A neutralizing antibody electrochemical sensor suitable for whole blood and its use method

Abstract

This invention discloses an electrochemical sensor for neutralizing antibodies applicable to whole blood and its usage method, comprising a foldable drainage region, a reaction region, and an adsorption region. The working electrode of the reaction region is coated with PEDOT:PSS conductive hydrogel material, which is then modified with PEG to immobilize specific biorecognition molecules. This invention's detection device can quickly and efficiently quantify neutralizing antibody concentrations, can be used for whole blood testing, can detect extremely low concentrations, and exhibits high sensitivity and good detection performance.

Description

Technical Field

[0001] This invention belongs to the field of medical testing, and relates to a timely quantitative electrochemical detection of neutralizing antibodies, and in particular to an electrochemical sensor for neutralizing antibodies applicable to whole blood and its method of use. Background Technology

[0002] After a person receives a vaccine (such as measles, polio, hepatitis B, hepatitis A, and COVID-19 vaccines), an immune response is induced, producing antibodies to prevent infection. Most antibodies produced by the immune system bind to antigens, stimulating white blood cells to engulf and destroy the foreign substance containing the antigen. Unlike ordinary antibodies, neutralizing antibodies are produced when pathogens invade the body. Pathogens invade cells by relying on specific molecules (antigens) expressed by the pathogen to bind to receptors on the cell surface in order to infect the cell and further proliferate. Neutralizing antibodies are certain antibodies produced by B lymphocytes that can bind to antigens on the surface of pathogens, thereby preventing the pathogen from adhering to target cell receptors and invading the cell. Therefore, neutralizing antibodies are an important specific defense against invading pathogens.

[0003] Therefore, quantitative detection of neutralizing antibody concentration is of great significance for assessing the effectiveness and durability of vaccination, especially in the face of emerging diseases where vaccine efficacy research is not yet fully developed (such as with COVID-19 vaccines). However, existing whole blood testing methods are insufficient for rapid quantitative testing of neutralizing antibodies. Therefore, there is a need to develop a miniaturized, portable, rapid quantitative point-of-care testing (POCT) device so that, after vaccination, testing at primary healthcare institutions can provide an objective assessment of vaccine protection, accurately and scientifically guide subsequent vaccination, improve disease control efficiency, and safeguard public health security.

[0004] Electrochemical biosensors capture target analytes in solution by immobilizing specific biorecognition molecules on the electrode surface, thereby achieving quantitative analysis of target molecules. Electrochemical detection methods have attracted increasing attention due to their simple equipment, convenient operation, rapid detection, and high sensitivity. They are widely used in the detection of small biomolecules, large protein molecules, tumor markers, and pathogenic bacteria. Furthermore, electrochemical methods can be easily combined with microfluidic chips, paper-based microelectrodes, and other technologies to achieve quantitative analysis of trace amounts of body fluids, meeting the needs of POCT (Point of Contact Testing) for on-site and real-time detection. Therefore, they have potential application value in pathogen infection and antibody detection. However, existing electrochemical sensors for antibody detection have complex fabrication processes, hindering large-scale production and application. Therefore, it is necessary to research an electrochemical detection electrode suitable for industrialization. Secondly, in the early and late stages of vaccination, the concentration of neutralizing antibodies in the body is very low. How to improve the sensitivity of the sensor to meet the testing requirements of low concentrations is also an urgent problem to be solved in this invention. Summary of the Invention

[0005] To overcome the aforementioned shortcomings of the existing technology, the first objective of this invention is to provide an electrochemical sensor for neutralizing antibodies applicable to whole blood. This sensor retains the advantages of rapid on-site detection of antibodies using traditional competitive methods, while also achieving high sensitivity and wide range of measurement for electrochemical detection through PEDOT:PSS (poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate)) hydrogel to make the paper fibers conductive. This allows for timely and quantitative neutralization detection.

