Electrochemical test paper, preparation method and application thereof

Abstract

The application provides an anti-electrochemical test paper and a preparation method and a detection method thereof. The test paper comprises an insulating base layer, a sample injection area, an anti-interference area and a reaction detection area which are sequentially arranged on the insulating base layer. The anti-interference area is provided with at least two regions, and each region is provided with an anti-interference reagent. The anti-interference reagents of adjacent regions are prepared from anti-interference solutions with different pH values. The sample injection area and the reaction detection area are connected with two different regions of the anti-interference area respectively. The test paper improves the repeatability of detection by dividing the anti-interference area into regions and making the sample enter the reaction area in a neutral environment.

Description

Technical Field

[0001] This invention belongs to the field of testing technology, specifically relating to an electrochemical test paper, its preparation method, and its application. Background Technology

[0002] Because ascorbic acid and uric acid have similar concentrations and oxidation potentials in the human body, ascorbic acid can be directly electro-oxidized on the electrode surface or oxidized by oxidizing electron mediators in electrochemical test strips that use oxidation current as the detection signal. In addition, other endogenous or exogenous reducing interfering substances in the blood can also participate in the current signal generation process, resulting in high background noise and inaccurate measurement results. For electrochemical test strips that use reduction current as the detection signal, ascorbic acid can react with hydrogen peroxide, leading to a negative bias in the measurement results.

[0003] In existing technologies, an anti-interference layer is typically added to the conventional test strip structure. For example, Chinese patents CN102507670B, CN208239340U, and CN205067415U use a non-water-soluble redox mediator, mixed with toner, resin, solvent, and additives, and screen-printed onto an insulating substrate. A reagent layer containing ascorbic acid oxidase is then dropped onto the surface. Because a non-water-soluble redox mediator is used, the reagent layer containing ascorbic acid oxidase added during manufacturing forms a water-soluble anti-interference enzyme layer and a non-water-soluble reaction enzyme layer. Therefore, when testing a sample, the sample first passes through the upper ascorbic acid oxidase layer, consuming the ascorbic acid in the sample. Then, when it passes through the bottom layer, i.e., the working electrode, the reaction signal is the uric acid reaction signal. However, this approach is not only complex in its process but also increases the thickness of the test strip, making it difficult to control repeatability during mass production. Furthermore, the use of a non-water-soluble electron mediator mixed with carbon paste results in a slow reaction process where uric acid in the blood sample reaches the electrode surface and reacts with the electron mediator, leading to long detection times and poor reaction specificity. Additionally, while the high oxidation potential eliminates interference from ascorbic acid, it does not prevent interference from other substances.

[0004] Existing technologies also include mixing interfering enzymes with the analyte enzyme to form a mixed enzyme, as mentioned in patent application CN101349667A. While this method avoids the superposition of water-soluble and water-insoluble interfaces in the aforementioned methods, thus avoiding slow reaction rates, long detection times, and poor reaction specificity, its anti-interference effect is very poor. Since the interfering substance and the analyte react with the electron mediator simultaneously, it does not significantly improve the test strip's resistance to ascorbic acid interference.

[0005] Therefore, it is of great significance to design and develop test strips that are easy to operate, small and portable, require little blood, are suitable for finger prick blood, are inexpensive, avoid interference from ascorbic acid and other interfering substances in the blood, and can accurately measure uric acid concentration. Summary of the Invention

[0006] To address the aforementioned problems in the prior art, this invention proposes an electrochemical test paper, its preparation method, and its application.

[0007] In a first aspect, the present invention provides an electrochemical test paper, comprising an insulating substrate layer, wherein a sample injection area, an anti-interference area, and a reaction detection area are sequentially connected on the insulating substrate layer;

[0008] The anti-interference zone is provided with at least two areas, each area is provided with an anti-interference reagent, and the anti-interference reagents of adjacent areas are formed by anti-interference solutions with different pH values;

[0009] The sample injection area and the reaction detection area are respectively connected to two different regions of the anti-interference area.

[0010] As a specific embodiment of the present invention, the anti-interference zone is provided with an alkaline anti-interference zone, an acidic anti-interference zone and a neutral anti-interference zone.

[0011] In a preferred embodiment of the present invention, the neutral anti-interference region is connected to the reaction detection region.

[0012] In a more preferred embodiment of the present invention, the alkaline anti-interference region is connected to the sample injection area, or the acidic anti-interference region is connected to the sample injection area.

[0013] As a specific embodiment of the present invention, the anti-interference reagent disposed on the alkaline anti-interference region is formed by an alkaline anti-interference solution with a pH value of 8.0-11.0; the anti-interference reagent disposed on the acidic anti-interference region is formed by an acidic anti-interference solution with a pH value of 3.0-6.0; and the anti-interference reagent disposed on the neutral anti-interference region is formed by a neutral anti-interference solution with a pH value of 6.0-8.0.

