Electrochemical sensing elisa reagent card

Abstract

The application provides an electrochemical sensing ELISA reagent card, comprising a microfluidic channel body and an electrochemical sensor body which are bonded together, wherein: a plurality of "annular" liquid flow channels are arranged on the microfluidic channel body, each "annular" liquid flow channel is formed by a working electrode covering flow channel and a counter electrode and a reference electrode covering flow channel in series, and the "annular" liquid flow channels are communicated through a common flow channel; the electrochemical sensor body comprises a flat plate substrate and a plurality of screen-printed electrodes which are printed in parallel on the flat plate substrate, and each screen-printed electrode comprises a working electrode, a counter electrode and a reference electrode. The electrochemical sensing ELISA reagent card combines an electrochemical biosensor and a microfluidic chip, and solves the technical problems of large sample / reagent consumption, single detection index and liquid volume influence on absorbance measurement of a traditional ELISA reagent kit.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostics, and more specifically to electrochemical sensing ELISA reagent cards. Background Technology

[0002] Enzyme-linked immunosorbent assay (ELISA) is a commonly used method in in vitro diagnostic immunological detection. It involves binding antigens or antibodies to a solid-phase carrier such as polystyrene and using the specific binding of antigens and antibodies to perform qualitative or quantitative detection of the immune reaction. Commonly used ELISA methods include: direct method, indirect method, double antibody sandwich method, and competitive method.

[0003] Traditional ELISA is widely used for antigen / antibody detection due to its good sensitivity, specificity, and reproducibility, as well as the good stability and ease of storage of the reagents used. However, traditional ELISA has many shortcomings: high sample / reagent consumption; limited detection indicators; absorbance measurement is easily affected by liquid volume; signal detection (absorbance) relies on expensive microplate readers; experimental procedures are cumbersome and time-consuming, requiring highly skilled operators; and the reagent kits are relatively expensive. These shortcomings limit the application of ELISA in resource-constrained areas.

[0004] With the development of new technologies and the integration of multiple disciplines, a new type of ELISA has been constructed by combining electrochemical biosensors and microfluidic chips. This not only overcomes many of the shortcomings of traditional ELISA, but also improves sensitivity, detection speed, detection throughput, and detection cost.

[0005] Electrochemical biosensors are sensors that use biological materials as sensing elements and electrodes (solid electrodes, ion-selective electrodes, gas-sensitive electrodes, etc.) as conversion elements, detecting signals characterized by potential or current. Due to their advantages such as high reliability, ease of operation, and low detection limits, and the ability to directly feedback and acquire electrical signals, they are highly beneficial for the miniaturization and integration of detection systems, and are now widely used in fields such as medical and health care, food supervision, and environmental monitoring.

[0006] Microfluidics refers to the science and technology involved in using microchannels (tens to hundreds of micrometers in size) to process or manipulate tiny fluids (volumes of microliters, nanoliters, or even attoliters). It has applications in fields such as medical diagnostics, environmental monitoring, food safety, forensic identification, and petrochemicals. Microfluidics offers numerous advantages, including low sample and reagent consumption, integrated miniaturization and automation, high throughput and efficiency, and low contamination. Summary of the Invention

[0007] The purpose of this invention is to provide an electrochemical sensing ELISA reagent card that combines an electrochemical biosensor and a microfluidic chip, solving the technical problems of traditional ELISA kits such as large sample / reagent consumption, single detection index, and absorbance measurement being easily affected by liquid volume.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0009] The electrochemical sensing ELISA reagent card of the present invention includes a microfluidic channel body and an electrochemical sensor body bonded together, wherein: the microfluidic channel body is provided with multiple "annular" liquid channels, each "annular" liquid channel is formed by a working electrode covered channel and a counter electrode and a reference electrode covered channel connected in series, and the "annular" liquid channels are connected through a common channel; the electrochemical sensor body includes a flat substrate and multiple screen-printed electrodes printed in parallel on the flat substrate, each screen-printed electrode including a working electrode, a counter electrode and a reference electrode.

[0010] Optionally, in the above-mentioned electrochemical sensing ELISA reagent card, the working electrode coverage channel completely covers the working electrode, and the counter electrode and reference electrode coverage channel completely covers the counter electrode and reference electrode.

