screen printed or a gold sputtered laser ablated sensor, by generating a self-assembled monolayer by addition of one or more reagents including the primary receptor (120).
  • the one or more reagents can include, but not be limited to, Mercapto Undecanic Acid (MUA), Mercapto Propanic Acid (MPA) or Protein G bioreagents, which can be activated by EDC-NHS (Carbodiimide N-hydroxysuccinimide) coupling for covalent immobilization of primary receptor (120) to the screen printed electrode or gold electrode.
  • MUA Mercapto Undecanic Acid
  • MPA Mercapto Propanic Acid
  • Protein G bioreagents which can be activated by EDC-NHS (Carbodiimide N-hydroxysuccinimide) coupling for covalent immobilization of primary receptor (120) to the screen printed electrode or gold electrode.
  • the primary receptor (120) can be any or a combination of an antibody, a protein, or an aptamer. Various other types of primary receptors can also be used.
  • the receiving stage (108) of the electrochemical immunoassay device (100) can be configured to receive a specimen.
  • the specimen can include, but not be limited to, blood, saliva, urine, or nasal swab effluent.
  • the specimen can be applied to the receiving stage (108) for the analysis of the specimen by the electrochemical immunoassay device (100).
  • the receiving stage (108) of the electrochemical immunoassay device (100) can be configured to include one or more membrane layers that can transport plasma component of the specimen through capillary action.
  • electrochemical immunoassay device (100) including electrochemical sensor (104) and receiving stage (108) can enable transport of specimen, applied on receiving stage (108), by partial overlap between the receiving stage (108) and the electrochemical sensor (104).
  • electrochemical immunoassay device (100) including electrochemical sensor (104), receiving stage (108) and first flow-through membrane (106), can enable transport of specimen, applied on receiving stage (108), by partial overlap of the first flow through membrane (106) with the receiving stage (108) on one side, and with electrochemical sensor (104) on other side.
  • the receiving stage (150) can be a separation membrane.
  • the first layer (130) can include a redox reagent (or mediator), to enable a redox reaction cycle in electrochemical immunoassay device (100).
  • the redox reaction cycles can be continuous in nature and can include continuous alternate reduction and oxidation cycle of the mediator.
  • the mediator can include, but is not limited to, Hexa- ammine ruthenium (III) Chloride. Various other mediators can also be used.
  • the first layer (130) can be made of nitrocellulose.
  • Various other membrane materials can also be used as a material for the first layer (130).
  • mediator can be applied on a surface of the first layer (130), which can be in direct contact with the reference electrode (112), for execution of continuous reduction and oxidation cycles of the mediator, present in the first layer (130).
  • the first flow-through membrane (106) can further include a second redox reagent (or mediator) for supporting continuous reduction and oxidation cycles of mediator, present in the first layer (130).
  • a second redox reagent or mediator for supporting continuous reduction and oxidation cycles of mediator, present in the first layer (130).
  • the first flow-through membrane (106) can include a secondary receptor.
  • the first flow-through membrane (106) can be made of nitrocellulose. Various other materials can also be used as a material for the first flow-through membrane (106).
  • the electrochemical immunoassay device (100) can include an electrochemical sensor (104) and a receiving stage (108), partially overlapping with each other.
  • the target analyte present in the specimen that needs to be detected can bind to the secondary receptor on the first flow through membrane (106), to obtain a complex which can get transported to the electrochemical sensor (104) through capillary action.
  • the complex can further bind with the primary receptor (120) immobilized on the electrochemical sensor (104) to form a second complex.
  • the second complex formation can allow the subsequent attachment of the secondary receptor on the electrode surface of the electrochemical sensor (104), which can form an insulator layer that can hinder access of the redox species (mediator and/or secondary mediator) to the electrode surface of the electrochemical sensor (104), thus limiting the rate of electron transfer.
  • the reduction in detection signal (or electric current) can be inversely proportional to the amount of target analyte bound to the electrode surface, which can be a representative of the presence and concentration of the target analyte in the specimen.
  • the electrochemical immunoassay device of the present disclosure can include plurality of electrochemical sensors, as shown in FIG. 2A.
