A blood glucose and uric acid electrochemical sensor and its preparation and detection method

Abstract

The present invention discloses a blood glucose and uric acid electrochemical sensor and a preparation and detection method. The electrochemical sensor includes a lower substrate layer, a printed electrode layer, a reagent layer and an insulating layer arranged in sequence from bottom to top, and the printed electrode layer includes a first electrode group consisting of a first working electrode and a first reference electrode, a second electrode group consisting of a second working electrode and a second reference electrode, and each electrode is connected to a corresponding wire; the reagent layer and the first electrode group form an enzyme electrode for measuring the blood glucose concentration of a blood sample, and the second electrode group is used for measuring the hematocrit and uric acid concentration of the blood sample, and the second working electrode is subjected to surface hydrophilization treatment. According to the signal measured by the electrochemical sensor, the blood glucose and uric acid concentrations can be obtained. The present invention can completely avoid the mutual interference of the two enzymes of blood glucose detection and uric acid detection, ensure the accuracy of the test results, and simultaneously achieve blood glucose, uric acid detection and hematocrit correction.

Description

Technical Field

[0001] The present invention belongs to the field of blood sugar and uric acid detection, and particularly relates to an electrochemical sensor for blood sugar and uric acid detection and a preparation and detection method thereof. Background Art

[0002] Hyperuricemia and diabetes are both metabolic diseases caused by overnutrition. Studies have shown that insulin resistance is a common pathogenic factor in both hyperuricemia and diabetes. Obese patients with typical insulin resistance are often the primary source of hyperuricemia and type 2 diabetes. Hyperuricemia is also an independent risk factor for type 2 diabetes. Monitoring uric acid and blood glucose biochemical markers is crucial for the prevention and diagnosis of hyperuricemia and diabetes.

[0003] Testing for uric acid and blood sugar biochemical indicators requires visits to specialized hospitals and specialized measuring instruments. These methods are inconvenient for individuals with suboptimal health or those suffering from related medical conditions. Consequently, some home-use test strips have become commercially available. These test strips are electrochemical sensors containing enzyme-modified electrodes. The sensor's electrode surface is modified with biological enzymes. Common enzymes used for blood sugar testing are glucose oxidase and glucose dehydrogenase, while uric acid testing uses urate oxidase. The enzymes in the sensors react with glucose or uric acid in the blood to generate an electric current, which is then converted into a glucose or uric acid concentration reading. Common test strips include single-function blood sugar test strips, single-function uric acid test strips, and dual-function blood sugar and uric acid test strips. Single-function blood sugar and uric acid test strips are particularly inconvenient to use, requiring separate drops of blood sample for blood sugar and uric acid testing. This is cumbersome, inconvenient for routine monitoring, and expensive. The electrode surface of a dual-function test strip (blood glucose and uric acid test strip) that simultaneously detects blood glucose and uric acid requires the presence of two enzymes. However, different enzymes have different characteristics and can interfere with each other, resulting in varying detection accuracy and anti-interference capabilities. To reduce this mutual interference, the two test items, blood glucose and uric acid, are typically placed on the front and back of the test strip, or the two test items are separated into separate test chambers to avoid interference from different enzyme reactions and achieve simultaneous detection of blood glucose and uric acid. However, these methods complicate the test strip manufacturing process, increase the number of electrodes, and easily lead to electrode and enzyme contamination. Since test strips are designed to be portable and compact, the precision requirements for electrode manufacturing also become stringent, leading to increased production costs. Furthermore, this can increase the amount of blood samples used, resulting in increased blood volume drawn from patients. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a blood glucose and uric acid electrochemical sensor and a preparation and detection method thereof.

[0005] The technical solution of the present invention is:

[0006] The present invention provides a blood glucose and uric acid electrochemical sensor, comprising a lower substrate layer, a printed electrode layer, a reagent layer and an insulating layer arranged in sequence from bottom to top, wherein the printed electrode layer comprises a first working electrode, a first reference electrode, a second working electrode and a second reference electrode, and the first working electrode, the first reference electrode, the second working electrode and the second reference electrode are connected to corresponding wires;

[0007] The first working electrode and the first reference electrode constitute a first electrode group, the second working electrode and the second reference electrode constitute a second electrode group, the reagent layer covers the first electrode group in the printed electrode layer, and the insulating layer covers part of the printed electrode layer and part of the reagent layer;

[0008] The reagent layer and the first electrode group form an enzyme electrode for measuring the blood glucose concentration of a blood sample. The second electrode group is used to measure the hematocrit and uric acid concentration of a blood sample, and the second working electrode is subjected to a surface hydrophilization treatment.

