Enzyme reagent, glucose electrochemical test paper and preparation method of glucose electrochemical test paper

Abstract

The invention relates to an enzyme reagent, glucose electrochemical test paper and a preparation method thereof, and belongs to the technical field of electrochemical detection. The glucose electrochemical test paper comprises an electrode substrate and an enzyme reagent arranged on the electrode substrate, the enzyme reagent is prepared from the following components in parts by mass: 5 to 7 parts of buffer solution, 0.01 to 0.2 part of hexamine ruthenium trichloride, 0.01 to 0.1 part of quinone compound, 0.1 to 1.2 parts of metal organic framework immobilized glucose dehydrogenase and 0.2 to 0.8 part of film-forming agent. According to the glucose electrochemical test paper, the detection accuracy of glucose can be improved, and the problems of large interference, small linear range, insensitive detection and the like in the prior art are improved or solved.

Description

Technical Field

[0001] The invention relates to an enzyme reagent, a glucose electrochemical test paper and a preparation method thereof, and belongs to the technical field of electrochemical detection. Background Art

[0002] Diabetes is a serious metabolic disease. Despite the continuous advancement of modern medical technology, the number of diabetic patients is still increasing. Therefore, monitoring blood glucose levels is of great significance for preventing and reducing the occurrence of diabetes and related diseases. Self-monitoring blood glucose devices that are reliable, easy to use, and efficient to test have been developed to quantify the blood glucose levels of diabetic patients. They provide a simple and rapid method for blood glucose quantification, so that patients can make important decisions related to their diet, drug dosage, and exercise, so as to achieve optimal blood glucose levels. At present, many companies on the market have developed glucose sensors based on biological enzymes (glucose oxidase and glucose dehydrogenase) and redox mediators for diabetic patients to self-monitor blood glucose concentrations in the body.

[0003] Traditional glucose testing methodology is mainly based on the photochemical color development principle and electrochemical current response to construct blood glucose sensors. However, the photochemical method based on the light intensity before and after the reaction usually has inherent disadvantages in the process of glucose detection, such as low accuracy, detection conditions are greatly affected by environmental factors, and background noise interference. In comparison, the electrochemical method based on converting the chemical reaction of glucose into a current signal has significant advantages such as higher accuracy and selectivity, low background signal and no interference from color changes, as well as faster response time and wider detection range.

[0004] Chen Zhencheng et al. (See: Chen Zhencheng, Li Lingyun, Deng Zhensheng. Research on a Glucose Oxidase Amperometric Sensor [J]. Journal of Sensor Technology, 2007, 20(4): 4. DOI: 10.3969 / j.issn.1004-1699.2007.04.006.) immobilized glucose oxidase on the surface of a carbon electrode, used potassium ferrocyanide as the medium of the electrochemical reaction, and used the amperometric method to indirectly measure glucose. The measurement range of glucose was 2.7-27 mmol / L. However, this technology uses potassium ferrocyanide as an electron transfer agent, and potassium ferrocyanide has poor thermal stability. The higher the temperature, the greater the background current. It is also easily interfered by reducing substances in the body, such as Vc, uric acid, bilirubin, creatinine, etc., and is greatly affected by individual factors. In addition, the linear range of glucose determination is small.

[0005] Chinese invention patent application CN1912626A discloses a method for preparing a disposable carbon-based glucose chip and an electrochemical detection method. The patent first uses screen printing technology to prepare a disposable electrode chip comprising a working electrode, an auxiliary electrode and a reference electrode on the surface of a PVC matrix. Combined with polymer membrane technology, a mixture of glucose oxidase and ferrocene acid is fixed on the surface of the working electrode, and the amperometric response of the electrochemically active substances produced by the enzyme's catalytic reaction of glucose is used to achieve rapid detection of glucose concentration in the blood. This method uses ferrocene as an electron transfer mediator, which has stability problems and may affect the long-term performance and reliability of glucose detection. In addition, during the detection process, other electroactive substances such as ascorbic acid and uric acid may be oxidized at high potential to generate oxidation current, which interferes with the determination and reduces sensitivity and selectivity.

[0006] Chinese invention patent application CN115893870A discloses a sensing electrode for detecting glucose and an electrode preparation method. The method uses a printable liquid metal on a substrate to synthesize multifunctional cuprous oxide to construct a cuprous oxide sensing electrode, which can oxidize glucose at a negative potential and thus be used to detect glucose. However, the sensor does not use glucose oxidase or glucose dehydrogenase that is highly specific for glucose. Therefore, it may be interfered by other substances during the detection process, and the low selectivity for glucose leads to inaccurate measurement results.