  • FIG. 2A illustrates an exemplary representation of an electrochemical immunoassay device (200) including plurality of electrochemical sensors (204-1, 204-2; herein individually and collectively designated 204) and a baseline sensor (202), arranged in T-shaped form, in accordance with an embodiment of the present disclosure.
  • FIG. 2B illustrates an exemplary representation of an exploded view (200A) depicting one of the electrochemical sensors (204-2) of FIG. 2A, showing an overlapping portion between a first layer (230-2) with reference electrode (212-2) comprising a primary receptor (220-2) immobilized thereon, in accordance with an embodiment of the present disclosure.
  • the present disclosure provides electrochemical immunoassay device (200) for effective and instant analysis of a specimen for detection of type and/or concentration of one or more target analytes.
  • the electrochemical immunoassay device (200) can include a baseline sensor (202), plurality of electrochemical sensors (204) and a receiving stage (208).
  • Each of the plurality of electrochemical sensors (204) can include first layer (230-1, 230-2; herein individually and collectively designated 230) that can be stacked over an area of reference electrode (212-1, 212-2; herein individually and collectively designated 212) coupled with working electrode (210-1, 210-2; herein individually and collectively designated 210), wherein the area of each of the reference electrode (212) can include a primary receptor (220-1, 220-2; herein individually and collectively designated 220).
  • the baseline sensor (202) can include a first layer that can be stacked over an area of reference electrode (212) coupled with working electrode (210), wherein the baseline sensor (202) can be devoid of any primary receptor and can be configured to provide a baseline correction for enhancement of signal-to-noise ratio of detection signal, as obtained by electrochemical immunoassay device of the present disclosure.
  • the electrochemical immunoassay device (200) can further include a plurality of first flow-through membrane (206), which can be positioned to overlap partially with the receiving stage (208) on one side, and with each of electrochemical sensors (204) and baseline sensor (202) on other side, in a T-shaped form.
  • each of electrochemical sensors (204) and baseline sensor (202) can be a gold sensor that can be prepared by laser ablation of a sputtered gold sheet, to obtain screen printed portion in the form of the working electrode (210) and the reference electrode (212).
  • Various other material and methods for the preparation of the baseline sensor (202) and the electrochemical sensors (204) can also be used.
  • the primary receptors (220) can be immobilized in area of each of the reference electrode (212), which can be screen printed, by generating a surface mono- layer by addition of one or more reagents including the primary receptors (220).
  • the one or more reagents can include, but not limited to, Mercapto Undecanic Acid (MUA), Mercapto Propanic Acid (MPA) or Protein G bioreagents, which can be activated by EDC-NHS for covalent immobilization of primary receptor (220) to the screen-printed electrode.
  • MUA Mercapto Undecanic Acid
  • MPA Mercapto Propanic Acid
  • Protein G bioreagents which can be activated by EDC-NHS for covalent immobilization of primary receptor (220) to the screen-printed electrode.
  • Various other immobilization methods and/or reagents can also be used.
  • the receiving stage (208) of the electrochemical immunoassay device (200) can be configured to receive a specimen.
  • the specimen can include, but not limited to, blood, saliva, urine, or nasal swab effluent.
  • the specimen can be applied to the receiving stage (208) for the analysis of the specimen by the electrochemical immunoassay device (200).
  • the receiving stage (208) of the electrochemical immunoassay device (200) can be configured to include one or more membrane layers that can transport plasma component of the specimen through capillary action to each of the electrochemical sensors (204-1, 204-2).
  • each of the first layer of the baseline sensor (202) and the electrochemical sensors (204) can include a mediator, to enable a redox reaction cycle in electrochemical immunoassay device (200).
  • the redox reaction cycles can be continuous in nature and can include continuous and alternate reduction and oxidation cycle of the mediator.
  • the mediator can include, but is not limited to, Hexa-ammine ruthenium (III) Chloride. Various other mediator, used in the art, can also be used.
  • the first layer of the baseline sensor (202) and the electrochemical sensors (204) can be made of nitrocellulose.
  • Various other materials in the art can also be used as a material for the first layer.
  • the first flow-through membrane (206) overlapping partially with each of the baseline sensor (202) and the electrochemical sensors (204) can further include second mediator for supporting continuous reduction and oxidation cycles of mediator.