[0011] Optionally, in the above-mentioned electrochemical sensing ELISA reagent card, the electrochemical sensing ELISA reagent card is inserted into the adapter of the matching instrument. The adapter includes a reagent card insertion port, a working electrode metal spring, a counter electrode metal spring, a reference electrode metal spring, and wires.

[0012] Optionally, in the above-mentioned electrochemical sensing ELISA reagent card, the electrochemical sensing ELISA reagent card is inserted into the adapter through the reagent card insertion port, and the working electrode conductive contact, counter electrode conductive contact and reference electrode conductive contact of the screen-printed electrode are in contact with the working electrode metal spring, counter electrode metal spring and reference electrode metal spring of the adapter, respectively.

[0013] Optionally, in the above-mentioned electrochemical sensing ELISA reagent card, a cleaning solution injection port is provided on the common channel, and a liquid inlet and outlet are provided on the working electrode covered channel.

[0014] Optionally, in the above-mentioned electrochemical sensing ELISA reagent card, the working electrode surface is immobilized with capture antibodies and blocked using a blocking solution.

[0015] Optionally, in the above-mentioned electrochemical sensing ELISA reagent card, the microfluidic channel body and the electrochemical sensor body are bonded together by one of the following methods: ultrasonic bonding, thermo-press bonding, plasma bonding, and adhesive bonding.

[0016] Optionally, in the above-mentioned electrochemical sensing ELISA reagent card, the material of the microfluidic channel body is one of polydimethylsiloxane (PDMS), polystyrene (PS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), and polyethylene (PE).

[0017] Optionally, in the above-mentioned electrochemical sensing ELISA reagent card, the substrate material is one of polyethylene terephthalate (PET), polystyrene (PS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), and polyethylene (PE).

[0018] Optionally, in the above-mentioned electrochemical sensing ELISA reagent card, the working electrode and the counter electrode are made of one of carbon, gold, platinum, graphene, and carbon nanotubes; the reference electrode is made of silver / silver chloride composite material.

[0019] The beneficial effects of this invention are as follows:

[0020] The electrochemical sensing ELISA reagent card of this invention combines an electrochemical biosensor and a microfluidic chip, resulting in minimal sample / reagent consumption and enabling simultaneous detection of multiple pathways and indicators. Due to the electrochemical sensing detection method, the accompanying instruments are compact, portable, and inexpensive, completely eliminating the reliance on microplate readers in traditional ELISA experiments. Compared to the absorbance measurement in traditional ELISA experiments, the electrochemical sensing-based detection method improves detection speed, sensitivity, and selectivity; the detection process is unaffected by liquid volume, sample color, or turbidity; and the experimental operation is simple, requiring no strict operator skills. Based on these advantages, the electrochemical sensing ELISA reagent card has broad application prospects. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0022] Figure 1A This is a schematic diagram of the electrochemical sensing ELISA reagent card and adapter of the present invention;

[0023] Figure 1B This is an exploded view of the electrochemical sensing ELISA reagent card of the present invention;

[0024] Figure 2 This is an overlay diagram of the electrochemical sensing ELISA reagent card of the present invention;

[0025] Figure 3 This is a bottom view of the microfluidic channel body of the electrochemical sensing ELISA reagent card of the present invention;

[0026] Figure 4 This is a schematic diagram of the structure of a "ring-shaped" liquid flow channel;

[0027] Figure 5 This is a top view of the electrochemical sensor body of the electrochemical sensing ELISA reagent card of the present invention;

[0028] Figure 6 This is a schematic diagram of the adapter of the present invention;

[0029] Figure 7 This is a top view of the electrochemical sensing ELISA reagent card insertion adapter of the present invention;

[0030] Figure 8 This is a schematic diagram of the electrochemical sensing ELISA reagent card and adapter of the present invention;

[0031] Figure 9 This is a schematic diagram illustrating the detection principle of the electrochemical sensing ELISA reagent card in an embodiment of the present invention;

[0032] Figure 10 This is a cyclic voltammogram of the antibody immobilization process captured on the surface of the working electrode in an embodiment of the present invention;

[0033] Figure 11 These are scanning electron microscope images of the working electrode surface before and after antibody immobilization in an embodiment of the present invention;

[0034] The attached figures are labeled as follows:

[0035] 1. Microfluidic channel body; 2. Electrochemical sensor body; 3. Adapter;

