CN116183906A - Electrochemical Sensing ELISA Reagent Card - Google Patents

Abstract

The invention provides an electrochemical sensing ELISA reagent card, which includes a microfluidic channel body and an electrochemical sensor body bonded together, wherein: the microfluidic channel body is provided with a plurality of "ring" liquid channels, Each "annular" liquid flow channel is formed in series by a working electrode covered flow channel and a counter electrode and a reference electrode covered flow channel, and the "annular" liquid flow channel is communicated through a common flow channel; A plurality of screen-printed electrodes printed in parallel on a flat substrate, each screen-printed electrode includes a working electrode, a counter electrode and a reference electrode. The electrochemical sensing ELISA reagent card of the present invention combines electrochemical biosensors and microfluidic chips, which solves the technical problems of large sample/reagent consumption, single detection index, and absorbance measurement easily affected by liquid volume in traditional ELISA kits. .

Description

Electrochemical Sensing ELISA Reagent Card

technical field

The invention relates to the field of in vitro diagnosis, in particular to an electrochemical sensing ELISA reagent card.

Background technique

Enzyme-linked immunosorbent assay (enzymelinkedimmunosorbentassay, ELISA) is a commonly used method in in vitro diagnostic immunological detection. Antigens or antibodies are bound to solid phase carriers such as polystyrene, and the specific binding of antigens and antibodies is used to carry out qualitative or quantitative analysis of immune responses. Quantitative detection methods, ELISA commonly used methods include: direct method, indirect method, double antibody sandwich method and competition method.

Traditional ELISA is widely used in antigen/antibody detection because of its good sensitivity, specificity, and repeatability, and because the reagents used have good stability and are easy to store. However, there are many shortcomings in traditional ELISA: large sample/reagent consumption; single detection index; absorbance measurement is easily affected by liquid volume; signal detection (absorbance) depends on expensive microplate reader; The requirements are higher; the price of the kit is more expensive. Many of the above deficiencies limit the application of ELISA to areas with limited resources.

With the development of new technologies and the integration of multiple disciplines, the combination of electrochemical biosensors and microfluidic chips to construct a new type of ELISA can not only get rid of the many shortcomings of the above-mentioned traditional ELISA, but also improve sensitivity, detection speed, detection throughput, Improvements have been made in aspects such as detection cost.

Electrochemical biosensor (Electrochemical Biosensor) refers to a sensor that uses biological materials as sensitive elements, electrodes (solid electrodes, ion-selective electrodes, gas-sensitive electrodes, etc.) as conversion elements, and detects signals characterized by potential or current. Because of its high reliability, easy operation, low detection limit, etc., and the characteristics of direct feedback of collected electrical signals, it is very conducive to the miniaturization and integration of the detection system, and has been widely used in medical health and food supervision. and environmental monitoring.

Microfluidics (Microfluidics) refers to the science and technology involved in the use of micropipes (tens to hundreds of microns in size) to process or manipulate tiny fluids (microliters, nanoliters or even Aliters in volume). It has applications in diagnosis, environmental monitoring, food safety, forensic identification, petrochemical and other fields. Microfluidics has many advantages, such as: less consumption of samples and reagents, integrated miniaturization and automation, high throughput and high efficiency, and less pollution.

Contents of the invention

The purpose of the present invention is to provide an electrochemical sensing ELISA reagent card, which combines electrochemical biosensors and microfluidic chips to solve the problem of large sample/reagent consumption, single detection index, and easy absorption measurement of traditional ELISA kits. Technical issues of liquid volume effects.

In order to solve the problems of the technologies described above, the technical solution of the present invention is:

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 a plurality of "ring" liquid channels, each " The "ring" liquid flow channel is formed by the working electrode covering flow channel and the counter electrode and reference electrode covering flow channel in series, and the "ring" liquid flow channel is connected through a common flow channel; A plurality of screen-printed electrodes printed in parallel, each screen-printed electrode includes a working electrode, a counter electrode and a reference electrode.