[0006] An electrochemical sensor for neutralizing antibodies applicable to whole blood is an integrally molded paper fiber substrate detection sheet; the paper fiber substrate detection sheet includes three functional areas: a drainage area (1), a reaction area (2) and an adsorption area (3), and creases are provided between adjacent functional areas;

[0007] The drainage area (1) includes a dripping area (11), a rinsing area (12), an infiltration area (13) and a microfluidic channel (14) arranged in sequence. The dripping area (11) and the rinsing area (12) are connected to each other, and the rinsing area (12) and the infiltration area (13) are connected to each other through the microfluidic channel (14).

[0008] The reaction zone (2) includes a microfluidic electrochemical electrode, a conductive strip (24), and a signal output interface (25). The microfluidic electrochemical electrode includes a reference electrode (21), a counter electrode (22), and a working electrode (23). The reference electrode (21) has an arc-shaped structure, and the counter electrode (22) has an arc-shaped structure. The reference electrode (21) and the counter electrode (22) form an outer ring. The working electrode (23) has a circular sheet-like structure and is located inside the ring formed by the reference electrode (21) and the counter electrode (22). A gap is left between the outer ring formed by the reference electrode (21) and the counter electrode (22) and the working electrode (23). The conductive strip (24) includes a first conductive strip, a second conductive strip, and a third conductive strip. The signal output interface includes a first signal output interface, a second signal output interface, and a third signal output interface; one end of the first conductive strip is connected to the reference electrode (21), and the other end is connected to the first signal output interface; one end of the second conductive strip is connected to the counter electrode (22), and the other end is connected to the second signal output interface; one end of the third conductive strip is connected to the working electrode (23), and the other end is connected to the third signal output interface; wherein the working electrode (23) is fixed with an antigen probe; the antigen probe is composed of a PEDOT:PSS hydrogel modified with -OH groups, which is fixed with a biorecognition molecule that can specifically bind to the antigen protein;

[0009] The PEDOT:PSS hydrogel modified with -OH groups and the paper fibers inside the working electrode (23) form a conductive three-dimensional network;

[0010] The adsorption region (3) includes an integrally formed adsorption region (31) and an extended region (32);

[0011] After being folded according to the creases, the permeation zone (13), the microfluidic electrochemical electrode and the adsorption zone (31) are arranged vertically aligned, and the droplet zone (11), the rinsing zone (12) and the extension zone (32) are all offset from the microfluidic electrochemical electrode; the upper surface of the microfluidic electrochemical electrode is in contact with the lower surface of the permeation zone (13).

[0012] Preferably, the dripping area (11), rinsing area (12) and penetration area (13) are all circular structures.

[0013] Preferably, the microfluidic channel (14) adopts a long strip structure.

[0014] Preferably, the adsorption region (31) has a circular structure and the extended region (32) has a long strip structure.

[0015] Preferably, the non-drip zone (11), rinsing zone (12), permeation zone (13), and microfluidic channel (14) of the drainage zone (1) are made of paper fiber and have wax layers on the upper and lower surfaces; the non-microfluidic electrochemical electrode of the reaction zone (2) is made of paper fiber and has wax layers on the upper and lower surfaces; the non-adsorption zone and the extension zone of the adsorption zone (3) are made of paper fiber substrate and have wax layers on the upper and lower surfaces.

[0016] Preferably, the paper fiber substrate detection sheet uses paper fibers with a pore size of 2-6 μm and a thickness of 700-1200 μm.

[0017] Preferably, the paper fiber substrate detection sheet is produced using a screen printing process.

[0018] Preferably, the outer ring formed by the reference electrode (21) and the counter electrode (22) has a window, and the third conductive strip passes through the window and is connected to the working electrode (23).

[0019] The second objective of this invention is to provide a method for using the above-mentioned neutralizing antibody electrochemical sensor applicable to whole blood, employing the following technical solution:

[0020] Step S1: Folding of the electrochemical sensor

[0021] The electrochemical sensor is folded along the crease so that the permeation area (13), the microfluidic electrochemical electrode and the adsorption area (31) are aligned vertically, and the droplet area (11), the rinsing area (12) and the extension area (32) are all offset from the microfluidic electrochemical electrode.