[0036] 1-1 Working electrode covering the flow channel; 1-2 Counter electrode and reference electrode covering the flow channel; 1-3 Common flow channel; 1-4 Liquid inlet and outlet; 1-5 Cleaning fluid inlet; 2-1 Working electrode; 2-2 Counter electrode; 2-3 Reference electrode; 2-4 Working electrode conductive contact; 2-5 Counter electrode conductive contact; 2-6 Reference electrode conductive contact; 2-7 Flat substrate; 3-1 Reagent card insertion port; 3-2 Working electrode metal spring; 3-3 Counter electrode metal spring; 3-4 Reference electrode metal spring; 3-5 Wire. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the electrochemical sensing ELISA reagent cards in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0038] like Figure 1A , 1B and Figure 2 As shown, the electrochemical sensing ELISA reagent card of the present invention includes: a microfluidic channel body 1 and an electrochemical sensor body 2 bonded together, wherein the microfluidic channel body 1 and the electrochemical sensor body 2 are bonded by a plasma bonding process.

[0039] like Figure 3 and 4 As shown, the microfluidic channel body 1 is provided with multiple "annular" liquid channels. Each "annular" liquid channel is composed of a working electrode covered channel 1-1 and counter electrode and reference electrode covered channels 1-2 connected in series. The "annular" liquid channels are connected by a common channel 1-3. A cleaning fluid injection port 1-5 is provided on the common channel 1-3, and a liquid inlet and outlet 1-4 are provided on the working electrode covered channel 1-1. The working electrode covered channel 1-1 is circular.

[0040] like Figure 5 As shown, the electrochemical sensor body 2 is fabricated by parallel printing multiple screen-printed electrodes on a flat substrate 2-7. The screen-printed electrodes form a three-electrode system, with each electrode including a working electrode 2-1, a counter electrode 2-2, and a reference electrode 2-3. The three conductive contacts of the screen-printed electrodes (working electrode conductive contact 2-4, counter electrode conductive contact 2-5, and reference electrode conductive contact 2-6) respectively contact the three metal springs (working electrode metal spring, counter electrode metal spring, and reference electrode metal spring) in the adapter. The working electrode covering channel 1-1 on the microfluidic channel body 1 completely covers the working electrode 2-1 printed on the surface of the electrochemical sensor body 2; the counter electrode and reference electrode covering channel 1-2 completely cover the counter electrode 2-2 and reference electrode 2-3. Furthermore, the surface of the working electrode 2-1 is immobilized with a capture antibody and blocked using a blocking solution, bovine serum albumin. The immobilization of the capture antibody will be described in detail below.

[0041] like Figure 6 , Figure 7 and Figure 8 As shown, the electrochemical sensing ELISA reagent card is inserted into the adapter 3 of the matching instrument and contacts the metal contacts in the adapter 3. The adapter 3 includes a reagent card insertion port 3-1, a working electrode metal contact 3-2, a counter electrode metal contact 3-3, a reference electrode metal contact 3-4, and a wire 3-5. The electrochemical sensing ELISA reagent card is inserted into the adapter 3 through the reagent card insertion port 3-1. The three conductive contacts of the screen-printed electrode (working electrode conductive contact 2-4, counter electrode conductive contact 2-5, and reference electrode conductive contact 2-6) contact the three metal contacts in the adapter 3 (working electrode metal contact 3-2, counter electrode metal contact 3-3, and reference electrode metal contact 3-4), respectively. The wire 3-5 of the adapter 3 is connected to the matching electrochemical instrument.

[0042] In this embodiment, the material of the microfluidic channel body 1 is one of polydimethylsiloxane (PDMS), polystyrene (PS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), and polyethylene (PE), preferably polydimethylsiloxane (PDMS).

[0043] The material of the flat substrate 2-7 is one of polyethylene terephthalate (PET), polystyrene (PS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), and polyethylene (PE), preferably polyethylene terephthalate (PET).

[0044] In the screen-printed electrodes printed on the surface of the electrochemical sensor body 2, the working electrode 2-1 and the counter electrode 2-2 are made of one of carbon, gold, platinum, graphene, and carbon nanotubes, preferably carbon; the reference electrode 2-3 is made of silver / silver chloride composite material.

[0045] Bonding of electrochemical sensing ELISA reagent cards:

[0046] 1. Perform surface treatment on the microfluidic channel body 1 and the electrochemical sensor body 2;

[0047] 2. The side of the microfluidic channel body 1 with the flow channel faces the side of the electrochemical sensor body 2 with the screen-printed electrode.