Optionally, in the above electrochemical sensing ELISA reagent card, the working electrode covering flow channel completely covers the working electrode, and the counter electrode and reference electrode covering flow channel completely covers the counter electrode and reference electrode.

Optionally, in the above-mentioned electrochemical sensing ELISA reagent card, the electrochemical sensing ELISA reagent card is inserted into an adapter of a supporting instrument, and the adapter includes a reagent card insertion port, a working electrode metal shrapnel, a counter electrode metal shrapnel, and a reference electrode metal shrapnel. Shrapnel and wire.

Optionally, in the above-mentioned electrochemical sensing ELISA reagent card, the electrochemical sensing ELISA reagent card is inserted into the adapter from the reagent card insertion port, and the conductive contact piece of the working electrode of the screen-printed electrode, the conductive contact piece of the counter electrode and the reference electrode The conductive contacts are respectively in contact with the metal shrapnel of the working electrode, the metal shrapnel of the counter electrode and the reference electrode of the adapter.

Optionally, in the above electrochemical sensing ELISA reagent card, the common flow channel is provided with a cleaning solution injection port, and the working electrode covered flow channel is provided with a liquid inlet and outlet.

Optionally, in the above electrochemical sensing ELISA reagent card, the surface of the working electrode is immobilized with a capture antibody and blocked with a blocking solution.

Optionally, in the above electrochemical sensing ELISA reagent card, the microfluidic channel body and the electrochemical sensor body are bonded by one of ultrasonic bonding, thermocompression bonding, plasma bonding, and adhesive bonding. combine together.

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

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

Optionally, in the above-mentioned electrochemical sensing ELISA reagent card, the material of the working electrode and the counter electrode is one of carbon, gold, platinum, graphene, carbon nanotube; the material of the reference electrode is silver/chloride Silver composite.

The beneficial effects of the present invention are:

The electrochemical sensing ELISA reagent card of the present invention combines an electrochemical biosensor and a microfluidic chip, which not only consumes very little sample/reagent, but also enables simultaneous detection of multiple channels/multiple indicators. Due to the use of electrochemical sensing and detection methods, the supporting instruments and equipment are small, portable, and cheap, completely getting rid of the dependence on microplate readers in traditional ELISA experiments. Compared with the measurement of absorbance in traditional ELISA experiments, the detection method based on electrical signals of electrochemical sensing can improve the detection speed, sensitivity and selectivity; the detection process is not affected by the liquid volume, sample color and deep turbidity; the experimental operation is simple , there are no strict requirements for operators. Based on the above advantages, the electrochemical sensing ELISA reagent card has broad application prospects.

Description of drawings

In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the specific embodiments or the prior art.

Fig. 1A is the structural representation of electrochemical sensing ELISA reagent card and adapter of the present invention;

Fig. 1 B is the explosion diagram of electrochemical sensing ELISA reagent card of the present invention;

Fig. 2 is the overlay diagram of electrochemical sensing ELISA reagent card of the present invention;

Fig. 3 is the bottom view of the microfluidic channel body of the electrochemical sensing ELISA reagent card of the present invention;

Fig. 4 is a structural schematic diagram of an "annular" liquid channel;

Fig. 5 is the top view of the electrochemical sensor body of the electrochemical sensing ELISA reagent card of the present invention;

Fig. 6 is the schematic diagram of adapter of the present invention;

Fig. 7 is the top view of electrochemical sensing ELISA reagent card insertion adapter of the present invention;

Fig. 8 is the outline schematic diagram of electrochemical sensing ELISA reagent card and adapter of the present invention;

9 is a schematic diagram of the detection principle of the electrochemical sensing ELISA reagent card in an embodiment of the present invention;

Fig. 10 is a cyclic voltammogram of the process of immobilizing the antibody captured on the surface of the working electrode in an embodiment of the present invention;

Fig. 11 is a scanning electron micrograph before and after immobilization of the capture antibody on the surface of the working electrode in an embodiment of the present invention;

Wherein, reference sign is:

1. Microfluidic channel body; 2. Electrochemical sensor body; 3. Adapter;

1-1, the working electrode covers the flow channel; 1-2, the counter electrode and the reference electrode cover the flow channel; 1-3, the public flow channel; 1-4, the liquid inlet and outlet; 1-5, the cleaning solution injection port; 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 base; 3-1, reagent card insertion port; 3-2, working electrode metal shrapnel; 3-3, counter electrode metal shrapnel; 3-4, reference electrode metal shrapnel; 3-5, wire.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the electrochemical sensing ELISA reagent card in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention .

As shown in Figure 1A, 1B and Figure 2, the electrochemical sensing ELISA reagent card of the embodiment of the present invention comprises: the microfluidic channel body 1 and the electrochemical sensor body 2 that are bonded together, wherein, the microfluidic channel The body 1 and the electrochemical sensor body 2 are bonded by a plasma bonding process.

As shown in Figures 3 and 4, the microfluidic channel body 1 is provided with a plurality of "annular" liquid flow channels, and each "annular" liquid flow channel is covered by a working electrode as well as a counter electrode and a reference electrode. Covering flow channels 1-2 are connected in series, and the "circular" liquid flow channels are connected through common flow channels 1-3. A cleaning liquid injection port 1-5 is provided on the common flow channel 1-3, and a liquid inlet and outlet 1-4 is provided on the working electrode covered flow channel 1-1, wherein the working electrode covered flow channel 1-1 is set as a circle shape.

As shown in FIG. 5 , the electrochemical sensor body 2 is made by printing a plurality of screen-printed electrodes in parallel on a flat substrate 2-7. The screen printing electrode is a three-electrode system, and each screen printing electrode includes a working electrode 2-1, a counter electrode 2-2 and a reference electrode 2-3, and three conductive contacts of the screen printing electrode (the working electrode conductive contact Sheet 2-4, counter electrode conductive contact 2-5 and reference electrode conductive contact 2-6) respectively with three metal shrapnel in the adapter (working electrode metal shrapnel, counter electrode metal shrapnel, reference electrode metal shrapnel) touch. The working electrode set on the microfluidic channel body 1 covers the flow channel 1-1 completely covering the working electrode 2-1 printed on the surface of the electrochemical sensor body 2; the counter electrode and the reference electrode cover the flow channel 1-2 completely covering the counter electrode 2-2 and reference electrode 2-3. And the surface of the working electrode 2-1 is immobilized with a capture antibody and blocked with a blocking solution, bovine serum albumin solution, wherein the immobilization of the capture antibody will be described in detail below.

As shown in FIG. 6 , FIG. 7 and FIG. 8 , the electrochemical sensing ELISA reagent card is inserted into the adapter 3 of the supporting instrument, and is in contact with the metal shrapnel in the adapter 3 . The adapter 3 includes a reagent card insertion port 3-1, a working electrode metal shrapnel 3-2, a counter electrode metal shrapnel 3-3, a reference electrode metal shrapnel 3-4 and a wire 3-5. The electrochemical sensing ELISA reagent card is inserted into the adapter 3 from the reagent card insertion port 3-1, and the three conductive contacts of the screen-printed electrodes (working electrode conductive contact 2-4, counter electrode conductive contact 2-5, reference The conductive contact piece 2-6 of the ratio electrode is respectively in contact with three metal shrapnels in the adapter 3 (the working electrode metal shrapnel 3-2, the counter electrode metal shrapnel 3-3, and the reference electrode metal shrapnel 3-4). Wires 3-5 are connected to supporting electrochemical instruments.

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

The material of flat base 2-7 is polyethylene terephthalate (PET), polystyrene (PS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP) 1. One of polyethylene (PE), preferably polyethylene terephthalate (PET).

Among the screen printing electrodes printed on the surface of the electrochemical sensor body 2, the material of the working electrode 2-1 and the counter electrode 2-2 is one of carbon, gold, platinum, graphene, and carbon nanotubes, preferably carbon; The material of the reference electrode 2-3 is silver/silver chloride composite material.