[0022] Step S2: Add the blood sample to be tested.

[0023] A blood sample is mixed with an antigen protein solution and pre-reacted for a period of time before being added dropwise to the droplet area (11). Due to the limitation of the fiber pore size, the blood cells in the mixture remain in the droplet area (11), while the remaining portion is adsorbed sequentially into the rinsing area (12) and the permeation area (13) through the microfluidic channel (14) by the capillary action of the paper fibers, and finally enters the reaction area (2). After incubation on the microfluidic electrochemical electrode for a period of time, the antigen proteins in the mixture that have not bound to the neutralizing antibodies are fully bound to the antigen probes in the working electrode. The antigen proteins can bind to the neutralizing antibodies in the blood or to the antigen probes fixed in the working electrode (23) of the reaction area. Therefore, when neutralizing antibodies are present in the blood, the antigen proteins will bind to the neutralizing antibodies in the blood during the pre-reaction stage, and the remaining antigen proteins will bind to the antigen probes in the working electrode (23) of the reaction area during the incubation stage. The lower the concentration of neutralizing antibodies in the blood, the more antigen proteins can bind to the antigen probe; conversely, the higher the concentration of neutralizing antibodies in the blood, the fewer antigen proteins remain, and therefore the fewer antigen proteins can bind to the antigen probe in the working electrode (23).

[0024] Step S3: Rinse with PBS rinsing solution

[0025] PBS rinsing solution is added to the rinsing zone (12). Under the capillary action of the paper fibers, the PBS rinsing solution is sequentially adsorbed into the rinsing zone (12) and the permeation zone (13) through the microfluidic channel (14), and then enters the reaction zone (2) to clean the microfluidic electrochemical electrode, removing interfering substances. It then enters the adsorption zone (3). During the cleaning process, the portion of the blood that cannot bind to the antigen probe enters the adsorption zone (3) with the cleaning solution, leaving only the antigen protein that binds to the antigen probe inside the working electrode. Since the amount of antigen protein that can bind to the antigen probe is inversely proportional to the concentration of neutralizing antibodies in the blood, the generated electrochemical signal reflects the concentration of neutralizing antibodies.

[0026] Step S4: Output of electrical signal results

[0027] The output interface (25) is connected to an external circuit and a corresponding control signal is applied. The microfluidic electrochemical electrode generates a corresponding output electrical signal, which is transmitted to the output interface (25) through the conductive strip (24). The output interface (25) outputs the output electrical signal to a signal display equipped with an LED screen through the external circuit. The correspondence and value between the output electrical signal and the neutralizing antibody concentration are calculated.

[0028] In this invention, the drainage zone (1) has the function of separating blood cells. The pore size of the paper fibers in the drainage zone (1) is about 2-6 μm, while the diameter of blood cells is about 6-10 μm. Therefore, when the mixture of blood and antigen protein solution is added to the dripping zone (11), larger blood cells will not be able to pass through the smaller pore size of the fibers and will be basically left in the dripping zone, thereby reducing the influence of blood cells on the sensor signal. The rinsing zone (12) is located between the dripping zone (11) and the permeation zone (13). When PBS rinsing solution is added, the blood cells located in the dripping zone (11) will not be flushed into the permeation zone (13).

[0029] After the surface of the working electrode (23) in the reaction zone (2) is modified with PEDOT:PSS hydrogel, the PEDOT:PSS hydrogel is wrapped on the paper fiber surface to form a conductive layer, and the paper fiber is used to form a three-dimensional conductive network, which greatly increases the effective area of ​​the working electrode (23), enabling it to fix more recognition molecules (including antibodies, enzymes, DNA and other specific recognition molecules), improve the recognition efficiency of target molecules, thereby improving the sensitivity of the sensor, reducing the detection limit of the sensor, and enabling it to detect ultra-low concentrations of target molecules.