[0014] In a preferred embodiment of the present invention, the anti-interference reagent disposed on the alkaline anti-interference region is formed by an alkaline anti-interference solution with a pH value of 8.5-9.5; and / or the anti-interference reagent disposed on the acidic anti-interference region is formed by an acidic anti-interference solution with a pH value of 4.5-5.5; and / or the anti-interference reagent disposed on the neutral anti-interference region is formed by a neutral anti-interference solution with a pH value of 6.5-7.5.

[0015] As a more preferred embodiment of the present invention, the alkaline anti-interference solution includes an alkaline buffer and a first strong oxidant; and / or, the acidic anti-interference solution includes an acidic buffer and a second strong oxidant; and / or, the anti-interference solution of the neutral anti-interference reagent includes a neutral buffer and a third strong oxidant.

[0016] As a specific embodiment of the present invention, the alkaline buffer is selected from one of phosphate buffer, borate buffer, and Tris-HCl buffer, preferably phosphate buffer; and / or,

[0017] As a specific embodiment of the present invention, the acidic buffer solution is selected from one of citrate buffer, acetate buffer, phosphate buffer, Tris-phosphate buffer, and MES buffer, preferably phosphate buffer; and / or,

[0018] As a specific embodiment of the present invention, the neutral buffer solution is selected from one of succinate buffer solution, phosphate buffer solution, and acetate buffer solution, preferably phosphate buffer solution; and / or,

[0019] As a specific embodiment of the present invention, the first strong oxidant, the second strong oxidant, and the third strong oxidant may be the same or different, and each is independently selected from at least one of sodium periodate, potassium iodate, potassium permanganate, potassium dichromate, and potassium chlorate, preferably potassium permanganate; and / or,

[0020] As a specific embodiment of the present invention, the mass ratio of the alkaline buffer solution to the first strong oxidant is 100:(3-8); and / or,

[0021] In a specific embodiment of the present invention, the mass ratio of the acidic buffer solution to the second strong oxidant is 100:(3-8); and / or,

[0022] As a specific embodiment of the present invention, the mass ratio of the neutral buffer solution to the third strong oxidant is 100:(3-8); and / or.

[0023] As a specific embodiment of the present invention, the alkaline anti-interference solution comprises an alkaline buffer, a strong oxidant, bovine serum albumin, Triton-100, and hydroxyethyl cellulose. Taking uric acid content test strips as an example, the mass ratio of the alkaline buffer, strong oxidant, bovine serum albumin, Triton-100, and hydroxyethyl cellulose is 100:(3-8):(1-3):(0.1-2):(3-8); preferably 100:5:2:1:5.

[0024] As a specific embodiment of the present invention, the acidic anti-interference solution comprises an acidic buffer solution, a strong oxidant, bovine serum albumin, Triton-100, and hydroxyethyl cellulose. Taking uric acid content test strips as an example, the mass ratio of the acidic buffer solution, strong oxidant, bovine serum albumin, Triton-100, and hydroxyethyl cellulose is 100:(3-8):(1-3):(0.1-2):(3-8); preferably 100:5:2:1:5.

[0025] As a specific embodiment of the present invention, the neutral anti-interference solution includes a neutral buffer solution, a strong oxidant, bovine serum albumin, Triton-100, and hydroxyethyl cellulose. Taking uric acid content test strips as an example, the mass ratio of the neutral buffer solution, strong oxidant, bovine serum albumin, Triton-100, and hydroxyethyl cellulose is 100:(3-8):(1-3):(0.1-2):(3-8); more preferably, it is 100:5:2:1:5.

[0026] As a specific embodiment of the present invention, the shape of the anti-interference zone is elliptical, linear, wavy, or annular; preferably, the shape is annular.

[0027] As a specific embodiment of the present invention, the alkaline buffer is selected from one of phosphate buffer, borate buffer and Tris-HCl buffer, preferably phosphate buffer.

[0028] As a specific embodiment of the present invention, the acidic buffer is selected from one of citrate buffer, acetate buffer, phosphate buffer, Tris-phosphate buffer and MES buffer, preferably phosphate buffer.

[0029] As a specific embodiment of the present invention, the neutral buffer solution is selected from one of succinate buffer solution, phosphate buffer solution and acetate buffer solution, preferably phosphate buffer solution.

[0030] As a specific embodiment of the present invention, the alkaline buffer solution is a phosphate buffer solution with a concentration of 0.1-0.3 mol / L and a pH value of 8.0-11.0, preferably 8.5-9.5.