Bonding of electrochemical sensing ELISA reagent card:

1. Surface treatment of the microfluidic channel body 1 and the electrochemical sensor body 2;

2. The side of the microfluidic channel body 1 provided with flow channels faces the side of the electrochemical sensor body 2 printed with screen-printed electrodes;

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

4. Using the plasma bonding process, the microfluidic channel body 1 and the electrochemical sensor body 2 are bonded. The bonding process is ultrasonic bonding, thermocompression bonding, plasma bonding, and adhesive bonding. A sort of.

Figure 9 is a schematic diagram of the detection principle of the electrochemical sensing ELISA reagent card, wherein (a) is the immobilization of the capture antibody, (b) is the capture of the antigen to be tested, and (c) is the formation of an immune sandwich complex with the enzyme-labeled detection antibody (ie, Enzyme-labeled detection antibody-antigen to be tested-capture antibody complex), (d) is to add a substrate to detect electrical signals. The specific operation steps of the electrochemical sensing ELISA reagent card are as follows.

Immobilization of the capture antibody includes the following steps:

1. Use a pipette gun to inject streptavidin solution into the working electrode covered flow channel 1-1 through the liquid inlet and outlet 1-4. After incubating at 37°C for 2 hours, the streptavidin is fixed on the surface by physical adsorption. The surface of the working electrode 2-1 and the working electrode made of PDMS cover the inner surface of the flow channel 1-1 (PDMS is a hydrophobic material);

2. Use a pipette gun to remove the streptavidin solution from the working electrode covered flow channel 1-1 through the liquid inlet and outlet 1-4;

3. Use a pipette gun to inject bovine serum albumin solution into the working electrode covered flow channel 1-1 through the liquid inlet and outlet 1-4, and after incubating at 37°C for 1 hour, the bovine serum albumin is fixed on the working electrode by physical adsorption The surface of 2-1 and the working electrode made of PDMS cover the inner surface of flow channel 1-1;

4. Use a pipette gun to remove the bovine serum albumin solution from the working electrode covered flow channel 1-1 through the liquid inlet and outlet 1-4;

5. Through the liquid inlet and outlet 1-4, use a pipette gun to inject the biotin-labeled capture antibody solution into the working electrode covered flow channel 1-1, and incubate at 37°C for 30 minutes, and the biotin will specifically bind to streptavidin , the capture antibody is indirectly immobilized on the surface of the working electrode 2-1 and the working electrode made of PDMS covers the inner surface of the flow channel 1-1;

6. Use a pipette gun to remove the biotin-labeled capture antibody solution from the working electrode covered flow channel 1-1 through the liquid inlet and outlet 1-4;

7. Seal the electrochemical sensing ELISA reagent card with capture 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.

Figure 10 is the cyclic voltammogram of the immobilization process of the capture antibody on the surface of the working electrode 2-1 (test base solution: 5mMK ₃ [Fe(CN) ₆ ]; scan rate: 0.05V/s), where curve (a) is the bare electrode , curve (b) is streptavidin, curve (c) is bovine serum albumin, and curve (d) is biotin-labeled capture antibody. Fig. 11 is a scanning electron microscope image of the surface of the working electrode 2-1 before and after immobilization of the capture antibody, wherein (a) is the bare electrode, and (b) is after the capture antibody is immobilized. Figure 10 and Figure 11 show the immobilization process of the capture antibody on the working electrode, and illustrate that the capture antibody is finally successfully immobilized on the working electrode. Among them, Figure 10 is an electrochemical characterization method, showing each step of the capture antibody immobilization; Figure 11 is a scanning electron microscope for characterization, in b, it can be seen that the capture antibody is immobilized on the surface of the working electrode.