[0030] The presence of the adsorption region (3) allows the PBS rinsing solution to penetrate downwards after passing through the working electrode (23), thereby rinsing the electrode. The target molecules captured by the recognition molecules in the working electrode (23) will not be washed away, while other interfering substances in the blood sample to be tested, which are not captured, will be adsorbed into the adsorption region (3) under the rinsing of the PBS rinsing solution. By controlling the amount of blood sample to be tested and the amount of PBS rinsing solution added, all the interfering substances in the reaction region (2) can be rinsed into the adsorption region (3).

[0031] The neutralizing antibody electrochemical sensor for whole blood disclosed herein has the following advantages:

[0032] (1) The antigen protein in the mixture can compete with the antigen probe inside the electrode for neutralizing antibody, thus changing the electrode signal. The higher the concentration of neutralizing antibody, the fewer antigen proteins can bind to the antigen probe in the working electrode, and the weaker the sensor signal. Therefore, the concentration of neutralizing antibody in serum can be quantitatively analyzed.

[0033] (2) PEDOT:PSS hydrogel was used to modify the surface of the paper fiber working electrode to form a conductive three-dimensional network, which fixed the protein on the surface of the conductive fiber. This not only greatly increased the amount of protein fixed, but also reduced the steric hindrance of the protein, thereby improving the sensitivity and minimum detection limit of the sensor.

[0034] (3) Paper-based microfluidics can achieve controllable flow of liquid in the electrode channel in both the lateral and longitudinal directions. It can not only accurately control the amount of solution required for the entire test process, but also facilitate the subsequent electrode cleaning and interference elimination process through the layered microfluidic channel.

[0035] (4) The present invention adopts a folded structure, and the microfluidic channel and electrochemical electrode can be formed on the filter paper in one step by screen printing process. It can be batch processed by screen printing process, which greatly reduces the process difficulty and manufacturing cost, and meets the requirements of large-scale production and processing. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the folded structure of the neutralizing antibody electrochemical sensor applicable to whole blood according to the present invention;

[0037] Figure 2 This is an enlarged schematic diagram of the internal structure of the modified working electrode in this invention;

[0038] Figure 3 This is a schematic diagram of the sensor structure before it is folded.

[0039] Figure 4 This is a schematic diagram of the liquid flow direction after the sensor of the present invention is folded.

[0040] The diagram is labeled as follows: Drainage zone 1, Reaction zone 2, Adsorption zone 3, Droplet zone 11, Rinse zone 12, Permeation zone 13, Microfluidic channel 14, Reference electrode 21, Counter electrode 22, Working electrode 23, Conductive strip 24, Signal output interface 25, Adsorption region 31, Extension region 32, Blood sample and antigen protein mixture 4, PBS rinse solution 5, Antigen probe 6. Detailed Implementation

[0041] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0042] like Figure 1As shown, the neutralizing antibody electrochemical sensor suitable for whole blood includes a drainage zone 1, a reaction zone 2, and an adsorption zone 3. The drainage zone 1 is used to drop blood samples and an antigen protein RBD (receptor-binding domain of the spike glycoprotein S on the surface of the SARS-CoV-2 virus) solution, ensuring thorough mixing before flowing to the reaction zone. After the blood and antigen protein RBD mixed solution sample is dropped into the droplet zone 11, larger blood cells are retained in the droplet zone 11 due to the pore size of the paper fiber substrate (2–6 μm), which is smaller than the diameter of blood cells. The serum, now free of blood cells, is guided by the capillary action of the paper fibers, passing through the microfluidic channel 14 and the permeation zone 13 of the device to rapidly reach the electrode's reaction zone 2.