[0031] As a specific embodiment of the present invention, the acidic buffer solution is a phosphate buffer solution with a concentration of 0.1-0.3 mol / L and a pH value of 3.0-6.0, preferably 4.5-5.5.

[0032] As a specific embodiment of the present invention, the neutral buffer solution is a phosphate buffer solution with a concentration of 0.1-0.3 mol / L and a pH value of 6.0-8.0, preferably 6.5-7.5.

[0033] As a specific embodiment of the present invention, the first strong oxidant, the second strong oxidant and the third strong oxidant may be the same or different, and each is independently selected from at least one of sodium periodate, potassium iodate, potassium permanganate, potassium dichromate and potassium chlorate, preferably sodium periodate.

[0034] In a specific embodiment of the present invention, the reaction detection zone is provided with a reaction reagent, which is formed from a reaction enzyme solution.

[0035] As a specific embodiment of the present invention, the reaction enzyme solution includes a neutral buffer, a biological reaction enzyme, an electron mediator, a binder, and a crosslinking agent; preferably, the reaction enzyme solution further includes a surfactant and a stabilizer; more preferably, the biological reaction enzyme is urate oxidase.

[0036] As a specific embodiment of the present invention, the electron mediator is selected from potassium ferricyanide, benzoquinone and ferrocene carboxylic acid, preferably potassium ferric chloride.

[0037] The adhesive is selected from hydroxyethyl cellulose, hydroxymethyl cellulose and polyethylene glycol, preferably hydroxyethyl cellulose.

[0038] In a specific embodiment of the present invention, the crosslinking agent is selected from malondialdehyde or glutaraldehyde.

[0039] As a specific embodiment of the present invention, the stabilizer is selected from at least one of bovine serum albumin, glycerol, gelatin, DTT, glycine, gentamicin, EDTA, and cyclodextrin.

[0040] As a specific embodiment of the present invention, the surfactant is selected from at least one of Triton X-100, Tween 80, and sodium dodecyl sulfate.

[0041] Taking the uric acid content test strip as an example, the reaction enzyme solution includes a neutral buffer solution, a biological reaction enzyme, potassium ferricyanide (an electron mediator), bovine serum albumin, Triton-100, hydroxyethyl cellulose, and glutaraldehyde.

[0042] Taking uric acid content test strips as an example, the reacting enzyme is one or more enzymes that react with uric acid, preferably uric acid oxidase.

[0043] As a specific embodiment of the present invention, the neutral buffer in the reaction enzyme solution can be selected from the neutral buffer described above, for example, it can be a phosphate buffer.

[0044] As a specific embodiment of the present invention, in the reaction enzyme solution, the mass ratio of the neutral buffer, biological reaction enzyme, electron mediator potassium ferricyanide, bovine serum albumin, Triton-100, hydroxyethyl cellulose, and glutaraldehyde is 100:(3-8):(3-8):(1-3):(0.1-2):(3-8):(1-3).

[0045] In a specific embodiment of the present invention, the test strip reaction detection area is divided into a reaction area and an electrode area, which are connected. The electrode area includes a working electrode, a reference electrode, a detection electrode, and a switching electrode. The electrodes in the electrode area are carbon ink with added multi-walled carbon nanotubes and carbon black. The amount of multi-walled carbon nanotubes added is 0.2-5% of the carbon ink, and the amount of carbon black added is 0.2-20% of the carbon ink.

[0046] According to the present invention, the addition of multi-walled carbon nanotubes and carbon black to carbon ink solves the problem of poor dispersibility of multi-walled carbon nanotubes and improves the conductivity of carbon black. The electrochemical film formation method not only ensures uniform and controllable film thickness but also improves the stability of the sensor. The resulting modified electrode is used for the electrochemical detection of uric acid in the presence of ascorbic acid. This method is simple and rapid. Furthermore, this modified electrode exhibits excellent electrocatalytic oxidation activity when used for uric acid detection.

[0047] In this invention, the electrode region can be formed by any method known in the art, and there are no special limitations on this.

[0048] As a specific embodiment of the present invention, the test strip further includes a covering layer, which is a covering film layer; the covering film layer is preferably a composite film layer; more preferably, the composite film layer includes a hydrophilic film layer and a non-hydrophilic film layer; even more preferably, the covering reaction area is a hydrophilic film layer and the covering anti-interference area is a non-hydrophilic film layer.

[0049] This invention does not have any special requirements for the materials of the hydrophilic and non-hydrophilic film layers, and any known hydrophilic or non-hydrophilic film layer materials in the art can be used.

[0050] As a specific embodiment of the present invention, the test paper further includes a base layer, which is an insulating material, preferably polyamide.