[0007] At present, glucose sensors constructed using electrochemical methods have problems that need to be solved, such as insufficient sensitivity, small linear range, and interference from many substances, resulting in low accuracy and repeatability. In addition, most of the current glucose sensors are developed based on glucose oxidase, which is easily affected by oxygen partial pressure, resulting in relative limitations in the detection of samples. Moreover, as a protein, the enzyme is extremely susceptible to environmental influences, which reduces the catalytic activity of the enzyme and thus affects the stability of the sensor. Summary of the invention

[0008] In view of the deficiencies in the prior art, one of the objects of the present invention is to provide an enzyme reagent, and the glucose electrochemical test paper constructed with the participation of the enzyme reagent can show excellent detection sensitivity; the second object of the present invention is to provide a glucose electrochemical test paper with high detection sensitivity; the third object of the present invention is to provide a method for preparing a glucose electrochemical test paper.

[0009] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0010] An enzyme reagent comprises, by weight, 5 to 7 parts of buffer, 0.01 to 0.2 parts of hexammineruthenium trichloride (CAS: 14282-91-8), 0.01 to 0.1 parts of quinone compounds, 0.1 to 1.2 parts of metal organic framework immobilized glucose dehydrogenase, and 0.2 to 0.8 parts of film-forming agent.

[0011] Furthermore, the enzyme reagent includes, by weight, 5.5 to 6.5 parts of buffer, 0.05 to 0.15 parts of hexammineruthenium trichloride, 0.05 to 0.08 parts of quinone compounds, 0.4 to 0.8 parts of metal organic framework-immobilized glucose dehydrogenase, and 0.4 to 0.6 parts of film-forming agent.

[0012] Furthermore, the quinone compound is one or both of 2,9-dimethyl-1,10-phenanthroline-5,6-dione and 1,10-phenanthroline-5,6-dione.

[0013] Preferably, the pH value of the buffer solution is 6.9-7.5, and further 7-7.2.

[0014] Preferably, the buffer is one of PBS buffer, HEPES buffer, TES buffer and Bis-Tris buffer.

[0015] Preferably, the film-forming agent is one or more of hydroxyethyl cellulose and polyvinyl pyrrolidone; more preferably, the film-forming agent is composed of hydroxyethyl cellulose and polyvinyl pyrrolidone in a mass ratio of 0.2-0.5:0.3-0.8; preferably, the weight average molecular weight of polyvinyl pyrrolidone is 7000-9000.

[0016] Preferably, the metal organic framework is one or more of Cu-TATB MOF, ZIF-8, ZIF-90, and MAF-7.

[0017] Preferably, the enzyme reagent further comprises 0.5 to 1 part of a non-ionic surfactant, and more preferably, the non-ionic surfactant is Triton X-100.

[0018] Based on the same inventive concept, the present invention also provides: a glucose electrochemical test paper, comprising an electrode substrate and an enzyme reagent arranged on the electrode substrate; measured by mass, the enzyme reagent contains 5 to 7 parts of buffer solution, 0.01 to 0.2 parts of hexaamineruthenium trichloride, 0.01 to 0.1 parts of quinone compounds, 0.1 to 1.2 parts of metal organic framework-immobilized glucose dehydrogenase, and 0.2 to 0.8 parts of a film-forming agent.

[0019] Further, the quinone compound is one of 2,9-dimethyl-1,10-phenanthroline-5,6-dione and 1,10-phenanthroline-5,6-dione;

[0020] Furthermore, the film-forming agent is one or more of hydroxyethyl cellulose and polyvinyl pyrrolidone; more preferably, the film-forming agent is composed of hydroxyethyl cellulose and polyvinyl pyrrolidone in a mass ratio of 0.2-0.5:0.3-0.8; even more preferably, the film-forming agent is composed of hydroxyethyl cellulose and polyvinyl pyrrolidone in a mass ratio of 0.3-0.4:0.4-0.7.

[0021] Furthermore, the metal organic framework is one or more of Cu-TATB MOF, ZIF-8, ZIF-90, and MAF-7.

[0022] Further, the enzyme reagent further contains 5.5-6.5 parts of a buffer solution, more preferably, the pH value of the buffer solution is 6.9-7.5, and more preferably 7-7.1; more preferably, the buffer solution is one of a PBS buffer solution, a HEPES buffer solution, a TES buffer solution and a Bis-Tris buffer solution;

[0023] Preferably, the enzyme reagent further contains 0.5-1 part (further 0.6-0.8 part) of a non-ionic surfactant, and more preferably, the non-ionic surfactant is Triton X-100.

[0024] Optionally, the preparation method of the enzyme reagent comprises the following steps: uniformly mixing a buffer solution, hexammineruthenium trichloride, a quinone compound, a metal organic framework-immobilized glucose dehydrogenase, and a film-forming agent to obtain the enzyme reagent.