  • the first flow-through membrane (206) overlapping partially with each of the electrochemical sensors (204), can be provided with a secondary receptor, which can enable formation of complex with target analyte, that can further complex with the primary receptor to form the insulating layer that can cause the reduction of the signal due to redox reaction of mediator.
  • the first flow-through membrane (206) can be made of nitrocellulose. Various other materials can also be used as a material for the first flow-through membrane (206).
  • the electrochemical immunoassay device of the present disclosure can have a modified configuration for effective and instant analysis of a specimen for detection of type and/or concentration of one or more target analytes, in accordance with an exemplary embodiment and as illustrated in FIG. 3A and FIG. 3B.
  • FIG. 3A illustrates an exemplary representation of an electrochemical immunoassay device (300) including plurality of electrochemical sensors (304-1, 304-2; herein individually and collectively designated 304) and a baseline sensor (302), arranged in Y-shaped form, in accordance with an embodiment of the present disclosure.
  • FIG. 3B illustrates an exemplary representation of an exploded view (300A) depicting one of the electrochemical sensors (304-2) of FIG. 3A, showing an overlapping portion between a first layer (330-2) with reference electrode (312-2) comprising a primary receptor (320-2) immobilized thereon, in accordance with an exemplary embodiment of the present disclosure.
  • the electrochemical immunoassay device (300) can include a baseline sensor (302), plurality of electrochemical sensors (304) and a receiving stage (308).
  • Each of the plurality of electrochemical sensors (304) and the baseline sensor (302) can include first layer that can be stacked over an area of reference electrode (312-1, 312-2; herein individually and collectively designated 312) coupled with working electrode (310-1, 310-2; herein individually and collectively designated 310), wherein the area of each of the reference electrode (312) can include primary receptor (320-1, 320-2; herein individually and collectively designated 320) immobilized thereon, as shown in FIG. 3B.
  • the baseline sensor (302) can include a first layer that can be stacked over an area of reference electrode (312) coupled with working electrode (310), wherein the baseline sensor (302) can be devoid of any primary receptor and can be configured to provide a baseline correction for enhancement of signal-to-noise ratio of detection signal, as obtained by electrochemical immunoassay device of the present disclosure.
  • the first layer of each of the plurality of electrochemical sensors (304) and the baseline sensor (302) can include a circular portion and an extending arm, as shown in FIG. 3B.
  • the first layer stacked on plurality of electrochemical sensors (304) and the baseline sensor (302) can be positioned such that the extending arm of each of the first layer can overlap partially with the receiving stage (308).
  • each of electrochemical sensors (304) and baseline sensor (302) can be a gold sensor that can be prepared by laser ablation of a sputtered gold sheet, to obtain screen printed portion in the form of the working electrode (310) and the reference electrode (312).
  • Various other material for the preparation of the baseline sensor (302) and the electrochemical sensors (304) can also be used.
  • the receiving stage (308) of the electrochemical immunoassay device (300) can be configured to receive a specimen.
  • the specimen can include, but not limited to, blood, saliva, urine, or nasal swab effluent.
  • the specimen can be applied to the receiving stage (308) for the analysis of the specimen by the electrochemical immunoassay device (300).
  • the receiving stage (308) of the electrochemical immunoassay device (300) can be configured to include one or more membrane layers that can transport plasma component of the specimen through capillary action to each of the electrochemical sensors (304).
  • each of the first layer of the baseline sensor (302) and the electrochemical sensors (304) can include a mediator, to enable a redox reaction cycle in electrochemical immunoassay device (300).
  • the redox reaction cycles can be continuous in nature and can include continuous and alternate reduction and oxidation cycle of the mediator.
  • the mediator can include, but is not limited to, Hexa-ammine ruthenium (III) Chloride. Various other mediator, used in the art, can also be used.
  • the first layer of the baseline sensor (302) and the electrochemical sensors (304) can be made of nitrocellulose.
  • mediator can be applied on a surface of the first layer, which can be in direct contact with the reference electrode (312), for execution of continuous reduction and oxidation cycles of the mediator, present in the first layer of the baseline sensor (302) and the electrochemical sensors (304).
  • the first layer of the baseline sensor (302) and the electrochemical sensors (304) can further include a second mediator for supporting continuous reduction and oxidation cycles of mediator.