[0051] Secondly, the present invention provides the application of the electrochemical test paper described in the first aspect in the preparation of a uric acid detection kit.

[0052] The uric acid mentioned refers to uric acid found in samples such as blood, quality control solution, urine, and saliva.

[0053] Thirdly, the present invention provides a uric acid detection system resistant to ascorbic acid interference, comprising the test strip described in the first aspect.

[0054] Fourthly, the present invention provides a method for preparing the electrochemical test paper, comprising the following steps:

[0055] S1: An anti-interference zone and a reaction detection zone are arranged on the insulating substrate layer in one step;

[0056] S2: Apply the reaction enzyme solution to the corresponding position in the reaction detection area and the anti-interference solution to the corresponding position in the anti-interference area; after drying, attach the insulating layer and the covering layer sequentially; thus obtaining the anti-interference electrochemical uric acid test paper.

[0057] In a preferred embodiment of the present invention, the coating of anti-interference liquid at the corresponding position of the anti-interference zone specifically involves coating an alkaline anti-interference liquid, an acidic anti-interference liquid, and a neutral anti-interference liquid sequentially from the injection port to the reaction zone; or coating an acidic anti-interference liquid, an alkaline anti-interference liquid, and a neutral anti-interference liquid sequentially.

[0058] As a specific embodiment of the present invention, the drying conditions are: drying temperature of 50-70℃ and drying time of 3-25min;

[0059] As a preferred embodiment of the present invention, the drying conditions are: drying temperature of 60°C and drying time of 5-20 min.

[0060] All of the above-mentioned raw materials used in this invention can be prepared in-house or purchased commercially; this invention does not impose any particular limitations on them.

[0061] Fifthly, the present invention provides a detection method for the electrochemical test paper, comprising the following steps:

[0062] S1: The sample to be tested enters the anti-interference zone through the sample injection zone, so that the interfering substances in the sample react with the three-stage anti-interference liquid in the anti-interference zone to eliminate the interfering substances;

[0063] S2: The test sample, after the interference substances have been eliminated, enters the reaction zone and reacts with the enzyme solution, while a voltage is applied through the electrodes;

[0064] S3: Reacts with the enzyme solution to generate an electric current value, which is then converted to obtain the corresponding uric acid value.

[0065] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0066] 1. The electrochemical test paper of the present invention comprises at least two anti-interference zones, wherein the anti-interference reagents in adjacent zones are formed by anti-interference solutions with different pH values. Specifically, the anti-interference zone is divided into three segments: an alkaline anti-interference zone, an acidic anti-interference zone, and a neutral anti-interference zone. The alkaline and acidic anti-interference zones react fully with ascorbic acid to eliminate interference, and their positions can be interchanged. The neutral anti-interference zone should be placed at the end to ensure a neutral environment before the blood enters the uric acid reaction zone, thereby improving the repeatability of the detection.

[0067] 2. The electrochemical test paper of the present invention, wherein the electrode is made by adding multi-walled carbon nanotubes and carbon black to carbon ink, which solves the problem of poor dispersibility of multi-walled carbon nanotubes and improves the conductivity of carbon black. Attached Figure Description

[0068] Figure 1 This is a schematic diagram of the anti-interference zone and reaction zone in the anti-interference electrochemical uric acid test paper in an embodiment of the present invention;

[0069] Figure 2 This is a schematic diagram showing the structural decomposition of the anti-interference electrochemical uric acid test paper in an embodiment of the present invention;

[0070] Figure 3 This is a schematic diagram of the shapes of different anti-interference zones in the anti-interference electrochemical uric acid test paper in an embodiment of the present invention.

[0071] Among them, 1-hydrophilic membrane, 2-non-hydrophilic membrane, 3-sample injection area; 4-reaction detection area; 5-anti-interference area, 6-electrode, 7-base layer;

[0072] 501 - Neutral anti-interference zone, 502 - Acidic anti-interference zone, 503 - Alkaline anti-interference zone. Detailed Implementation

[0073] The present invention will be further described below with reference to specific embodiments, but this does not constitute any limitation on the present invention.

[0074] Example 1

[0075] This embodiment provides an electrochemical test paper and its preparation method, such as... Figure 1 and Figure 2 As shown:

[0076] The test strip has an anti-interference zone and a reaction detection zone arranged sequentially on its insulating base layer. The sample enters the anti-interference zone through the sample injection zone. The anti-interference zone is connected to the reaction detection zone. The anti-interference zone is coated with an anti-interference solution, and the reaction detection zone is coated with a reaction enzyme solution. An anti-interference zone is designed before the sample enters the reaction detection zone. The anti-interference zone contains a reaction substance that can consume interfering substances.