[0025] Optionally, the electrode substrate comprises a substrate on which a working electrode and a counter electrode are provided.

[0026] Optionally, the working electrode is made of carbon, silver-carbon, gold or palladium-carbon.

[0027] Optionally, the counter electrode is made of carbon, silver-carbon, gold or palladium-carbon.

[0028] Furthermore, the preparation method of metal organic framework immobilized glucose dehydrogenase comprises the following steps:

[0029] (1) Providing Cu-TATB MOF powder;

[0030] (2) immersing the Cu-TATB MOF powder in a buffer solution containing a biological enzyme, culturing, solid-liquid separation, washing, and drying to obtain a metal organic framework immobilized glucose dehydrogenase (GDH@Cu-TATB MOF);

[0031] The pH value of the buffer solution is 6.8-7.2. Preferably, the buffer solution is one of HEPES buffer, PBS buffer, TES buffer and Bis-Tris buffer.

[0032] Further, the preparation method of Cu-TATB MOF powder is as follows: 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine (CAS: 61414-16-2), water-soluble copper salt and 2-fluorobenzoic acid, organic solvent, low-carbon alcohol, acid solution are mixed in a ratio of 4-6 mg: 4-12 mg: 50-70 mg: 4-6 mL: 8-12 mL: 40-60 μL to form a mixed solution; then ultrasonically treated in an ice water bath for 30-60 min, reacted at 180-220° C. for 4-8 h, washed with an alcohol solution, and dried to obtain Cu-TATB MOF powder;

[0033] Wherein, the low-carbon alcohol is one or more of methanol, ethanol, and propanol; preferably, the organic solvent is DMF; the alcohol solution is an aqueous solution of ethanol; preferably, H + The concentration is 10-14 mol / L; preferably, the water-soluble copper salt is one or more of copper nitrate, copper chloride, and copper sulfate; preferably, the acid solution contains one or more of HCl, H2SO4, and HNO3.

[0034] Furthermore, the ratio of 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine, water-soluble copper salt and 2-fluorobenzoic acid, organic solvent, low carbon alcohol, and HCl solution is 4.5-5.5 mg: 6-10 mg: 55-65 mg: 5 mL: 9-11 mL: 45-55 μL.

[0035] Based on the same inventive concept, the present invention also provides a method for preparing the glucose electrochemical test paper as described above, wherein the enzyme reagent is dripped onto the electrode substrate, dried, coated, and cut to obtain the glucose electrochemical test paper. Optionally, the enzyme reagent is dripped onto the electrode substrate by a liquid dot method.

[0036] Optionally, the samples that can be tested by the glucose electrochemical test paper include one or more of apical blood, venous blood, and arterial blood.

[0037] The glucose electrochemical test paper of the present invention can improve the accuracy of glucose testing, and improve or solve the problems of large interference, small linear range and insensitive detection existing in the prior art.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) The enzyme reagent of the present invention contains a dual redox mediator, a quinone compound as the first redox mediator, and hexaamine ruthenium trichloride as the second electron mediator. Its working principle in the glucose electrochemical test paper is as follows: during the glucose dehydrogenase catalyzing glucose, the quinone compound can act as a bridge, enter the enzyme active center inside the protein to accept electrons, and after the double bond of the conjugated quinone in its structure is opened, it can accept all the electrons in the enzyme catalysis process and store them, and then the bipyridine structure further transfers the stored electrons to hexaamine ruthenium trichloride through a complex reaction, and finally the redox reaction of hexaamine ruthenium trichloride triggers a response current on the surface of the electron transfer electrode. Through the synergistic effect of the two, at least the following performances of the test paper for detecting glucose are enhanced: ① The anti-interference ability of the test paper is improved, and it is not interfered by 16 typical substances; ② The linear range of the test paper for detecting glucose is increased to 0.5-40mmol / L; ③ The detection current response signal is improved, and the sensitivity of the current response is enhanced.