[0009] Preferably, the first working electrode, the second working electrode and the first reference electrode are carbon electrodes;

[0010] The second reference electrode is a silver / silver chloride electrode, which can be prepared using conventional silver / silver chloride electrode materials without special restrictions;

[0011] The wire is a silver wire, and conventional silver electrode materials can be used without special restrictions;

[0012] The reagent layer contains glucose reaction enzyme and electron mediator. The reagent layer and the first electrode group form an enzyme electrode, which is the area where blood glucose undergoes electrochemical reaction under the action of enzyme, and is used to measure the blood glucose concentration (i.e., glucose concentration) of the blood sample.

[0013] Preferably, the carbon electrodes used in 0-3 of the first working electrode, the second working electrode and the first reference electrode are carbon electrodes containing a doping material, and the doping material is a mixture of one or more of single-walled carbon nanotubes, multi-walled carbon nanotubes, platinum-carbon nanoparticles and micron-sized platinum-carbon particles;

[0014] The slurry for preparing a carbon electrode containing a doping material is a mixture of the doping material and a conductive carbon paste for screen printing. The mass ratio of the conductive carbon paste to the doping material is 1:0.01 to 0.06. The conductive carbon paste can be a commercially available product and does not contain an electron mediator. The carbon electrode can also be a carbon electrode without a doping material, and the slurry used to prepare the conductive carbon paste is a conductive carbon paste for screen printing.

[0015] Preferably, the surface hydrophilization treatment is plasma cleaning or laser etching.

[0016] Preferably, the plasma cleaning is medium frequency oxygen plasma cleaning, the oxygen intake volume is 50 to 200 SCCM, the cleaning power is 300 to 1000 W, and the cleaning time is 3 to 10 minutes.

[0017] The insulating layer, located above the reagent layer and printed electrode layer, is printed using an insulating mixture. It prevents short circuits while allowing electrochemical reactions to occur in specific areas, isolating current interference between electrodes and ensuring the accuracy and stability of the test signal.

[0018] Preferably, it further comprises an intermediate substrate and an upper substrate, wherein the intermediate substrate is sandwiched between the upper substrate and the lower substrate layer;

[0019] The insulating layer is provided with a first channel, and the intermediate substrate is provided with a sample suction port and a second channel, the second channel corresponding to the first channel, the second channel and the first channel are combined to form a fine reaction channel, and the blood sample is sucked into the reaction channel through the sample suction port through a siphon effect;

[0020] The upper substrate is provided with an air outlet, and the end of the reaction channel is connected to the air outlet to achieve a siphon effect. When the blood sample flows into the reaction channel, the air in the reaction channel can be discharged through the air outlet;

[0021] The bottom of the reaction channel is the first electrode group, the second electrode group and the reagent layer in the printed electrode layer. The electrodes are the second working electrode, the second reference electrode, the first working electrode and the first reference electrode in order from near to far from the sample suction port.

[0022] Preferably, the first reference electrode and the second reference electrode are connected to each other for starting the electrochemical tester.

[0023] Preferably, the lower substrate layer is made of one of polyethylene terephthalate, polyvinyl chloride, polypropylene, polycarbonate, polyethylene, polystyrene, and polyimide.

[0024] Preferably, the intermediate substrate is a double-sided tape with adhesive force on both sides.

[0025] Preferably, the upper substrate is made of a transparent or translucent material, such as polyethylene terephthalate (PET), so as to facilitate observation of the blood sample entering the reaction channel.

[0026] The present invention also provides a method for preparing the above-mentioned blood glucose and uric acid electrochemical sensor, comprising the following steps:

[0027] S1. Making printed electrode layer

[0028] (1) Preparing a lower substrate as a lower substrate layer;

[0029] (2) preparing a first working electrode, a first reference electrode, a second working electrode, a second reference electrode and a wire on the lower substrate;

[0030] S2. Surface hydrophilic treatment

[0031] Performing surface hydrophilization treatment on the second working electrode of the product obtained in step S1;

[0032] S3. Make reagent layer

[0033] forming a reagent layer on the product obtained in step S2, wherein the reagent layer covers the first electrode group in the printed electrode layer;

[0034] S4. Make insulation layer

[0035] An insulating layer is formed on the product obtained in step S3, and the insulating layer covers part of the printed electrode layer and part of the reagent layer.

[0036] Preferably, it also includes:

[0037] S5, assemble with the middle substrate and upper substrate

[0038] The middle substrate is pasted on the insulating layer, and the upper substrate and the lower substrate are assembled together.

[0039] Preferably, in step S2, the surface hydrophilization treatment is medium frequency oxygen plasma cleaning, the oxygen intake volume is 50 to 200 SCCM, the cleaning power is 300 to 1000 W, and the cleaning time is 3 to 10 minutes.

[0040] Preferably, in step S2, a lower plate with holes is used to press on the lower substrate, leaving only the second working electrode exposed for oxygen plasma cleaning.

[0041] Preferably, in step S3, a reagent layer composition containing glucose reactive enzyme, electron mediator and buffer matrix is ​​applied to the first working electrode and the first reference electrode, and the reagent layer is formed by heating and drying at a temperature of 50-70° C. for 3-10 minutes.