[0048] 3. The working electrode set on the microfluidic channel body 1 covers the flow channel 1-1 and completely covers the working electrode 2-1 printed on the surface of the electrochemical sensor body 2; the counter electrode and reference electrode set on the microfluidic channel body 1 cover the flow channel 1-2 and completely cover the counter electrode 2-2 and reference electrode 2-3 printed on the surface of the electrochemical sensor body 2.

[0049] 4. The microfluidic channel body 1 and the electrochemical sensor body 2 are bonded using a plasma bonding process. One of the following bonding processes is selected: ultrasonic bonding, thermo-press bonding, plasma bonding, or adhesive bonding.

[0050] Figure 9 This is a schematic diagram of the detection principle of the electrochemical sensing ELISA reagent card, where (a) is the capture antibody immobilization, (b) is the capture of the analyte antigen, (c) is the formation of an immune sandwich complex with the enzyme-labeled detection antibody (i.e., enzyme-labeled detection antibody-analyte antigen-capture antibody complex), and (d) is the addition of substrate and detection of the electrical signal. The specific operating steps of the electrochemical sensing ELISA reagent card are as follows.

[0051] The fixation of captured antibodies includes the following steps:

[0052] 1. Using a pipette, inject streptavidin solution into the working electrode covering channel 1-1 through liquid inlet / outlet 1-4. After incubation at 37°C for 2 hours, streptavidin is fixed on the surface of the working electrode 2-1 and the inner surface of the PDMS material working electrode covering channel 1-1 through physical adsorption (PDMS is a hydrophobic material).

[0053] 2. Remove the streptavidin solution from the working electrode covered flow channel 1-1 using a pipette through liquid inlet / outlet 1-4;

[0054] 3. Using a pipette, inject bovine serum albumin solution into the working electrode covered channel 1-1 through liquid inlet / outlet 1-4. After incubation at 37°C for 1 hour, bovine serum albumin is fixed on the surface of the working electrode 2-1 and the inner surface of the PDMS material working electrode covered channel 1-1 through physical adsorption.

[0055] 4. Remove the bovine serum albumin solution from the working electrode covered flow channel 1-1 using a pipette through liquid inlet / outlet 1-4;

[0056] 5. Using a pipette, inject biotin-labeled capture antibody solution into the working electrode covered channel 1-1 through liquid inlet / outlet 1-4. After incubation at 37°C for 30 minutes, biotin specifically binds to streptavidin, and the capture antibody is indirectly fixed on the surface of the working electrode 2-1 and the inner surface of the PDMS material working electrode covered channel 1-1.

[0057] 6. Using a pipette, remove the biotin-labeled capture antibody solution from the working electrode covered channel 1-1 through liquid inlet / outlet 1-4;

[0058] 7. Seal the electrochemical sensing ELISA reagent card with the captured antibody immobilized on the surface of the working electrode 2-1 in an aluminum foil bag and store it at 4°C for later use.

[0059] Figure 10 Cyclic voltammetry (test substrate: 5mM K3 [Fe(CN)6]; scan rate: 0.05V / s) is used to measure the antibody immobilization process on the surface of working electrode 2-1. Curve (a) represents the bare electrode, curve (b) represents streptavidin, curve (c) represents bovine serum albumin, and curve (d) represents biotin-labeled capture antibody. Figure 11 Scanning electron microscopy (SEM) images of the working electrode 2-1 before and after antibody immobilization, where (a) is the bare electrode and (b) is the electrode after antibody immobilization. Figure 10 and Figure 11 The immobilization process of the captured antibody on the working electrode is demonstrated, and it is shown that the captured antibody was ultimately successfully immobilized on the working electrode. Among other things, Figure 10 It is an electrochemical characterization method that demonstrates each step of the capture antibody immobilization process; Figure 11The image was characterized using scanning electron microscopy, and in image b, the captured antibody was seen immobilized on the surface of the working electrode.