[0040] (2) The present invention uses a metal organic framework to immobilize glucose dehydrogenase, which has the following advantages: ① The glucose dehydrogenase is encapsulated in the large pores of metal organic frameworks such as Cu-TATB MOF by the permeation channel method. The high surface area of ​​the metal organic framework ensures a high enzyme loading amount, and the effect of the pore-enzyme interface avoids the leaching of enzyme molecules; ② The metal organic frameworks such as Cu-TATB MOF have a hierarchical pore structure. When glucose dehydrogenase is encapsulated in the large pores, the enzyme molecules will open part of the tertiary structure to form a conformation that is different from its natural conformation and denatured protein, so that the enzyme active center is no longer hidden inside the protein, promoting the channelization of the medium, which is beneficial to the electron transfer between the redox medium and the enzyme active center. The small pores allow the diffusion of redox medium, reactants and products, increase the contact specific surface area between the enzyme reagent and the sample, and help to increase the rate of the redox reaction; ③ The special scaffolding effect of metal organic frameworks such as Cu-TATB MOF can adjust the distance between the electron and the electrode surface, thereby improving the electron transfer efficiency. The enzyme reagent containing glucose dehydrogenase immobilized on a metal organic framework such as GDH@Cu-TATB MOF is applied to glucose electrochemical test paper, which can improve at least the following properties of the prepared test paper: ① reduce the background current of the test paper (<100nA); ② improve the precision of the test paper (CV<5%); ③ the presence of metal organic frameworks such as Cu-TATBMOF enhances the stability of glucose dehydrogenase and increases the shelf life of the test paper (accelerated aging for 30 days). BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The figure is an overall structural diagram of a glucose electrochemical test paper of the present invention.

[0042] Figure 2The figure is a schematic diagram of the structural decomposition of a glucose electrochemical test paper of the present invention.

[0043] Figure 3 It is the fixed code equation of the glucose electrochemical test paper of Example 1.

[0044] In the figure, 1, electrode substrate; 2, enzyme reagent; 3, double-sided tape; 4, hydrophilic film; 1-1, PET substrate; 1-2, working electrode; 1-3, counter electrode; 1-4, insulating layer. DETAILED DESCRIPTION

[0045] The present invention will be described in detail below in conjunction with the embodiments. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0046] Example 1

[0047] A) Preparation of metal organic framework immobilized enzyme: 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine (5 mg), copper nitrate (8 mg) and 2-fluorobenzoic acid (60 mg) were dissolved in 5 mL of DMF, and 10 mL of ethanol and 50 μL of HCl solution (the concentration of HCl was 12 mol / L, the same below) were added to form a mixed solution; after ultrasonic treatment in an ice water bath for 45 minutes, the mixture was placed in a hydrothermal reactor and reacted at a high temperature of 200° C. for 6 hours, solid-liquid separation was performed, and the solid phase was washed several times with a mixed solution of ethanol and deionized water, and finally dried in a drying oven to constant weight to obtain Cu-TATB MOF solid powder. Then, the synthesized Cu-TATBMOF solid powder was immersed in HEPES buffer (pH 7.0) containing glucose dehydrogenase and placed in an incubator at 37°C for 24 hours. After solid-liquid separation, the obtained solid phase was washed several times with fresh buffer solution to obtain GDH@Cu-TATB MOF for later use.

[0048] B) Preparation of enzyme reagent: 0.4 parts of hydroxyethyl cellulose, 0.6 parts of polyvinyl pyrrolidone (Mw 8000), 1.0 parts of GDH@Cu-TATB MOF, 0.1 parts of hexaamine ruthenium trichloride, 0.06 parts of 1,10-phenanthroline-5,6-dione and 0.5 parts of Triton X-100 were added in sequence to a TES buffer solution with a pH of 7.0, and the mixture was thoroughly shaken and mixed to obtain the product.

[0049] C) Preparation of Glucose Electrochemical Test Paper: See Figure 1-2 After assembling the working electrode (made of silver-carbon, the same below), the counter electrode (made of silver-carbon, the same below), and the insulating layer on the PET substrate in sequence, the enzyme reagent is drop-coated on the working area of ​​the electrode substrate (i.e. Figure 2The notch area of ​​the insulating layer shown in the figure is coated with 5 μL of enzyme reagent on the working area of ​​each test paper, the same below, and a programmed temperature is adopted, wherein the temperature rise program is 30°C for 2 min, 45°C for 5 min, and 35°C for 3 min. After drying in a drying oven, double-sided tape and a hydrophilic film are successively affixed to the paper and assembled into a large card. The paper is then rolled and cut into small strips and finally bottled to obtain the finished product.

[0050] Example 2

[0051] A) Preparation of metal organic framework immobilized enzyme: 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine (5 mg), copper nitrate (8 mg) and 2-fluorobenzoic acid (60 mg) were dissolved in 5 mL of DMF, 10 mL of ethanol and 50 μL of HCl were added to form a mixed solution, and after 45 minutes of ultrasonic treatment in an ice water bath, it was placed in a hydrothermal reactor and reacted at 200°C for 6 hours, solid-liquid separation, and the solid phase was washed several times with a mixed solution of ethanol and deionized water, and finally dried in a drying oven to constant weight to obtain Cu-TATB MOF solid powder. The synthesized Cu-TATB MOF was soaked in HEPES buffer (pH 7.0) containing glucose dehydrogenase and placed in an incubator at 37°C for 24 hours, followed by solid-liquid separation, and the obtained solid phase was washed several times with fresh buffer solution to obtain GDH@Cu-TATB MOF for use.