[0042] Preferably, the glucose-responsive enzyme is glucose dehydrogenase or glucose oxidase;

[0043] The electron mediator is at least one selected from potassium ferrocyanide, hexaammineruthenium trichloride, ferrocene, benzoquinone, benzoquinone derivatives, organic conductive salts, potassium ferrocyanide, dimethylferrocene, ferrocenium ion, ferrocenecarboxylic acid, dimethylaniline, o-toluidine, 2,4-dichlorophenol, 4-aminoantipyrine, benzidine and Prussian blue;

[0044] The buffer matrix is ​​phosphate buffered saline (PBS), citrate buffer or a phosphate and citrate complex buffer solution.

[0045] Preferably, in step S4, a mixture composed of insulating materials is printed on a lower substrate layer provided with a printed electrode layer and a reagent layer to form an insulating layer so as to cover part of the printed electrode layer and part of the reagent layer, and is heated and dried. The heating and drying temperature of the insulating layer should be selected to be an appropriate temperature that does not damage the glucose reaction enzyme in the reagent layer.

[0046] The present invention also provides a method for detecting blood glucose and uric acid, using the above-mentioned blood glucose and uric acid electrochemical sensor, the detection method comprises the following steps:

[0047] (1) Connect the blood glucose and uric acid electrochemical sensor to the electrochemical tester, turn on the electrochemical tester, and then apply a trigger voltage between the first working electrode and the first reference electrode. The trigger voltage is a DC voltage. Blood enters the reaction channel, and the first working electrode and the first reference electrode form a current, causing the electrochemical tester to start testing and start timing.

[0048] (2) applying a first voltage to the second working electrode and the second reference electrode, wherein the first voltage is a DC voltage, the potential of which is less than the oxidation potential of uric acid, and measuring the current signal I1 of the interfering substances such as ascorbic acid and acetaminophen in the blood sample flowing through the reaction channel by the second working electrode and the second reference electrode using the redox characteristics of the interfering substances such as ascorbic acid and acetaminophen in the blood sample;

[0049] (3) applying a second voltage to the second working electrode and the second reference electrode, wherein the second voltage is a DC voltage, and measuring the current signal I2 of uric acid in the blood sample flowing through the reaction channel by the second working electrode and the second reference electrode using the redox characteristics of uric acid in the blood sample;

[0050] (4) applying a third voltage to the first working electrode and the first reference electrode, where the third voltage is a DC voltage, and measuring a current signal I3 of blood glucose in the blood sample flowing through the reaction channel by an enzyme electrode formed by the first working electrode, the first reference electrode, and the reagent layer;

[0051] (5) applying a fourth voltage to the second working electrode and the second reference electrode, the fourth voltage being an AC voltage, and measuring the hematocrit (HCT) of the blood sample flowing through the reaction channel by the second working electrode and the second reference electrode, and obtaining a maximum value of the AC current electrical signal being S1;

[0052] (6) First, according to the current signal I 1、 I2 calculates the initial concentration of uric acid, and calculates the initial concentration of blood glucose based on the current signal I3. The initial concentrations of uric acid and blood glucose are then corrected to obtain the final concentrations of uric acid and blood glucose.

[0053] The first working electrode and the first reference electrode are located at the end of the reaction channel and act as trigger electrodes. When blood enters the sample inlet, it must reach the first electrode group located at the end of the reaction channel to generate current. This can avoid inaccurate measurement values ​​caused by blood not filling the entire reaction channel.

[0054] Preferably, in step (1), the trigger voltage ranges from 100 to 700 mV;

[0055] In step (2), the voltage range of the first voltage is 0.05 to 0.25 V, and the duration of applying the voltage is 1 to 3 seconds;

[0056] In step (3), the second voltage has a voltage range of 0.3 to 0.6 V, and the duration of applying the voltage is 1 to 4 seconds;

[0057] In step (4), the voltage range of the third voltage is 0.2 to 0.6 V, and the duration of applying the voltage is 5 to 6 seconds;

[0058] In step (5), the fourth voltage has a voltage range of 0.05 to 1 V, a frequency of 10 to 1000 Hz, and a duration of applying the voltage of 1 to 10 seconds.

[0059] Preferably, the final concentration of uric acid and the final concentration of blood glucose in step (6) are obtained by the following method:

[0060] a. Substitute I2 and I1 of the test blood sample into equation I u =I2-k1*I1, and the uric acid response current intensity I is obtained. u , where k1 ranges from 0.8 to 1.2;

[0061] b. Then, the I obtained in step a is u Substitute Cua=a*I u +b, obtain the corresponding initial concentration of uric acid Cua, substitute I3 into Cglu=c*I3+d, and obtain the corresponding initial concentration of blood glucose Cglu;

[0062] c. Substituting the electrical signal S1 obtained by the fourth voltage into the linear equation HCT*100=k2+k3*S1 to obtain the hematocrit HCT, where k2 ranges from 50 to 150, k3 ranges from -50 to 0, and the unit of S1 is μA;

[0063] d. Obtain the final concentration of uric acid Cuat and blood glucose Cglut:

[0064] Cuat=Cua / (k4*HCT+k5), where k4 ranges from -5 to 0 and k5 ranges from 0 to +5;

[0065] Cglut=Cglu / (k6*HCT+k7), wherein k6 ranges from -5 to 0 and k7 ranges from 0 to +5.