[0060] The use of electrochemical sensing ELISA reagent cards includes the following steps:

[0061] 1. Remove the electrochemical sensing ELISA reagent card from the aluminum foil bag and place it horizontally with the side with liquid inlets / outlets 1-4 and cleaning solution inlets 1-5 facing upwards;

[0062] 2. Clean the inner surfaces of the flow channels (working electrode covering channel 1-1, counter electrode and reference electrode covering channel 1-2, and common channel 1-3) and the surfaces of the screen-printed electrodes (working electrode 2-1, counter electrode 2-2, and reference electrode 2-3) of the electrochemical sensing ELISA reagent card using phosphate-buffered saline (PBST) solution. Inject PBST solution into the electrochemical sensing ELISA reagent card through cleaning solution inlet 1-5. The PBST solution flows sequentially through common channel 1-3, counter electrode and reference electrode covering channel 1-2, and working electrode covering channel 1-1, finally exiting from liquid inlet / outlet 1-4. After cleaning, inject air into the electrochemical sensing ELISA reagent card through cleaning solution inlet 1-5 to remove residual PBST solution from the flow channels.

[0063] 3. Add the sample solution to be tested. Through the liquid inlet / outlet 1-4, use a pipette to inject the sample solution to be tested into the working electrode covered channel 1-1. After incubation at 37°C for 30 minutes, the antigen to be tested and the capture antibody specifically bind to each other, forming an antigen-capture antibody complex on the surface of the working electrode 2-1 and the inner surface of the PDMS material working electrode covered channel 1-1.

[0064] 4. Remove the sample solution from the working electrode covering channel 1-1 and clean the surface of working electrode 2-1 and the inner surface of PDMS material working electrode covering channel 1-1. Inject PBST solution into the electrochemical sensing ELISA reagent card through cleaning solution injection port 1-5. The PBST solution flows sequentially through common channel 1-3, counter electrode and reference electrode covering channel 1-2, and working electrode covering channel 1-1, finally flowing out from liquid inlet / outlet 1-4. This process removes the sample solution from working electrode covering channel 1-1 and cleans the surface of working electrode 2-1 and the inner surface of PDMS material working electrode covering channel 1-1. After removing the sample solution and cleaning, inject air into the electrochemical sensing ELISA reagent card through cleaning solution injection port 1-5 to remove residual PBST solution from the channels.

[0065] 5. Add enzyme-labeled detection antibody solution. Using a pipette, inject the enzyme-labeled detection antibody solution into the working electrode covered channel 1-1 through liquid inlet / outlet 1-4. After incubation at 37°C for 30 minutes, the detection antibody specifically binds to the antigen to be tested, forming an immune sandwich structure (enzyme-labeled detection antibody-antigen to be tested-capture antibody complex) on the surface of the working electrode 2-1 and the inner surface of the PDMS material working electrode covered channel 1-1.

[0066] 6. Remove the enzyme-labeled detection antibody solution from the working electrode covering channel 1-1, and clean the surface of working electrode 2-1 and the inner surface of the PDMS material working electrode covering channel 1-1. Inject PBST solution into the electrochemical sensing ELISA reagent card through the cleaning solution injection port 1-5. The PBST solution flows sequentially through the common channel 1-3, the counter electrode and reference electrode covering channel 1-2, and the working electrode covering channel 1-1, finally flowing out from the liquid inlet / outlet 1-4. This process removes the enzyme-labeled detection antibody solution from the working electrode covering channel 1-1 and cleans the surface of working electrode 2-1 and the inner surface of the PDMS material working electrode covering channel 1-1. After removing the enzyme-labeled detection antibody solution and cleaning, inject air into the electrochemical sensing ELISA reagent card through the cleaning solution injection port 1-5 to remove residual PBST solution from the channels.

[0067] 7. Add substrate solution. Inject the substrate solution into the electrochemical sensing ELISA reagent card through the cleaning solution inlet 1-5. The substrate solution flows sequentially through the common channel 1-3, the counter electrode and reference electrode covering channel 1-2, and the working electrode covering channel 1-1, finally filling the channels of the electrochemical sensing ELISA reagent card (working electrode covering channel 1-1, counter electrode and reference electrode covering channel 1-2, and common channel 1-3). The substrate solution contacts the inner surface of the PDMS material working electrode covering channel 1-1, while simultaneously covering the surfaces of the working electrode 2-1, counter electrode 2-2, and reference electrode 2-3.

[0068] 8. Insert the electrochemical sensing ELISA reagent card into adapter 3 through reagent card insertion port 3-1. The three conductive contacts of the screen-printed electrode (working electrode conductive contact 2-4, counter electrode conductive contact 2-5, and reference electrode conductive contact 2-6) are in contact with the three metal springs in adapter 3 (working electrode metal spring 3-2, counter electrode metal spring 3-3, and reference electrode metal spring 3-4), respectively. Connect the lead wire 3-5 of adapter 3 to the matching electrochemical instrument. Use the three-electrode detection system to measure the current, quantify the antigen to be tested, and complete the simultaneous detection of multiple pathways.