[0052] B) Preparation of enzyme reagent: 0.4 parts of hydroxyethyl cellulose, 0.6 parts of polyvinyl pyrrolidone (Mw 8000), 1.0 parts of GDH@Cu-TATB MOF, 0.1 parts of hexaamine ruthenium trichloride, 0.06 parts of 2,9-dimethyl-1,10-phenanthroline-5,6-dione and 0.5 parts of Triton X-100 were added to a TES buffer solution with a pH of 7.0 in sequence, and the mixture was thoroughly shaken and mixed.

[0053] C) Preparation of glucose electrochemical test paper: After assembling the working electrode, counter electrode and insulating layer on the PET substrate in sequence, the enzyme reagent is drop-coated on the working area of ​​the electrode substrate by dot-coating, and the temperature is programmed to heat at 30°C for 2 min, 45°C for 5 min, and 35°C for 3 min. The test paper is dried in a drying oven, and then affixed with double-sided tape and a hydrophilic film to form a large card. The test paper strips are then rolled and cut into small strips, and finally bottled to obtain the finished product.

[0054] Example 3

[0055] A) Preparation of metal organic framework immobilized enzyme: 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine (5 mg), copper nitrate (8 mg) and 2-fluorobenzoic acid (60 mg) were dissolved in 5 mL of DMF, 10 mL of ethanol and 50 μL of HCl were added to form a mixed solution, and after ultrasonic treatment in an ice water bath for 45 minutes, it was placed in a hydrothermal reactor and reacted at 200°C for 6 hours. The resulting suspension was washed several times with a mixed solution of ethanol and deionized water, and finally dried in a drying oven to constant weight to obtain Cu-TATB MOF solid powder. The synthesized Cu-TATB MOF was soaked in HEPES buffer (pH 7.0) containing glucose dehydrogenase and placed in an incubator at 37°C for 24 hours, followed by solid-liquid separation, and the resulting solid phase was washed several times with fresh buffer solution to obtain GDH@Cu-TATB MOF for use.

[0056] B) Preparation of enzyme reagent: 0.4 parts of hydroxyethyl cellulose, 0.6 parts of polyvinyl pyrrolidone (Mw 8000), 1.0 parts of GDH@Cu-TATB MOF, 0.1 parts of hexaamine ruthenium trichloride, 0.01 parts of 1,10-phenanthroline-5,6-dione and 0.5 parts of Triton X-100 were added to a TES buffer solution with a pH of 7.0 in sequence, and the mixture was thoroughly shaken and mixed.

[0057] C) Preparation of glucose electrochemical test paper: After assembling the working electrode, counter electrode and insulating layer on the PET substrate in sequence, the enzyme reagent is drop-coated on the working area of ​​the electrode substrate by dot-coating, and the temperature is programmed to heat at 30°C for 2 min, 45°C for 5 min, and 35°C for 3 min. The test paper is dried in a drying oven, and then affixed with double-sided tape and a hydrophilic film to form a large card. The test paper strips are then rolled and cut into small strips, and finally bottled to obtain the finished product.

[0058] Example 4

[0059] A) Preparation of metal organic framework immobilized enzyme: 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine (5 mg), copper nitrate (8 mg) and 2-fluorobenzoic acid (60 mg) were dissolved in 5 mL of DMF, 10 mL of ethanol and 50 μL of HCl were added to form a mixed solution, and after ultrasonic treatment in an ice water bath for 45 minutes, it was placed in a hydrothermal reactor and reacted at 200°C for 6 hours. The resulting suspension was washed several times with a mixed solution of ethanol and deionized water, and finally dried in a drying oven to constant weight to obtain Cu-TATB MOF solid powder. The synthesized Cu-TATB MOF was soaked in HEPES buffer (pH 7.0) containing glucose dehydrogenase and placed in an incubator at 37°C for 24 hours, followed by solid-liquid separation, and the resulting solid phase was washed several times with fresh buffer solution to obtain GDH@Cu-TATB MOF for use.

[0060] B) Preparation of enzyme reagent: 0.4 parts of hydroxyethyl cellulose, 0.6 parts of polyvinyl pyrrolidone (Mw 8000), 1.0 parts of GDH@Cu-TATB MOF, 0.1 parts of hexaamine ruthenium trichloride, 0.1 parts of 1,10-phenanthroline-5,6-dione and 0.5 parts of Triton X-100 were added to a TES buffer solution with a pH of 7.0 in sequence, and the mixture was thoroughly shaken and mixed.