The use of the electrochemical sensing ELISA reagent card includes the following steps:

1. Take out the electrochemical sensing ELISA reagent card from the aluminum foil bag, place it horizontally, and set the liquid inlet and outlet 1-4, and the side of the cleaning solution inlet 1-5 facing up;

2. Use phosphate-buffered saline (PBST) solution to clean the channels of the electrochemical sensing ELISA reagent card (the working electrode covers the channel 1-1, the counter electrode and the reference electrode cover the channel 1-2, and the common channel 1-3 ) inner surface and screen-printed electrode (working electrode 2-1, counter electrode 2-2, reference electrode 2-3) surface. Inject PBST solution into the electrochemical sensing ELISA reagent card through the cleaning solution injection port 1-5, the PBST solution flows through the common flow channel 1-3 in sequence, the counter electrode and reference electrode cover the flow channel 1-2, and the working electrode covers The flow channel 1-1 finally flows out from the liquid inlet and outlet 1-4; after cleaning, inject air into the electrochemical sensing ELISA reagent card through the cleaning solution injection port 1-5, thereby removing the residual PBST solution in the flow channel ;

3. Add the sample solution to be tested. Through the liquid inlet and outlet 1-4, use a pipette gun to inject the sample solution to be tested into the working electrode covered flow channel 1-1, and after incubating at 37°C for 30 minutes, the antigen to be tested will specifically bind to the capture antibody, and the working electrode 2- 1 The surface and the working electrode made of PDMS cover the inner surface of the flow channel 1-1 to form the antigen-capture antibody complex to be tested;

4. Remove the sample solution to be tested in the working electrode covered flow channel 1-1, and clean the surface of the working electrode 2-1 and the inner surface of the PDMS working electrode covered flow 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 through the common flow channel 1-3 in sequence, the counter electrode and reference electrode cover the flow channel 1-2, and the working electrode covers The flow channel 1-1 finally flows out from the liquid inlet and outlet 1-4. On the one hand, this process removes the sample solution to be tested in the flow channel 1-1 covered by the working electrode, and on the other hand, cleans the surface of the working electrode 2-1 The working electrode made of PDMS covers the inner surface of the flow channel 1-1; after the sample solution to be tested is removed and cleaned, air is injected into the electrochemical sensing ELISA reagent card through the cleaning solution injection port 1-5 to remove the flow channel. The residual PBST solution in the channel;

5. Add enzyme-labeled detection antibody solution. Through the liquid inlet and outlet 1-4, use a pipette gun to inject the enzyme-labeled detection antibody solution into the working electrode covered flow channel 1-1, and incubate at 37°C for 30 minutes. The surface of -1 and the working electrode made of PDMS cover the inner surface of flow channel 1-1 to form an immune sandwich structure (enzyme-labeled detection antibody-antigen to be tested-capture antibody complex);

6. Remove the enzyme-labeled detection antibody solution in the working electrode covering flow channel 1-1, and clean the surface of the working electrode 2-1 and the inner surface of the PDMS material working electrode covering flow 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 through the common flow channel 1-3 in sequence, the counter electrode and reference electrode cover the flow channel 1-2, and the working electrode covers The flow channel 1-1 finally flows out from the liquid inlet and outlet 1-4. On the one hand, this process removes the enzyme-labeled detection antibody solution covered by the working electrode in the flow channel 1-1, and on the other hand, cleans the working electrode 2-1. The surface and the PDMS material working electrode cover the inner surface of the flow 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, thereby removing Remove the residual PBST solution in the flow channel;

7. Add substrate solution. Inject the substrate solution into the electrochemical sensing ELISA reagent card through the cleaning solution injection port 1-5, and the substrate solution flows through the common flow channel 1-3 in sequence, the counter electrode and the reference electrode cover the flow channel 1-2, and the working The electrode covers the flow channel 1-1, and finally fills the flow channel of the electrochemical sensing ELISA reagent card (the working electrode covers the flow channel 1-1, the counter electrode and reference electrode cover the flow channel 1-2, and the common flow channel 1-3) . The substrate solution covers the surfaces of the working electrode 2-1, the counter electrode 2-2 and the reference electrode 2-3 while contacting the PDMS material working electrode to cover the inner surface of the flow channel 1-1;