[0043] Reaction zone 2 includes a microfluidic paper-based electrochemical electrode prepared by screen printing, a conductive strip 24, and a signal output interface 25. The microfluidic paper-based electrochemical electrode includes a reference electrode 21, a counter electrode 22, and a working electrode 23, used for the generation and measurement of electrochemical signals. The conductive strip 24 and the signal output interface 25 are used for the transmission and conversion of electrical signals. The counter electrode 22, the conductive strip 24, and the signal output interface 25 are all prepared by screen printing carbon paste material on the surface of a paper fiber substrate. The reference electrode 21 is prepared by screen printing silver chloride material on the surface of a paper fiber substrate. The working electrode 23 is formed by casting a PEDOT:PSS solution onto the surface of a paper fiber substrate. The PEDOT:PSS solution is uniformly diffused on the surface of the paper fiber, forming a porous conductive layer of PEDOT:PSS hydrogel through self-assembly, thereby realizing effective electron transport and mass diffusion within the paper fiber. To immobilize the specific antigen probe ACE-2 (angiotensin-converting enzyme 2) inside the working electrode 23, polyethylene glycol (PEG) can be used to modify the -OH groups of PEDOT:PSS, making it suitable for subsequent amylation and protein immobilization. PEG has good hydrophilicity; each PEG backbone can firmly bind water molecules, forming a tight hydration layer by connecting each chain via ether oxygen, thereby physically preventing protein adsorption and improving the electrode's anti-interference capability. After attaching -OH groups to the conductive fiber surface by linking PEG, 3-aminopropyltriethoxysilane (APTES) can react with the -OH groups to modify the conductive fiber surface with amino groups that can bind to proteins. Then, through the condensation reaction between the carboxyl groups on the protein surface and the amino groups on the electrode surface, the antigen probe ACE-2 is immobilized on the electrode surface, forming an antigen protein RBD capture site. Figure 2This is an enlarged schematic diagram of the internal structure of the modified working electrode in this invention. The working electrode in this invention utilizes the three-dimensional structure of the paper fiber itself, and forms a conductive three-dimensional network after being modified with PEDOT:PSS. The antigen probe 6 (ACE-2) is fixed on the surface of the conductive fiber, which not only greatly increases the amount of protein immobilized, but also reduces the steric hindrance of the protein. When the serum flows through the working area of ​​the electrode, it can efficiently capture the antibody, thereby improving the sensitivity and the lowest detection limit of the sensor.

[0044] This invention employs a competitive method to detect neutralizing antibodies in blood: the blood sample to be tested is first mixed with an antigen protein RBD solution for a pre-reaction. If neutralizing antibodies are present in the blood, the antigen protein RBD will bind to the neutralizing antibodies, thereby reducing the concentration of free antigen protein RBD in the mixture. When the mixture of serum (without blood cells) and antigen protein RBD passes through drainage zone 1 and reaches reaction zone 2, the free antigen protein RBD in the serum that has not been bound by neutralizing antibodies can be captured by the recognition molecule antigen probe 6 (ACE-2) immobilized inside the working electrode, generating a corresponding electrochemical signal. Simultaneously, since the amount of antigen protein RBD that can bind to antigen probe 6 (ACE-2) is inversely proportional to the concentration of neutralizing antibodies in the blood, the generated electrochemical signal reflects the concentration of neutralizing antibodies. The electrical signal generated by the electrochemical electrode is connected to an external circuit via conductive strip 24 and output interface 25, and transmitted to a visualization device for calculation and display, facilitating observation.

[0045] Adsorption zone 3 consists of adsorption region 31 and extension region 32, used to hold the reacted substances. After the mixture of serum and antigen protein RBD reaches electrode reaction zone 2 for incubation reaction, the electrode can be rinsed by adding an appropriate amount of PBS solution to rinsing zone 12. During rinsing, only antigen protein RBD bound to antigen probe 6 (ACE-2) can remain inside the working electrode. Other substances in the serum, such as interfering proteins and other small molecules, will continue to reach adsorption zone 31 under the PBS solution rinsing after the reaction, and then reach the extension region 32 at the end under capillary action. The electrode cleaning process can remove the influence of interfering substances on the electrode signal and improve the electrode's anti-interference ability.