[0061] C) Preparation of glucose electrochemical test paper: After assembling the working electrode, counter electrode and insulating layer on the PET substrate in sequence, the enzyme reagent is drop-coated on the working area of ​​the electrode substrate by dot-coating, and the temperature is programmed to heat at 30°C for 2 min, 45°C for 5 min, and 35°C for 3 min. The test paper is dried in a drying oven, and then affixed with double-sided tape and a hydrophilic film to form a large card. The test paper strips are then rolled and cut into small strips, and finally bottled to obtain the finished product.

[0062] Comparative Example 1

[0063] A) Preparation of metal organic framework immobilized enzyme: 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine (5 mg), copper nitrate (8 mg) and 2-fluorobenzoic acid (60 mg) were dissolved in 5 mL of DMF, 10 mL of ethanol and 50 μL of HCl were added to form a mixed solution, and after ultrasonic treatment in an ice water bath for 45 minutes, it was placed in a hydrothermal reactor and reacted at 200°C for 6 hours. The resulting suspension was washed several times with a mixed solution of ethanol and deionized water, and finally dried in a drying oven to constant weight to obtain Cu-TATB MOF solid powder. The synthesized Cu-TATB MOF was soaked in HEPES buffer (pH 7.0) containing glucose dehydrogenase and placed in an incubator at 37°C for 24 hours, followed by solid-liquid separation, and the resulting solid phase was washed several times with fresh buffer solution to obtain GDH@Cu-TATB MOF for use.

[0064] B) Preparation of enzyme reagent: 0.4 parts of hydroxyethyl cellulose, 0.6 parts of polyvinyl pyrrolidone (Mw 8000), 1.0 parts of GDH@Cu-TATB MOF, 0.1 parts of hexaamine ruthenium trichloride and 0.5 parts of Triton X-100 were added to a TES buffer solution with a pH of 7.0 in sequence, and the mixture was thoroughly shaken and mixed.

[0065] C) Preparation of glucose electrochemical test paper: After assembling the working electrode, counter electrode and insulating layer on the PET substrate in sequence, the enzyme reagent is drop-coated on the working area of ​​the electrode substrate by dot-coating, and the temperature is programmed to heat at 30°C for 2 min, 45°C for 5 min, and 35°C for 3 min. The test paper is dried in a drying oven, and then affixed with double-sided tape and a hydrophilic film to form a large card. The test paper strips are then rolled and cut into small strips, and finally bottled to obtain the finished product.

[0066] Comparative Example 2

[0067] Example 1 was repeated, except that in step B), hexaamineruthenium trichloride was not added.

[0068] Comparative Example 3

[0069] A) Preparation of enzyme reagent: 0.4 parts of hydroxyethyl cellulose, 0.6 parts of polyvinyl pyrrolidone (Mw 8000), 1.0 parts of GDH, 0.1 parts of hexaamine ruthenium trichloride, 0.06 parts of 1,10-phenanthroline-5,6-dione and 0.5 parts of Triton X-100 were added to a TES buffer solution with a pH of 7.0 in sequence, and the mixture was thoroughly shaken and mixed.

[0070] B) Preparation of glucose electrochemical test paper: After assembling the working electrode, counter electrode and insulating layer on the PET substrate in sequence, the enzyme reagent is drop-coated on the working area of ​​the electrode substrate by dot-coating, and the temperature is programmed to heat at 30°C for 2 min, 45°C for 5 min, and 35°C for 3 min. The test paper is dried in a drying oven, and then affixed with double-sided tape and a hydrophilic film to form a large card. The test paper is then rolled and cut into small strips, and finally bottled to obtain the finished product.

[0071] Inspection process:

[0072] 1. Code determination and code verification: The test strips prepared in Examples 1 to 4 and Comparative Examples 1 to 3 were used in conjunction with a matching electrochemical tester. First, whole blood samples with different concentration gradients of glucose were prepared, with concentrations of 0.5, 1.1, 5, 10, 20, 30 and 40 mmol / L. The current values ​​of different glucose blood samples and physiological saline were tested and processed by EXCEL to obtain the current-concentration curve equation, i.e., the code determination equation ( Figure 1 The fixed code equation of Example 1) is used, and the sensitivity between current and concentration is calculated. The data are shown in Tables 1 to 2. The corresponding fixed code equation is then burned into the electrochemical tester, and whole blood samples with different concentrations of glucose are tested. The measurement results are recorded, and the accuracy and precision are calculated. The specific data are shown in Tables 3 to 4.

[0073] 2. Anti-interference substance screening: 16 typical interfering substances were selected, including ① exogenous interfering substances (common drugs, anticoagulants and food-derived substances), ② endogenous interfering substances (common substances and metabolites in the human body), and ③ other sugar substances in the blood. Blank control tests were performed to test the anti-interference ability of the test papers of Examples 1 to 4 and Comparative Examples 1 to 3. The specific summary data are shown in Table 5.