[0066] Among them, k1, a and b are obtained by the multivariate linear regression method, and c, d and k2~k7 are obtained by the unit linear regression method.

[0067] Linear Equations I u =I2-k1*I1 can be obtained by the following method: prepare a series of blood samples with different interfering substance concentrations (for example, a series of blood samples with different ascorbic acid concentrations), measure the corresponding I1 and I2 according to the methods of steps (2) and (3), analyze that the current I1 has a linear relationship with the interfering substance concentration, and the current I2 has a linear relationship with the uric acid and interfering substance concentrations, and the correlation I can be obtained by deducting them proportionally. u =I2-k1*I1, and finally this relationship was substituted into the mixed solution of uric acid and interfering substances for verification;

[0068] Taking the initial concentration of uric acid as an example, a series of blood samples with different uric acid concentrations were prepared, and the corresponding I1 and I2 were measured according to the methods of steps (2) and (3), and substituted into I u =I2-k1*I1 to obtain the corresponding I u , for multiple groups of uric acid concentrations and multiple groups of I u Perform linear fitting to obtain the corresponding standard curve Cua=a*I u +b=a*(I2-k1*I1)+b;

[0069] Taking the initial measurement of blood glucose concentration as an example, a series of blood samples with different blood glucose concentrations are prepared, and the corresponding blood glucose current signal I3 is obtained according to the operation of step (4). A linear fit is performed on multiple groups of blood glucose concentrations and multiple groups of current signals I3 to obtain the corresponding standard curve Cglu=c*I3+d;

[0070] HCT*100=k2+k3*S1 is obtained by the following method: a series of blood samples with different hematocrits (HCT) are prepared, S1 is measured according to the method in step (5), and linear fitting is performed on multiple groups of hematocrits and multiple groups of S1 to obtain the linear equation HCT*100=k2+k3*S1;

[0071] The two correction equations were obtained by the following method: a series of blood samples with different hematocrits (HCT) were prepared, and the uric acid and blood glucose concentrations of the samples were measured using professional instruments (recorded as the final uric acid concentration Cuat and the final blood glucose concentration Cglut). Then, the initial concentrations of different samples were measured according to the method in step b (recorded as the initial uric acid concentration Cua and the initial blood glucose concentration Cglu). The values ​​of multiple groups of Cua / Cuat were linearly fitted with the values ​​of multiple groups of HCT to obtain the equation Cuat = Cua / (k4*HCT+k5). The values ​​of multiple groups of Cglu / Cuat were linearly fitted with the values ​​of multiple groups of HCT, and Cglut = Cglu / (k6*HCT+k7).

[0072] The beneficial effects of the present invention are:

[0073] (1) The present invention uses a shared reaction channel for the uric acid detection electrode and the blood glucose detection electrode to achieve joint measurement without introducing additional reaction channels or electrodes. This reduces the amount of blood sample required, reduces the amount of blood drawn from the patient, and reduces pain. Furthermore, the present invention has the advantages of a simple manufacturing process and avoids a substantial increase in production costs.

[0074] (2) The present invention can realize the detection of uric acid and hematocrit correction on the same electrode, completely avoiding the mutual interference between the two enzymes of blood glucose detection and uric acid detection, ensuring the accuracy of the test results, and realizing blood glucose, uric acid detection and hematocrit correction at the same time, thereby improving the detection efficiency of the test paper. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0076] Figure 1 This is an exploded view of the blood glucose and uric acid electrochemical sensor according to Example 1 of the present invention;

[0077] Figure 2 This is a partial structural diagram of the blood glucose and uric acid electrochemical sensor according to Example 1 of the present invention;

[0078] Figure 3 This is a schematic diagram of the working circuit principle of the blood glucose and uric acid electrochemical sensor and its supporting instruments of the present invention;

[0079] Figure 4 It is a test timing diagram of the detection process of the present invention;

[0080] Figure 5This is a linear graph of blood glucose and uric acid detected by the electrochemical sensor involved in Example 2 of the present application.