8. Insert the electrochemical sensing ELISA reagent card from the reagent card insertion port 3-1 into the adapter 3, and the three conductive contacts of the screen-printed electrodes (the working electrode conductive contact 2-4, the counter electrode conductive contact 2-5 , reference electrode conductive contact piece 2-6) are respectively in contact with three metal shrapnel in adapter 3 (working electrode metal shrapnel 3-2, counter electrode metal shrapnel 3-3, reference electrode metal shrapnel 3-4), and the adapter The wires 3-5 of 3 are connected to supporting electrochemical instruments, and the three-electrode detection system is used to measure the current, quantify the antigen to be tested, and complete the simultaneous detection of multiple channels.

9. After use, pull out the electrochemical sensing ELISA reagent card from the reagent card insertion port 3-1 of the adapter 3, and discard the electrochemical sensing ELISA reagent card into a biochemical waste bin.

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 examples mentioned in this application are illustrative only and not limiting.

Although the present application has been described in conjunction with specific embodiments, those skilled in the art may make appropriate replacements, modifications and changes under the inventive spirit of the present application, and such replacements, modifications and changes still belong to the protection scope of the present application.

Claims (10)

1. An electrochemical sensing ELISA reagent card, characterized in that, comprises a bonded microfluidic channel body and an electrochemical sensor body, wherein: The body of the microfluidic channel is provided with a plurality of "ring-shaped" liquid flow channels, each of which is composed of a working electrode covering flow channel and a counter electrode and a reference electrode covering flow channel in series. The above-mentioned "annular" liquid flow channels are connected through a common flow channel; The electrochemical sensor body includes a flat substrate and a plurality of screen-printed electrodes printed in parallel on the flat substrate, and each of the screen-printed electrodes includes a working electrode, a counter electrode and a reference electrode. 2. electrochemical sensing ELISA reagent card according to claim 1, is characterized in that, wherein, described working electrode covers flow channel to cover described working electrode completely, and described counter electrode and reference electrode cover flow channel to cover completely the counter electrode and the reference electrode. 3. electrochemical sensing ELISA reagent card according to claim 1, is characterized in that, wherein, described electrochemical sensing ELISA reagent card is inserted in the adapter of supporting instrument, and described adapter comprises reagent card insertion port, working electrode Metal shrapnel, counter electrode shrapnel, reference electrode shrapnel and wire. 4. electrochemical sensing ELISA reagent card according to claim 3, is characterized in that, described electrochemical sensing ELISA reagent card inserts described adapter from described reagent card insertion port, the work of described screen printing electrode The conductive contact piece of the electrode, the conductive contact piece of the counter electrode and the conductive contact piece of the reference electrode are respectively in contact with the metal shrapnel of the working electrode, the metal shrapnel of the counter electrode and the reference electrode of the adapter. 5 . The electrochemical sensing ELISA reagent card according to claim 1 , wherein a cleaning liquid injection port is provided on the common flow channel, and a liquid inlet and outlet is provided on the working electrode covering flow channel. 6 . 6 . The electrochemical sensing ELISA reagent card according to claim 1 , wherein a capture antibody is immobilized on the surface of the working electrode and blocked with a blocking solution. 7. The electrochemical sensing ELISA reagent card according to claim 1, wherein the microfluidic channel body and the electrochemical sensor body are bonded by ultrasonic bonding, thermocompression bonding, plasma bonding, One of the adhesive bonds that bond together. 8. The electrochemical sensing ELISA reagent card according to claim 1, wherein the material of the microfluidic channel body is polydimethylsiloxane (PDMS), polystyrene (PS), One of polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE). 9. electrochemical sensing ELISA reagent card according to claim 1, is characterized in that, the material of described plate substrate is polyethylene terephthalate (PET), polystyrene (PS), polyformaldehyde One of methyl acrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE). 10. electrochemical sensing ELISA reagent card according to claim 1, is characterized in that, the material of working electrode and counter electrode is the one in carbon, gold, platinum, graphene, carbon nanotube; The material is silver/silver chloride composite.

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