[0074] 3. Accelerated stability study on the 30th day: The same batch of glucose electrochemical test strips were prepared according to Examples 1 to 4 and Comparative Examples 1 to 3 and divided into two equal parts, one for storage at room temperature and the other for accelerated aging in a 60°C constant temperature box. On the 30th day, a comparative experiment was conducted on the aged test strips and the room temperature test strips. The specific summary data are shown in Tables 6 to 7.

[0075] Referring to Table 1-2, compared with Comparative Example 1-2, the current tested by the test paper in Example 1-4 is larger, and the detection sensitivity of the test paper increases by about 15% compared with Comparative Example 1, and the detection sensitivity of the test paper increases by about 35% compared with Comparative Example 2. The possible reason is that the synergistic effect of the quinone compounds added to the enzyme reagent and hexamethyleneruthenium trichloride promotes the electron transfer in the enzyme catalysis process, improves the response current signal of the test paper detecting glucose, and also significantly enhances the current sensitivity. This is attributed to the fact that quinone compounds, as a small molecule compound without charge, have a large electron self-exchange rate constant and the conjugated diketone in the structure has a high electron acceptance efficiency, which can shuttle the protein channel and the active site embedded in the center of the protein to directly undergo redox reaction, and transfer electrons, and further undergo redox reaction with hexamethyleneruthenium trichloride to induce a response current on the surface of the electron transfer electrode. Compared with Comparative Example 3, the background current of the test paper test in Examples 1 to 4 is smaller, and the detection sensitivity of the test paper increases by about 8%, indicating that the addition of metal organic frameworks such as Cu-TATB MOF in the enzyme reagent can reduce the background current signal. At the same time, the exposure of the enzyme active center will be promoted during the enzyme encapsulation process, which can improve the sensitivity of the detection current and also has a certain auxiliary effect on the amplification of the current signal.

[0076] Table 1 Summary of sensitivity of Examples 1-4

[0077]

[0078] Table 2 Summary of sensitivity of comparative examples 1-3

[0079]

[0080]

[0081] Referring to Tables 3-4, compared with Comparative Examples 1 to 3, the relative deviations of the test strips in Examples 1 to 4 are smaller, all below 8%, and the current precision is all below 5%, which indirectly illustrates that the synergistic effect of quinone compounds and hexaammonium ruthenium trichloride can effectively improve the sensitivity, precision and accuracy of the test strips in detecting glucose; in addition, the hierarchical porous structure of Cu-TATB MOF promotes substrate channelization, accelerates the diffusion rate of redox mediators and glucose, expands the contact area with glucose dehydrogenase, and its special scaffolding effect can adjust the distance between electrons and the electrode surface, fully ensuring the efficiency of electron transfer.

[0082] Table 3 Summary of accuracy and precision of Examples 1-4

[0083]

[0084] Table 4 Accuracy and precision summary of comparative examples 1-3

[0085]

[0086]

[0087] The analysis results in Table 5 show that, compared with Comparative Examples 1 to 3, Examples 1 to 4 use quinone compounds as the first redox medium and hexaamineruthenium trichloride as the second redox medium. The synergistic effect of the two redox mediums can reduce the influence of interfering substances in the blood on the measurement results.

[0088] Table 5 Summary of interference screening data of Examples 1-4 and Comparative Examples 1-2

[0089]

[0090] Referring to Tables 6 to 7, compared with Comparative Example 3, the absolute deviations of the currents of the accelerated aging test papers and the room temperature test papers on the 30th day in Examples 1 to 4 are all less than 50 nA, and the relative deviations are all less than 3%, indicating that the Cu-TATB MOF has a highly ordered porous network structure, which can provide a stable microenvironment for glucose dehydrogenase, protect the enzyme molecules from the external environment, thereby maintaining its active structure, and its confinement effect greatly improves the enzyme loading rate and limits the loss of enzyme molecules, thereby extending the service life of the enzyme. Therefore, the stability of the test paper can be ensured and the validity period of the test paper can be extended.

[0091] Table 6 Summary of the 30th day accelerated stability of Examples 1-4

[0092]

[0093] Table 7 Summary of the 30th day accelerated stability of comparative examples 1-3

[0094]

[0095] In summary, the enzyme reagent of the present invention can effectively improve the sensitivity, precision and accuracy of glucose test strips, reduce the influence of interfering substances on current, enhance the stability of test strips and extend the validity period of test strips, and the test range reaches 0.5-40mmol / L.

[0096] The contents explained in the above embodiments should be understood as these embodiments are only used to more clearly illustrate the present invention, and are not used to limit the scope of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.