  • the first layer of the baseline sensor (302) and the electrochemical sensors (304) can further include a secondary receptor, which can enable formation of complex with target analyte, that can further complex with the primary receptor to form the insulating layer that can cause the reduction of the signal due to redox reaction of mediator.
  • FIG. 4 illustrates an exemplary flow diagram for a method to manufacture an electrochemical immunoassay device (100), in accordance with an embodiment of the present disclosure.
  • the method 400 includes:
  • the electrochemical immunoassay device of the present disclosure is not limited to the above-mentioned configuration, and several other configurations are possible.
  • the device of the present disclosure can be implemented to detect C-reactive protein (CRP).
  • CRP C-reactive protein
  • the following section shall describe the detection of C-reactive proteins by the device of the present disclosure. It may be appreciated that the detection of C-reactive proteins by the device is an exemplary embodiment and is provided here to illustrate the working of the device. The device may be used for other applications as persons skilled in the art would appreciate, and the other applications shall be under the scope of the application of the proposed device.
  • DUV Differential pulse voltammetry
  • the reagents and materials used for the assay are,
  • Table-1 provides the assay parameters
  • FIG. 5 illustrates an exemplary plot of the graphical results of trial 1, in accordance with an exemplary embodiment of the present disclosure. It can be seen that, as expected, sensor current linearly decreases with linear increase in concentration of CRP.
  • the electrochemical immunoassay device of the present disclosure provides a single device for the direct measurement of specimen such as whole blood samples, which is needed in small amount, with no requirement of multiple washing steps or pre-treatment procedures, unlike conventional methods.
  • the device can be used to perform instant detection of the type and/or concentration of the target analyte in the specimen.
  • the device requires no pre-treatment procedures, thus making it a cost-effective approach as well as effective in terms of reproducibility and sensitivity.
  • the present disclosure provides an electrochemical immunoassay device for detecting an analyte in a biological fluid sample.
  • the present disclosure provides an electrochemical immunoassay device that can be used label-free and wash free.
  • the present disclosure provides an electrochemical immunoassay device that can be directly used at a remote location.
  • the present disclosure provides an electrochemical immunoassay device that can provide quick and accurate results.
  • the present disclosure provides an electrochemical immunoassay device that is economical.

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Abstract

La présente invention concerne un dispositif de dosage immunologique électrochimique sans lavage et sans marqueur pour une analyse efficace et instantanée d'un échantillon pour la détection du type et/ou de la concentration d'un ou de plusieurs analytes cibles. Le dispositif de dosage immunologique électrochimique peut comprendre un capteur électrochimique et une platine de réception. Le capteur électrochimique peut comprendre une première couche qui peut être empilée sur une zone d'une électrode de référence couplée à une électrode de travail, la zone de l'électrode de référence pouvant comprendre un récepteur primaire. Le dispositif de dosage immunologique électrochimique peut en outre comprendre une première membrane à écoulement continu, qui peut être positionnée pour se chevaucher partiellement avec la platine de réception sur un côté, et avec un capteur électrochimique sur l'autre côté. Le dispositif de dosage immunologique électrochimique peut comprendre un unique capteur électrochimique ou de multiples capteurs électrochimiques dans un unique dispositif et peut fournir une approche directe, efficace et instantanée pour la détection d'analytes cibles.
PCT/IB2021/050523 2020-04-23 2021-01-23 Dispositif de dosage immunologique électrochimique Ceased WO2021214555A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8425745B2 (en) * 2006-10-06 2013-04-23 Nanomix, Inc. Electrochemical nanosensors for biomolecule detection
US9910007B2 (en) * 2011-05-23 2018-03-06 Roche Diabetes Care, Inc. Sensor device for detecting an analyte
US20190154625A1 (en) * 2017-11-17 2019-05-23 ProbiusDx All-electronic high-throughput analyte detection system

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8425745B2 (en) * 2006-10-06 2013-04-23 Nanomix, Inc. Electrochemical nanosensors for biomolecule detection
US9910007B2 (en) * 2011-05-23 2018-03-06 Roche Diabetes Care, Inc. Sensor device for detecting an analyte
US20190154625A1 (en) * 2017-11-17 2019-05-23 ProbiusDx All-electronic high-throughput analyte detection system

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