[0077] The reaction detection region comprises an interconnected reaction region and an electrode region. The electrode region includes a working electrode, a reference electrode, a detection electrode, and a switching electrode. The electrodes in this region are carbon ink with added multi-walled carbon nanotubes and carbon black. Adding multi-walled carbon nanotubes and carbon black to the carbon ink solves the problem of poor dispersibility of multi-walled carbon nanotubes and improves the conductivity of carbon black. The electrodes in this region are formed using an electrochemical film-forming method, which not only ensures uniform and controllable film thickness but also improves the stability of the sensor. This modified electrode exhibits excellent electrocatalytic oxidation activity when used to detect uric acid.

[0078] The test strip is covered with an insulating layer, such as a double-sided adhesive layer, which has a reagent window. By attaching a cover layer to the test strip, a sample injection area is formed with the reagent window. A small hole is set at the end of the reagent window away from the opening. The reagent sample can flow through the reagent window first to form a siphon pool through the siphon effect. The interfering substances in the sample react quickly with the reagent in the anti-interference area, effectively eliminating the interference of the interfering substances.

[0079] The portion of the cover layer corresponding to the sample injection area and the anti-interference area is a non-hydrophilic membrane, while the portion of the cover layer corresponding to the reaction detection area and the electrode area is a hydrophilic membrane. Because the membrane covering the anti-interference area (cover layer) is non-hydrophilic, the ascorbic acid reaction is sufficient, and the membrane covering the reaction detection area (cover layer) is hydrophilic, which makes it easier for blood samples to flow from the anti-interference area to the reaction detection area, thus accelerating the reaction speed and detection speed.

[0080] To protect the electrodes, polyamide, a high-molecular insulating material, is used as the insulating base layer.

[0081] The anti-interference zone is designed as a ring, comprising at least two regions. Each region is coated with an anti-interference reagent, and the reagents in adjacent regions are formed from anti-interference solutions with different pH values. This formation method involves covering the anti-interference solution with a dispensing agent, followed by heat treatment and drying at 60°C for 10 minutes. While the anti-interference zone is designed as a ring, it can also be elliptical, linear, wavy, etc., for details. Figure 3 The anti-interference zone can be set sequentially from the sample injection zone to the reaction detection zone as an alkaline anti-interference zone, an acidic anti-interference zone, and a neutral anti-interference zone, or sequentially as an acidic anti-interference zone, an alkaline anti-interference zone, and a neutral anti-interference zone.

[0082] Example 2

[0083] This embodiment provides an electrochemical test strip for uric acid detection, and the specific preparation steps are as follows:

[0084] Substrate preparation: Polyamide, a high-molecular insulating material, is used as the substrate.

[0085] Electrode preparation: 3% multi-walled carbon nanotubes and 12% carbon black are added to carbon ink to serve as working electrode, reference electrode, sample injection detection electrode and turn-on electrode; specifically, it is prepared by screen printing.

[0086] Preparation of neutral buffer: Prepare a phosphate buffer solution with a concentration of 0.2M and a pH of 7.0.

[0087] Preparation of acidic buffer solution: Prepare a phosphate buffer solution with a concentration of 0.2M and a pH of 5.0.

[0088] Preparation of alkaline buffer solution: Prepare a phosphate buffer solution with a concentration of 0.2M and a pH of 9.0.

[0089] Preparation of the reaction enzyme solution: Take the above neutral buffer solution as the solvent, add 5% urate oxidase, 5% potassium ferricyanide (electron mediator), 2% bovine serum albumin, 1% Triton-100, 5% hydroxyethyl cellulose, and 2% glutaraldehyde, and mix and stir until completely dissolved.

[0090] Preparation of anti-interference solutions: For the neutral anti-interference solution, use the above-mentioned neutral basal buffer as the solvent, add 5% potassium permanganate, 2% bovine serum albumin, 1% Triton-100, and 5% hydroxyethyl cellulose, and mix and stir until completely dissolved. The preparation methods for acidic and alkaline anti-interference solutions are the same as for the neutral anti-interference solution, using different basal buffers as solvents.

[0091] Preparation of uric acid test strips: A layer of reactive enzyme solution is applied to the reaction zone of the electrode using a dispensing method. An anti-interference solution is then applied to the anti-interference zone of the electrode using a dispensing method, in the following order from the sample inlet to the reaction zone: alkaline anti-interference solution, acidic anti-interference solution, neutral anti-interference solution (or acidic anti-interference solution, alkaline anti-interference solution, neutral anti-interference solution). The strips are then heat-treated at 60℃ for 10 minutes to dry. After drying, a hydrophilic / non-hydrophilic film layer is applied. Finally, after pressing and cutting, the finished anti-interference electrochemical uric acid test strips are stored in a sealed plastic container with a desiccant.