[0069] 9. After use, remove the electrochemical sensing ELISA reagent card from the reagent card insertion port 3-1 of adapter 3, and discard the electrochemical sensing ELISA reagent card into the biochemical waste bin.

[0070] Unless otherwise defined, all technical and / or scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The materials, methods, and embodiments mentioned in this application are illustrative only and not restrictive.

[0071] Although this application has been described in conjunction with specific embodiments, those skilled in the art can make appropriate substitutions, modifications and changes under the inventive spirit of this application, and such substitutions, modifications and changes still fall within the protection scope of this application.

Claims

1. An electrochemical sensing ELISA reagent card, characterized in that, This includes a microfluidic channel body and an electrochemical sensor body bonded together, wherein: The microfluidic channel body is provided with multiple annular liquid channels. Each annular liquid channel is composed of a working electrode covered channel and counter electrode and reference electrode covered channels connected in series. The annular liquid channels are connected to each other through a common channel. The electrochemical sensor body includes a flat substrate and a plurality of screen-printed electrodes printed in parallel on the flat substrate, each of the screen-printed electrodes including a working electrode, a counter electrode and a reference electrode; The working electrode covering channel completely covers the working electrode, and the counter electrode and reference electrode covering channel completely cover the counter electrode and the reference electrode; The common flow channel is provided with a cleaning fluid injection port, and the working electrode covered flow channel is provided with a liquid inlet and outlet, through which an enzyme-labeled detection antibody-antigen-capture antibody complex with an immune sandwich structure is formed; Streptavidin and bovine serum albumin are injected into the working electrode covered channel via the liquid inlet and outlet, and immobilized on the working electrode surface and the inner surface of the working electrode covered channel through physical adsorption. Biotin-labeled capture antibody is injected into the working electrode covered channel via the liquid inlet and outlet, and specifically binds to streptavidin, thereby indirectly immobilizing the biotin-labeled capture antibody on the working electrode surface and the inner surface of the working electrode covered channel. After the test liquid is injected into the working electrode covered channel via the liquid inlet and outlet, the test antigen in the test liquid specifically binds to the biotin-labeled capture antibody, forming a test antigen-capture antibody complex on the working electrode surface and the inner surface of the working electrode covered channel. After the enzyme-labeled detection antibody is injected into the working electrode covered channel via the liquid inlet and outlet, the enzyme-labeled detection antibody specifically binds to the test antigen, forming the enzyme-labeled detection antibody-test antigen-capture antibody complex on the working electrode surface and the inner surface of the working electrode covered channel.

2. The electrochemical sensing ELISA reagent card according to claim 1, characterized in that, in, The electrochemical sensing ELISA reagent card is inserted into the adapter of the matching instrument. The adapter includes a reagent card insertion port, a working electrode metal spring, a counter electrode metal spring, a reference electrode metal spring, and wires.

3. The electrochemical sensing ELISA reagent card according to claim 2, characterized in that, The electrochemical sensing ELISA reagent card is inserted into the adapter through the reagent card insertion port, and the working electrode conductive contact, counter electrode conductive contact, and reference electrode conductive contact of the screen-printed electrode are in contact with the working electrode metal spring, counter electrode metal spring, and reference electrode metal spring of the adapter, respectively.

4. The electrochemical sensing ELISA reagent card according to claim 1, characterized in that, The microfluidic channel body and the electrochemical sensor body are bonded together by one of the following methods: ultrasonic bonding, thermo-press bonding, plasma bonding, and adhesive bonding.

5. The electrochemical sensing ELISA reagent card according to claim 1, characterized in that, The microfluidic channel body is made of one of the following materials: polydimethylsiloxane, polystyrene, polymethyl methacrylate, polycarbonate, polypropylene, and polyethylene.

6. The electrochemical sensing ELISA reagent card according to claim 1, characterized in that, The material of the flat substrate is one of polyethylene terephthalate, polystyrene, polymethyl methacrylate, polycarbonate, polypropylene, and polyethylene.

7. The electrochemical sensing ELISA reagent card according to claim 1, characterized in that, The working electrode and the counter electrode are made of one of the following materials: carbon, gold, platinum, graphene, and carbon nanotubes; the reference electrode is made of silver / silver chloride composite material.

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