[0081] The markings in the figure are: 1. Lower substrate layer; 2. Printed electrode layer; 201. First working electrode; 202. Second working electrode; 203. First reference electrode; 204. Second reference electrode; 205. Wire; 3. Reagent layer; 4. Insulating layer; 401. First channel; 5. Intermediate substrate; 501. Sample suction port; 502. Second channel; 6. Upper substrate; 601. Air outlet. DETAILED DESCRIPTION

[0082] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are merely exemplary and are not intended to limit the scope of the present invention. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessary confusion of the concepts of the present invention.

[0083] like Figure 1 and 2 As shown, a blood glucose and uric acid electrochemical sensor includes a lower substrate layer 1, a printed electrode layer 2, a reagent layer 3, an insulating layer 4, an intermediate substrate 5 and an upper substrate 6 which are sequentially distributed from bottom to top.

[0084] The lower substrate layer 1 is made of one of polyethylene terephthalate, polyvinyl chloride, polypropylene, polycarbonate, polyethylene, polystyrene and polyimide.

[0085] The printed electrode layer 2 is provided with a test electrode group, including a first electrode group consisting of a first working electrode 201 and a first reference electrode 203, and a second electrode group consisting of a second working electrode 202 and a second reference electrode 204. Each electrode is connected to a corresponding wire 205. The second electrode group is used to measure the hematocrit (HCT) and uric acid concentration of a blood sample.

[0086] The electrodes of the printed electrode layer 2 are, in order from near to far from the sample suction port 501, the second working electrode 202, the second reference electrode 204, the first working electrode 201, and the first reference electrode 203. The first working electrode 201 and the first reference electrode 203 are carbon electrodes, the second working electrode 202 is a carbon electrode with a surface hydrophilic treatment, the second reference electrode 204 is a silver / silver chloride electrode, and the wire 205 is a silver wire. The surface hydrophilic treatment process of the second working electrode 202 is preferably plasma cleaning or laser etching. The first reference electrode 203 and the second reference electrode 204 are connected to each other for starting the electrochemical tester.

[0087] Reagent layer 3 overlies the first electrode group in printed electrode layer 2. Reagent layer 3 contains glucose-responsive enzyme and an electron mediator. Together with the first electrode group, reagent layer 3 forms the enzyme electrode, where the enzyme-induced electrochemical reaction of glucose occurs. This electrode is used to measure the glucose concentration (i.e., glucose level) in a blood sample.

[0088] During the preparation process, a reagent layer composition containing glucose reaction enzyme, electron mediator and buffer matrix is ​​applied to the first working electrode and the first reference electrode, and a reagent layer is formed by heating and drying at a temperature of 50-70°C for 3-10 minutes.

[0089] The glucose reaction enzyme is glucose dehydrogenase or glucose oxidase; the electron mediator is selected from at least one of potassium ferrocyanide, hexaammineruthenium trichloride, ferrocene, benzoquinone, benzoquinone derivatives, organic conductive salts, potassium ferrocyanide, dimethylferrocene, ferrocenium ion, ferrocenylcarboxylic acid, dimethylaniline, o-toluidine, 2,4-dichlorophenol, 4-aminoantipyrine, benzidine and Prussian blue; and the buffer matrix is ​​phosphate buffered saline (PBS), citrate buffer or a phosphate and citrate composite buffer solution.

[0090] An insulating layer 4 is positioned above the reagent layer 3 and printed electrode layer 2. A first channel 401 is formed on the insulating layer 4 and is formed by printing an insulating mixture. Specifically, the mixture, composed of an insulating material, is printed onto the lower substrate layer having the printed electrode layer and reagent layer to form the insulating layer, partially covering the printed electrode layer and the reagent layer. The insulating layer is then heated and dried at a temperature that does not damage the glucose reactive enzyme in the reagent layer.

[0091] Insulating layer 4 prevents short circuits while allowing electrochemical reactions to occur in specific areas, isolating current interference between electrodes and ensuring the accuracy and stability of the detection signal. Portions of first working electrode 201, first reference electrode 203, second working electrode 202, and second reference electrode 204 corresponding to first channel 401 are not covered by insulating layer 4.

[0092] The intermediate substrate 5 can be made of double-sided tape with adhesive strength on both sides, which is used to bond the lower substrate layer 1 to the upper substrate 6. The intermediate substrate 5 is provided with a sample suction port 501 and a second channel 502. The second channel 502 corresponds to the first channel 401. The second channel 502 and the first channel 401 form a fine reaction channel. The bottom of the reaction channel is the first electrode group, the second electrode group, and the reagent layer 3 of the printed electrode layer 2.

[0093] The upper substrate 6 covers the reaction channel and a portion of the printed electrode layer 2. It is provided with an air vent 601, which is connected to the end of the reaction channel. This creates a siphon effect. When a blood sample flows into the reaction channel, air in the reaction channel can be discharged through the air vent 601. The upper substrate 6 is made of a transparent or translucent material, such as polyethylene terephthalate (PET), to facilitate observation of the blood sample entering the reaction channel.