[0046] like Figure 3The image shows the sensor structure before folding. The sensor described in this invention employs a folded structure, allowing the microfluidic channels and electrochemical electrodes to be formed on filter paper in a single step via screen printing. This eliminates the need for cumbersome processing steps, significantly reducing the complexity and cost of manufacturing and meeting the requirements for large-scale production. After the paper-based electrodes are mass-produced using screen printing, three-dimensional microfluidic channels can be fabricated according to the corresponding functional areas through cutting and folding. This enables controllable lateral and longitudinal flow of liquid within the electrode channels, facilitating functions such as liquid addition, reaction incubation, and waste liquid adsorption in microfluidic reactions. During the folding process, attention must be paid to the correspondence between the upper and lower layers of the functional areas to ensure the correct flow direction of the liquid.

[0047] The diagram showing the liquid flow direction after folding is as follows: Figure 4 As shown, the blood sample and antigen protein (RBD) mixture 4 is first added dropwise to the droplet area 11. After separation from blood cells in the droplet area, the serum and antigen protein RBD mixture flows along the direction indicated by the arrow through the microfluidic channel 14 and the permeation area 13, reaching the upper part of the reaction area 2, and further flowing from top to bottom into the electrochemical electrode area under capillary action. By controlling the amount of solution added, the liquid can be made to reach the electrochemical electrode area during incubation without reaching the adsorption area. After the incubation reaction is completed, PBS rinsing solution 5 is added dropwise to the rinsing area 12. Similarly, under capillary action, the liquid continues to advance along the paper-based microfluidic channel, and the rinsing solution and the reaction solution reach the adsorption area 31 of the adsorption area 3 from top to bottom, and then reach the extension area 32 along the direction indicated by the arrow. By controlling the amount of rinsing solution added and the size of the adsorption area, it can be ensured that interfering substances inside the working electrode are completely rinsed away. Therefore, the generated electrochemical signal comes only from the antigen protein BRD that binds to the antigen probe 6 (ACE-2), and is unrelated to other substances in the blood. This invention, through the ingenious design of microfluidic channels and test electrodes, enables the entire microfluidic process to proceed without external power, resulting in a simple structure and convenient operation. The paper-based microfluidic channel not only allows for precise control of the solution volume required throughout the testing process, enabling quantitative analysis of trace samples, but the presence of adsorption zone 3 also reduces contamination from reaction residues, facilitating subsequent electrode cleaning and interference removal.

[0048] The invention has been detailed and described in the accompanying drawings and the foregoing description, but these descriptions should be considered illustrative or exemplary rather than restrictive. The invention is not limited to the disclosed embodiments. Other variations of the disclosed embodiments will be understood and implemented by those skilled in the art through study of the drawings, the specification, and the appended claims and practice of the claimed invention.

Claims

1. A neutralizing antibody electrochemical sensor suitable for whole blood, characterized in that, It is an integrally formed paper fiber substrate detection sheet; the paper fiber substrate detection sheet includes three functional areas: a drainage area (1), a reaction area (2) and an adsorption area (3), and creases are provided between adjacent functional areas; The drainage area (1) includes a dripping area (11), a rinsing area (12), an infiltration area (13) and a microfluidic channel (14) arranged in sequence. The dripping area (11) and the rinsing area (12) are connected to each other, and the rinsing area (12) and the infiltration area (13) are connected to each other through the microfluidic channel (14). The reaction zone (2) includes a microfluidic electrochemical electrode, a conductive strip (24), and a signal output interface (25). The microfluidic electrochemical electrode includes a reference electrode (21), a counter electrode (22), and a working electrode (23). The reference electrode (21) has an arc-shaped structure, and the counter electrode (22) has an arc-shaped structure. The reference electrode (21) and the counter electrode (22) form an outer ring. The working electrode (23) has a circular sheet-like structure and is located inside the ring formed by the reference electrode (21) and the counter electrode (22). A gap is left between the outer ring formed by the reference electrode (21) and the counter electrode (22) and the working electrode (23). The conductive strip (24) includes a first conductive strip, a second conductive strip, and a third conductive strip. The signal output interface includes a first signal output interface, a second signal output interface, and a third signal output interface; one end of the first conductive strip is connected to the reference electrode (21), and the other end is connected to the first signal output interface; one end of the second conductive strip is connected to the counter electrode (22), and the other end is connected to the second signal output interface; one end of the third conductive strip is connected to the working electrode (23), and the other end is connected to the third signal output interface; wherein the working electrode (23) is fixed with an antigen probe; the antigen probe is composed of a PEDOT:PSS hydrogel modified with -OH groups, which is fixed with a biorecognition molecule that can specifically bind to the antigen protein; -OH group-modified PEDOT:PSS hydrogel and the paper fibers inside the working electrode (23) form a conductive three-dimensional network; The adsorption region (3) includes an integrally formed adsorption region (31) and an extended region (32); After being folded according to the creases, the permeation zone (13), the microfluidic electrochemical electrode and the adsorption zone (31) are arranged vertically aligned, and the droplet zone (11), the rinsing zone (12) and the extension zone (32) are all offset from the microfluidic electrochemical electrode; the upper surface of the microfluidic electrochemical electrode is in contact with the lower surface of the permeation zone (13).