[0092] Example 3

[0093] This embodiment provides the detection method of the electrochemical uric acid test strip in Example 2, and the specific details are as follows:

[0094] When using the test strip of this invention, the exposed conductive end of the test strip is inserted into the connector of the matching detection device, and the blood sample is dripped in from the inlet of the siphon chamber.

[0095] The blood is first drawn into the anti-interference layer within the siphon chamber via a siphon effect. As the blood passes through this zone, it reacts with ascorbic acid, gradually consuming the main interfering substance, ascorbic acid. Afterward, the blood enters the reaction zone.

[0096] A voltage of 0.3V is applied, and an electrochemical reaction occurs with the enzyme solution and electron mediator to generate a current value, which is then converted to obtain the corresponding uric acid value.

[0097] Example 4

[0098] This embodiment uses the electrochemical uric acid test strip provided in Example 2 and the detection method provided in Example 3 for testing. Specific details are as follows:

[0099] Different concentrations of uric acid were prepared by adding different amounts of uric acid solution to venous blood samples. The concentrations measured by the biochemical analyzer were 256 μmol / L, 344 μmol / L, 489 μmol / L, 637 μmol / L, 781 μmol / L, 952 μmol / L, and 1178 μmol / L. The applied voltage was set to 0.3V, and each concentration was tested 10 times. The uric acid values, repeatability, and accuracy of different samples were obtained, and the results are shown in Table 1.

[0100] Table 1. Test results of the anti-interference electrochemical uric acid test strip of the present invention at different uric acid concentrations.

[0101]

[0102] In Example 4, the CV values ​​of all tested concentrations were below 4%, and the deviations from those detected by the biochemical analyzer were all less than 5%. The uric acid test strips prepared by this method have high accuracy and good repeatability, and can accurately test the concentration of uric acid in whole blood.

[0103] Example 5

[0104] This example compares the performance of the following three anti-interference liquids in different region sequences. The test scheme is the same as in Example 2.

[0105] Sequence 1: From the injection port to the reaction zone, the solutions are alkaline interfering solution, acidic interfering solution, and neutral interfering solution. The test results are shown in Table 2.

[0106] Sequence 2: From the injection port to the reaction zone, the solutions were acidic interference solution, alkaline interference solution, and neutral interference solution. The test results are shown in Table 3.

[0107] Sequence 3: From the injection port to the reaction zone, the solutions were neutral interfering solution, alkaline interfering solution, and acidic interfering solution, respectively. The test results are shown in Table 4.

[0108] Table 2. Results of Interference Resistance Tests for Sequence 1 Uric Acid Test Strips

[0109]

[0110] Table 3. Results of anti-interference test on sequence 2 uric acid test strips.

[0111]

[0112]

[0113] Table 4. Results of anti-interference test on uric acid test strips in sequence 3.

[0114]

[0115]

[0116] Sequence 1 and Sequence 2 both exhibit good anti-interference performance and good repeatability. In contrast, Sequence 3 shows poor repeatability because the anti-interference area connecting the reaction zones is not a neutral environment, and the blood sample entering the reaction zone has a certain impact on the enzyme system for uric acid detection. Therefore, preferably, the neutral anti-interference area should be located at the last connection of the reaction zones.

[0117] Example 6

[0118] The anti-interference zone in this embodiment is defined as follows.

[0119] Option 1: The anti-interference zone is a single area: the acidic anti-interference zone;

[0120] Option 2: The anti-interference zone is a single area: the alkaline anti-interference zone;

[0121] Option 3: From the injection port to the reaction zone, the zones are sequentially acidic anti-interference zone and alkaline anti-interference zone;

[0122] Option 4: From the injection port to the reaction zone, the zones are arranged in sequence as follows: acidic anti-interference zone, alkaline anti-interference zone, and neutral anti-interference zone.

[0123] The test plan is the same as in Example 2.

[0124] Table 5

[0125]

[0126]

[0127] Example 7

[0128] This embodiment compares the performance of ring-shaped, wavy, and straight anti-interference zones. Schematic diagrams of test strips with different anti-interference zone shapes are shown below. Figure 3 As shown in Table 6, Table 7, and Table 8, the test plan is the same as in Example 2.