[0094] The above-mentioned blood glucose and uric acid electrochemical sensor can be used to detect blood glucose and uric acid. The specific detection method includes the following steps:

[0095] (1) Connect the blood glucose and uric acid electrochemical sensor to the electrochemical tester, turn on the electrochemical tester, and then apply a trigger voltage between the first working electrode and the first reference electrode. The trigger voltage is a DC voltage with a voltage range of 100 to 700 mV. Blood enters the reaction channel, and the first working electrode and the first reference electrode form a current, causing the electrochemical tester to start testing and start timing.

[0096] (2) applying a first voltage to the second working electrode and the second reference electrode, wherein the first voltage is a DC voltage having a potential lower than the oxidation potential of uric acid, and the voltage range of the first voltage is 0.05 to 0.25 V. The duration of applying the voltage is 1 to 3 seconds. The second working electrode 202 and the second reference electrode 204 use the redox characteristics of the interfering substances such as ascorbic acid and acetaminophen in the blood sample to measure the current signal I1 of the interfering substances such as ascorbic acid and acetaminophen in the blood sample flowing through the reaction channel;

[0097] (3) applying a second voltage to the second working electrode and the second reference electrode, wherein the second voltage is a DC voltage, the voltage range of the second voltage is 0.3 to 0.6 V, and the duration of applying the voltage is 1 to 4 seconds, and the second working electrode and the second reference electrode use the redox characteristics of uric acid in the blood sample to measure the current signal I2 of uric acid in the blood sample flowing through the reaction channel;

[0098] (4) applying a third voltage to the first working electrode and the first reference electrode, wherein the third voltage is a DC voltage, has a voltage range of 0.2 to 0.6 V, and is applied for 5 to 6 seconds. The enzyme electrode formed by the first working electrode, the first reference electrode, and the reagent layer measures a current signal I3 of blood glucose in the blood sample flowing through the reaction channel;

[0099] (5) applying a fourth voltage to the second working electrode and the second reference electrode, the fourth voltage being an AC voltage, having a voltage range of 0.05 to 1 V, a frequency of 10 to 1000 Hz, and a voltage application duration of 1 to 10 s, and measuring the hematocrit (HCT) of the blood sample flowing through the reaction channel by the second working electrode and the second reference electrode, and obtaining a maximum value of the AC current electrical signal being S1;

[0100] (6) a. Substitute I2 and I1 of the test blood sample into the linear equation I u =I2-k1*I1, and the uric acid response current intensity I is obtained. u , where k1 ranges from 0.8 to 1.2;

[0101] Linear Equations I u =I2-k1*I1 is obtained by the following method: prepare a series of blood samples with different ascorbic acid concentrations, use electrochemical sensors to measure the corresponding I1 and I2 according to the method of steps (2) and (3), analyze that the current I1 has a linear relationship with the concentration of the interferent, and the current I2 has a linear relationship with the concentration of uric acid and ascorbic acid. The correlation can be obtained by deducting them proportionally, I u =I2-k1*I1, and finally this relationship is substituted into the mixed solution of uric acid and ascorbic acid for verification;

[0102] b. Then, the I obtained in step a is u Substitute Cua=a*I u +b, obtain the corresponding initial concentration of uric acid Cua, substitute I3 into Cglu=c*I3+d, and obtain the corresponding initial concentration of blood glucose Cglu;

[0103] a, b, c, and d are coefficients determined by the standard curve method experiment. Taking the initial blood glucose concentration as an example, a series of blood samples with different blood glucose concentrations are prepared. The electrochemical sensor is used to obtain the corresponding blood glucose current signal I3 according to the operation of step (4). The multiple groups of blood glucose concentrations and the multiple groups of current signals I3 are linearly fitted to obtain the corresponding standard curve Cglu=c*I3+d;

[0104] Taking the initial concentration of uric acid as an example, a series of blood samples with different uric acid concentrations were prepared, and the corresponding I1 and I2 were measured using an electrochemical sensor according to steps (2) and (3). u =I2-k1*I1 to obtain the corresponding I u , for multiple groups of uric acid concentrations and multiple groups of I u Perform linear fitting to obtain the corresponding standard curve Cua=a*I u +b=a*(I2-k1*I1)+b;

[0105] c. Substituting the electrical signal S1 obtained by the fourth voltage into the linear equation HCT*100=k2+k3*S1 to obtain the hematocrit HCT, where k2 ranges from 50 to 150, k3 ranges from -50 to 0, and the unit of S1 is μA;

[0106] HCT*100=k2+k3*S1 is obtained by the following method: a series of blood samples with different hematocrits (HCT) are prepared, S1 is measured using an electrochemical sensor according to the method of step (5), and linear fitting is performed on multiple groups of hematocrits and multiple groups of S1 to obtain the linear equation HCT*100=k2+k3*S1;

[0107] d. Obtain the final concentration of uric acid Cuat and blood glucose Cglut:

[0108] Cuat=Cua / (k4*HCT+k5), where k4 ranges from -5 to 0 and k5 ranges from 0 to +5;

[0109] Cglut=Cglu / (k6*HCT+k7), wherein k6 ranges from -5 to 0 and k7 ranges from 0 to +5.