2. The sensor according to claim 1, characterized in that... The dripping area (11), rinsing area (12) and penetration area (13) are all circular structures.

3. The sensor according to claim 1, characterized in that... The microfluidic channel (14) adopts a long strip structure.

4. The sensor according to claim 1, characterized in that... The adsorption region (31) has a circular structure, and the extended region (32) has a long strip structure.

5. The sensor according to claim 1, characterized in that... Wax layers are laid on the upper and lower surfaces of the non-drip zone (11), rinsing zone (12), permeation zone (13), and microfluidic channel (14) in the drainage zone (1); wax layers are laid on the upper and lower surfaces of the non-microfluidic electrochemical electrode in the reaction zone (2); and wax layers are laid on the upper and lower surfaces of the non-adsorption zone and the extension zone in the adsorption zone (3).

6. The sensor according to claim 1, characterized in that... The paper fiber substrate detection sheet uses paper fibers with a pore size of 2-6 μm and a thickness of 700-1200 μm.

7. The sensor according to claim 1, characterized in that... The paper fiber substrate test strip is manufactured using a screen printing process.

8. The sensor according to claim 1, characterized in that... A window is opened on the outer ring formed by the reference electrode (21) and the counter electrode (22), and the third conductive strip passes through the window and is connected to the working electrode (23).

9. The method of using the neutralizing antibody electrochemical sensor applicable to whole blood according to any one of claims 1-8, characterized in that... The method includes the following steps: Step S1: Folding of the electrochemical sensor The electrochemical sensor is folded along the crease so that the permeation area (13), the microfluidic electrochemical electrode and the adsorption area (31) are aligned vertically, and the droplet area (11), the rinsing area (12) and the extension area (32) are all offset from the microfluidic electrochemical electrode. Step S2: Add the blood sample to be tested. The blood sample is mixed with the antigen protein solution and pre-reacted for a period of time before being added dropwise to the droplet area (11). Due to the limitation of the fiber pore size, the blood cells in the mixture remain in the droplet area (11), while the rest are adsorbed sequentially into the rinsing area (12) and the permeation area (13) through the microfluidic channel (14) under the capillary action of the paper fiber, and finally enter the reaction area (2). After incubation on the microfluidic electrochemical electrode for a period of time, the antigen protein in the mixture that has not bound to the neutralizing antibody is fully bound to the antigen probe in the working electrode. Step S3: Rinse with PBS rinsing solution PBS rinsing solution is added to the rinsing zone (12). Under the capillary action of the paper fibers, the PBS rinsing solution is adsorbed into the rinsing zone (12) and the permeation zone (13) through the microfluidic channel (14) in sequence, and enters the reaction zone (2) to clean the microfluidic electrochemical electrode, remove interfering substances, and then enters the adsorption zone (3). Step S4: Output of electrical signal results The output interface (25) is connected to an external circuit and a corresponding control signal is applied. The microfluidic electrochemical electrode generates an output electrical signal, which is transmitted to the output interface (25) through the conductive strip (24). The output interface (25) outputs the output electrical signal to a signal display equipped with an LED screen through the external circuit. The correspondence and value between the output electrical signal and the neutralizing antibody concentration are calculated.

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