[0129] Table 6 Test Results of the Annular Anti-interference Zone Detection Device

[0130]

[0131]

[0132] Table 7 Test Results of the Wave-Shaped Anti-Interference Zone Detection Device

[0133]

[0134] Table 8. Test Results of Straight-Line Anti-Interference Zone Test Paper

[0135]

[0136]

[0137] When the anti-interference zone is circular, it exhibits better anti-interference performance. However, when the anti-interference zone is wavy or straight, the blood sample spends a shorter time passing through it, resulting in insufficient ascorbic acid consumption and thus inferior anti-interference performance compared to a circular anti-interference zone. Therefore, the anti-interference zone should preferably be circular.

[0138] Comparative Example 1

[0139] In this comparative example, the anti-interference electrochemical uric acid test paper of the present invention is compared with the non-interference electrochemical uric acid test paper to verify the anti-interference ability of the anti-interference electrochemical uric acid test paper of the present invention against different concentrations of ascorbic acid in blood samples.

[0140] The preparation method of the interference-free uric acid test strip is the same as that in Example 2, except that no interference-resistant area is set. The preparation of other areas is the same as in Example 2 of this invention.

[0141] The interference resistance of the device of the present invention to different concentrations of ascorbic acid in blood samples was verified. Three different concentrations of uric acid were prepared by adding uric acid to the blood samples. Each sample was then divided into four portions, and ascorbic acid was added to each portion to prepare interference samples with ascorbic acid concentrations of 0 mg / dL, 3 mg / dL, 6 mg / dL, and 12 mg / dL, for a total of 12 samples. The applied voltage was set to 0.3V, and each sample was tested 10 times. The uric acid values, repeatability, and deviations from the uric acid values ​​of samples without added ascorbic acid were obtained. The results are shown in Table 9.

[0142] Table 9 Results of Ascorbic Acid Interference Tests Using Electrochemical Uric Acid Test Strips Without Anti-Interference

[0143]

[0144] Comparative Example 2

[0145] In this comparative example, the electrochemical uric acid test paper of the present invention is compared with the uric acid electrochemical test paper coated with a mixture of anti-interference solution and reaction enzyme solution to verify the anti-interference ability of the electrochemical test paper of the present invention against different concentrations of ascorbic acid in blood samples.

[0146] The preparation of anti-interference uric acid test strips with mixed coating of anti-interference solution and reaction enzyme solution involves mixing the anti-interference reagent with uric acid reaction enzyme and other reagents, and then covering the reaction area together. There is no independent anti-interference area. The preparation of other areas is the same as in Example 2 of this invention.

[0147] The interference resistance of the device of the present invention to different concentrations of ascorbic acid in blood samples was verified. Three different concentrations of uric acid were prepared by adding uric acid to the blood samples. Each sample was then divided into four portions, and ascorbic acid was added to each portion to prepare interference samples with ascorbic acid concentrations of 0 mg / dL, 3 mg / dL, 6 mg / dL, and 12 mg / dL, for a total of 12 samples. The applied voltage was set to 0.3V, and each sample was tested 10 times. The uric acid values, repeatability, and deviations from the uric acid values ​​of samples without added ascorbic acid were obtained. The results are shown in Table 10.

[0148] Table 10 Results of Ascorbic Acid Interference Test on Uric Acid Electrochemical Test Strips with Mixed Coating of Anti-interference Solution and Reactive Enzyme Solution

[0149]

[0150]

[0151] The test results of the uric acid electrochemical test paper coated with a mixture of anti-interference solution and reactive enzyme solution are shown in Table 4. The measured uric acid concentration of the sample increased significantly with the increase of ascorbic acid concentration in the sample, resulting in inaccurate test results. The anti-interference ability results of the anti-interference test paper of the present invention are shown in Table 2. With the increase of ascorbic acid concentration, the measured uric acid concentration remained basically unchanged. Using this method can effectively reduce the influence of ascorbic acid on the test results.

[0152] In summary, the anti-interference electrochemical test paper of the present invention comprises three anti-interference regions: an alkaline anti-interference region, an acidic anti-interference region, and a neutral anti-interference region. The alkaline and acidic anti-interference regions react fully with ascorbic acid to eliminate interference, and their positions can be interchanged. The neutral anti-interference region should be placed at the end to ensure a neutral environment before the blood enters the reaction zone, thereby improving the repeatability of the detection.

[0153] Any numerical value mentioned in this invention, if there is only a two-unit interval between any minimum and any maximum value, includes all values ​​that increase by one unit each time from the minimum to the maximum value. For example, if the amount of a component, or the value of a process variable such as temperature, pressure, or time, is stated as 50-90, in this specification it means specifically listing values ​​such as 51-89, 52-88… and 69-71 and 70-71, etc. For non-integer values, it may be appropriately considered that a unit is 0.1, 0.01, 0.001, or 0.0001. These are merely some specifically specified examples. In this application, in a similar manner, all possible combinations of numerical values ​​between the listed minimum and maximum values ​​are considered to have been disclosed.