[0110] The two correction equations were obtained by the following method: a series of blood samples with different hematocrits (HCT) were prepared, and the uric acid and blood glucose concentrations of the samples were measured using professional instruments (denoted as the final uric acid concentration Cuat and the final blood glucose concentration Cglut). Then, an electrochemical sensor was used to obtain the initial concentrations of different samples according to the method in step b (denoted as the initial uric acid concentration Cua and the initial blood glucose concentration Cglu). The values ​​of multiple groups of Cua / Cuat were linearly fitted with the values ​​of multiple groups of HCT to obtain the equation Cuat = Cua / (k4*HCT+k5). The values ​​of multiple groups of Cglu / Cuat were linearly fitted with the values ​​of multiple groups of HCT, and Cglut = Cglu / (k6*HCT+k7).

[0111] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0112] Multi-walled carbon nanotubes (short, 10-20 nm) were purchased from Nanjing Xianfeng Nanomaterial Technology Co., Ltd., 50% platinum-carbon catalyst powder was purchased from Wuhan Lanabai Pharmaceutical Chemical Co., Ltd., and the carbon paste used was a conductive carbon paste produced by Japan Jujo Ink Co., Ltd., model CH-8.

[0113] Example 1

[0114] A method for preparing a blood glucose and uric acid electrochemical sensor comprises the following steps:

[0115] S1. Making printed electrode layer

[0116] (1) Prepare the lower substrate;

[0117] The material is polyethylene terephthalate (PET).

[0118] (2) preparing a first working electrode 201, a first reference electrode 203, a second working electrode 202, a second reference electrode 204, and a wire 205 on the lower substrate by screen printing and then curing;

[0119] The preparation method of the slurry of the first working electrode 201, the second working electrode 202 and the first reference electrode 203 is as follows: add 15 g of multi-walled carbon nanotube material to 500 g of carbon slurry and mix thoroughly.

[0120] The second reference electrode 204 is a silver / silver chloride electrode, which can be made of conventional silver / silver chloride electrode materials without special restrictions;

[0121] The wire 205 is a silver wire, and can be made of conventional silver electrode materials without any special restrictions.

[0122] S2, oxygen plasma cleaning

[0123] The product obtained in step S1 is placed in an oxygen plasma machine for medium-frequency oxygen plasma cleaning. During use, a perforated lower plate is pressed against the lower substrate, exposing the corresponding second working electrode 202 for oxygen plasma cleaning. The oxygen intake volume is 100 SCCM, the cleaning power is 600 W, and the cleaning time is 5 minutes.

[0124] S3. Make reagent layer

[0125] A reagent layer composition containing glucose reactive enzyme, electron mediator and buffer matrix is ​​applied on the first working electrode 201 and the first reference electrode 203, and then heated and dried to form a reagent layer 3.

[0126] The glucose-responsive enzyme is glucose dehydrogenase; the electron mediators are hexaammineruthenium trichloride, benzidine and Prussian blue; and the buffer matrix is ​​phosphate buffered saline (PBS).

[0127] The drying temperature was 70°C and the drying time was 3 minutes.

[0128] S4. Make insulation layer

[0129] A mixture composed of insulating materials is printed on the lower substrate layer 1 provided with a printed electrode layer 2 and a reagent layer 3 to form an insulating layer 4, so that the insulating layer 4 covers part of the printed electrode layer 2 and part of the reagent layer 3. The purpose of covering part of the reagent layer 3 is to ensure that the area of ​​the exposed (uncovered) reagent layer 3 is constant. The insulating layer 4 is heated and dried. The heating and drying temperature should be selected to be an appropriate temperature that does not damage the glucose reaction enzyme in the reagent layer. The specific drying temperature is 70°C and the drying time is 4 minutes.

[0130] S5, assembling with the intermediate substrate 5 and the upper substrate 6

[0131] The middle substrate 5 is made of double-sided tape, and the upper substrate 6 is pasted on the middle substrate 5. The upper substrate 6 is assembled with the lower substrate layer to form a blood glucose and uric acid electrochemical sensor.

[0132] The blood glucose and uric acid electrochemical sensor of Example 1 is used to detect blood glucose and uric acid. The specific process is as follows:

[0133] Connect the blood glucose and uric acid electrochemical sensor to the electrochemical tester, turn on the electrochemical tester, and then apply a trigger voltage between the first working electrode 201 and the first reference electrode 203. Blood is sucked into the reaction channel through the sample suction port 501 by the siphon effect. The first working electrode 201 and the first reference electrode 203 form a current, causing the electrochemical tester to start testing and start timing (the trigger voltage is a DC voltage of 500 mV).