Claims

1. An enzyme reagent, characterized in that Calculated by weight, it includes 5 to 7 parts of buffer solution, 0.01 to 0.2 parts of hexammineruthenium trichloride, 0.01 to 0.1 parts of quinone compounds, 0.1 to 1.2 parts of metal organic framework immobilized glucose dehydrogenase, and 0.2 to 0.8 parts of film-forming agent.

2. The enzyme reagent according to claim 1, characterized in that The quinone compound is one or both of 2,9-dimethyl-1,10-phenanthroline-5,6-dione and 1,10-phenanthroline-5,6-dione; Preferably, the pH value of the buffer is 6.9-7.5; Preferably, the buffer is one of PBS buffer, HEPES buffer, TES buffer and Bis-Tris buffer; Preferably, the film-forming agent is one or more of hydroxyethyl cellulose and polyvinyl pyrrolidone; more preferably, the film-forming agent is composed of hydroxyethyl cellulose and polyvinyl pyrrolidone in a mass ratio of 0.2-0.5:0.3-0.8; Preferably, the metal organic framework is one or more of Cu-TATB MOF, ZIF-8, ZIF-90, and MAF-7; Preferably, the enzyme reagent further comprises 0.5 to 1 part of a non-ionic surfactant, and more preferably, the non-ionic surfactant is Triton X-100.

3. A glucose electrochemical test paper, comprising an electrode substrate and an enzyme reagent disposed on the electrode substrate; characterized in that: The enzyme reagent contains 5 to 7 parts of buffer solution, 0.01 to 0.2 parts of hexammineruthenium trichloride, 0.01 to 0.1 parts of quinone compounds, 0.1 to 1.2 parts of metal organic framework-immobilized glucose dehydrogenase, and 0.2 to 0.8 parts of film-forming agent in parts by mass.

4. The glucose electrochemical test paper according to claim 3, characterized in that: The quinone compound is one or both of 2,9-dimethyl-1,10-phenanthroline-5,6-dione and 1,10-phenanthroline-5,6-dione.

5. The glucose electrochemical test paper according to claim 3, characterized in that: The film-forming agent is one or more of hydroxyethyl cellulose and polyvinyl pyrrolidone; more preferably, the film-forming agent is composed of hydroxyethyl cellulose and polyvinyl pyrrolidone in a mass ratio of 0.2-0.5:0.3-0.8; preferably, the weight average molecular weight of polyvinyl pyrrolidone is 7000-9000.

6. The glucose electrochemical test paper according to claim 3, characterized in that: The metal organic framework is one or more of Cu-TATBMOF, ZIF-8, ZIF-90 and MAF-7.

7. The glucose electrochemical test paper according to claim 3, characterized in that: The pH value of the buffer is 6.9-7.5; more preferably, the buffer is one of PBS buffer, HEPES buffer, TES buffer and Bis-Tris buffer; Preferably, the enzyme reagent further contains 0.5 to 1 part of a non-ionic surfactant, and more preferably, the non-ionic surfactant is Triton X-100.

8. The glucose electrochemical test paper according to claim 3, characterized in that: The preparation method of metal organic framework immobilized glucose dehydrogenase comprises the following steps: (1) Providing Cu-TATB MOF powder; (2) immersing the Cu-TATB MOF powder in a buffer solution containing a biological enzyme, culturing, solid-liquid separation, washing, and drying to obtain a metal organic framework-immobilized glucose dehydrogenase; The pH value of the buffer solution is 6.8-7.

2. Preferably, the buffer solution is one of HEPES buffer, PBS buffer, TES buffer and Bis-Tris buffer.

9. The glucose electrochemical test paper according to claim 8, characterized in that: The preparation method of Cu-TATB MOF powder is as follows: 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine, water-soluble copper salt and 2-fluorobenzoic acid, organic solvent, low-carbon alcohol, and acid solution are mixed in a ratio of 4-6 mg:4-12 mg:50-70 mg:4-6 mL:8-12 mL:40-60 μL to form a mixed solution; then ultrasonically treated in an ice water bath for 30-60 min, reacted at 180-220° C. for 4-8 h, solid-liquid separation, washed with an alcohol solution, and dried to obtain Cu-TATB MOF powder; Wherein, the low-carbon alcohol is one or more of methanol, ethanol, and propanol; preferably, the organic solvent is DMF; the alcohol solution is an aqueous solution of ethanol; preferably, H + The concentration is 10-14 mol / L; preferably, the water-soluble copper salt is one or more of copper nitrate, copper chloride, and copper sulfate; preferably, the acid solution contains one or more of HCl, H2SO4, and HNO3.

10. The method for preparing a glucose electrochemical test paper according to any one of claims 3 to 9, characterized in that: The enzyme reagent is drop-coated on the electrode substrate, dried, coated, and cut to obtain the glucose electrochemical test paper.

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