[0154] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. An electrochemical test paper comprising an insulating base layer, characterized in that, The sample injection area, the anti-interference area and the reaction detection area are sequentially arranged on the insulating substrate layer; The anti-interference area is provided with at least two regions, each of which is provided with an anti-interference reagent, and the anti-interference reagents of adjacent regions are formed by anti-interference solutions with different pH values; The sample injection area and the reaction detection area are respectively connected with two different regions of the anti-interference area; The anti-interference area is provided with a basic anti-interference region, an acidic anti-interference region and a neutral anti-interference region; The neutral anti-interference region is connected with the reaction detection area; The anti-interference reagent arranged on the basic anti-interference region is formed by a basic anti-interference solution with a pH value of 8.0-11.0; the anti-interference reagent arranged on the acidic anti-interference region is formed by an acidic anti-interference solution with a pH value of 3.0-6.0; the anti-interference reagent arranged on the neutral anti-interference region is formed by a neutral anti-interference solution with a pH value of 6.0-8.0; the pH values of the basic anti-interference solution and the neutral anti-interference solution are different from 8.0, and the pH values of the acidic anti-interference solution and the neutral anti-interference solution are different from 6.0; The basic anti-interference solution for forming the anti-interference reagent on the basic anti-interference region comprises a basic buffer and a first strong oxidant; The acidic anti-interference solution for forming the anti-interference reagent on the acidic anti-interference region comprises an acidic buffer and a second strong oxidant; The neutral anti-interference solution for forming the anti-interference reagent on the neutral anti-interference region comprises a neutral buffer and a third strong oxidant.

2. The test paper according to claim 1, characterized in that, The basic anti-interference region is connected with the sample injection area, or the acidic anti-interference region is connected with the sample injection area.

3. The test paper according to claim 2, characterized in that The anti-interference reagent arranged on the basic anti-interference region is formed by a basic anti-interference solution with a pH value of 8.5-9.5; and / or the anti-interference reagent arranged on the acidic anti-interference region is formed by an acidic anti-interference solution with a pH value of 4.5-5.5; and / or the anti-interference reagent arranged on the neutral anti-interference region is formed by a neutral anti-interference solution with a pH value of 6.5-7.

5.

4. The test paper according to claim 1, characterized by The shape of the anti-interference area is oval, linear, wavy or annular.

5. The test paper according to claim 1, characterized by The basic buffer is selected from one of phosphate buffer, borate buffer and Tris-HCl buffer; and / or, The acidic buffer is selected from one of citric acid buffer, acetic acid buffer, phosphate buffer, Tris-phosphate buffer and MES buffer; and / or, The neutral buffer is selected from one of succinic acid buffer solution, phosphate buffer solution and acetic acid buffer solution; and / or, The first strong oxidant, the second strong oxidant and the third strong oxidant are the same or different, and each is independently selected from at least one of sodium periodate, potassium iodate, potassium permanganate, potassium dichromate and potassium chlorate; and / or, The mass ratio of the basic buffer to the first strong oxidant is 100:(3-8); and / or, The mass ratio of the acidic buffer to the second strong oxidant is 100:(3-8); and / or, The mass ratio of the neutral buffer to the third strong oxidant is 100:(3-8).

6. The test paper according to any one of claims 1 to 5, characterized by The reaction detection area is provided with a reaction reagent formed by a reaction enzyme solution.

7. The test paper according to claim 6, characterized in that The reaction enzyme solution comprises a neutral buffer, a biological reaction enzyme, an electronic mediator, an adhesive and a cross-linking agent.

8. The test paper according to claim 7, characterized in that The reaction enzyme solution further comprises a surfactant and a stabilizer.

9. The test paper according to claim 7, characterized by The biological reaction enzyme is uric acid oxidase.

10. The test paper according to claim 8, characterized in that The electronic mediator is selected from one of potassium ferricyanide, benzoquinone and ferrocene formic acid; and / or, the adhesive is selected from one of hydroxyethyl cellulose, hydroxymethyl cellulose and polyethylene glycol; and / or, the cross-linking agent is selected from one of malondialdehyde or glutaraldehyde; and / or, the stabilizer is selected from at least one of bovine serum albumin, glycerol, gelatin, DTT, glycine, gentamicin, EDTA, cyclodextrin; and / or, the surfactant is selected from at least one of Triton X-100, Tween 80, sodium dodecyl sulfate.

11. Use of the test paper according to any one of claims 1-10 in the preparation of a kit for detecting uric acid.

12. An ascorbic acid interference resistant uric acid detection system, characterized by, The kit comprises the test paper according to any one of claims 1-10.

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