[0134] First, a first voltage is applied to the second working electrode 202 and the second reference electrode 204. Specifically, the first voltage is a DC voltage, the potential of which is less than the oxidation potential of uric acid, and the DC voltage is 200 mV. The voltage is applied for 1.0 s. The second working electrode 202 and the second reference electrode 204 use the redox properties of the interfering substances such as ascorbic acid and acetaminophen in the blood sample to measure the current signal I1 of the interfering substances such as ascorbic acid and acetaminophen in the blood sample flowing through the reaction channel.

[0135] Next, a second voltage is applied to the second working electrode 202 and the second reference electrode 204. Specifically, the second voltage is a DC voltage of 400 mV, and the voltage is applied for 1.0 second. The second working electrode 202 and the second reference electrode 204 use the redox characteristics of uric acid in the blood sample to measure the current signal I2 of uric acid in the blood sample flowing through the reaction channel.

[0136] Then, a third voltage is applied to the first working electrode 201 and the first reference electrode 203. Specifically, the third voltage is a DC voltage of 200 mV, and the voltage is applied for 5.0 seconds. The enzyme electrode formed by the first working electrode 201, the first reference electrode 203, and the reagent layer 3 measures the current signal I3 of the blood glucose in the blood sample flowing through the reaction channel.

[0137] Finally, a fourth voltage is applied to the second working electrode 202 and the second reference electrode 204. Specifically, the fourth voltage is an AC voltage with a voltage of 200 mV and a frequency of 200 Hz. The voltage is applied for 1.0 s. The second working electrode 202 and the second reference electrode 204 measure the hematocrit (HCT) of the blood sample flowing through the reaction channel. The maximum value of the AC current electrical signal obtained is S1.

[0138] During the above detection process, the electrochemical tester first 1、 I2 calculates the initial concentration of uric acid, and calculates the initial concentration of blood glucose based on the current signal I3. Then, using the test value of hematocrit (HCT), the initial concentration of uric acid and the initial concentration of blood glucose are corrected by hematocrit (HCT) to obtain more accurate final concentrations of uric acid and blood glucose.

[0139] Initial uric acid concentration and current signal I 1、 The calculation equation between I2 can be obtained by Cua=a*(I2-k1*I1)+b. The specific equation is Cua=1.414*(I2-0.82*I1)-0.0771, where the units of I1 and I2 are μA and the unit of Cua is mmol / L.

[0140] The initial blood glucose concentration and current signal I3 can be obtained by Cglu=c*I3+d. The specific equation is Cglu=5.863*I3-0.4514, where I3 is in μA and Cua is in mmol / L.

[0141] The hematocrit (HCT) is determined by the electrical signal S1 obtained by the fourth voltage. The relevant equation is: HCT*100=k2+k3*S1. The specific equation is HCT*100=-12.12*S1+88.49, and the unit of S1 is μA.

[0142] The final concentration of the analyte at room temperature (final concentration of uric acid Cuat, final concentration of blood glucose Cglut) is calculated by correcting the initial concentration of uric acid and blood glucose by hematocrit (HCT) as follows:

[0143] Cuat = Cua / (k4*HCT+k5), specifically Cuat = Cua / (-0.0116*HCT+0.934);

[0144] Cglut=Cglu / (k6*HCT+k7), specifically Cglut=Cglu / (-0.0214*HCT+0.9165).

[0145] Verify the accuracy of blood glucose and uric acid monitoring:

[0146] 1. Blood glucose monitoring accuracy

[0147] (1) Fresh venous blood from healthy individuals was used as the matrix, with a hematocrit range of 35% to 50%. Blood samples with different glucose concentrations (2.2 mM, 5.6 mM, 11.1 mM, 16.7 mM, and 25.0 mM) were prepared. The uric acid concentration in the blood samples was within the normal range and low (0.30 mM). The glucose and uric acid concentrations of the samples were measured using a Cobas c111 automatic biochemical analyzer after centrifugation of the plasma.

[0148] (2) The blood glucose and uric acid electrochemical sensor of Example 1 was used to test blood samples of different concentrations, and the blood glucose and uric acid concentration data were recorded, as shown in Table 1.

[0149] (3) Add a high-concentration uric acid solution to the blood sample from step 1 to increase the uric acid concentration in the blood sample to 0.80 mM. Measure blood glucose and uric acid concentrations using an automatic biochemical analyzer.

[0150] (4) The blood glucose and uric acid electrochemical sensor of Example 1 was used to test samples of different concentrations, and the blood glucose and uric acid data were recorded, as shown in Table 1.

[0151] Table 1

[0152]

[0153] 2. Uric acid monitoring accuracy

[0154] (1) Fresh venous blood from healthy individuals was used as the matrix, with a hematocrit range of 35% to 50%. Blood samples were prepared with different uric acid concentrations (0.30 mM, 0.50 mM, 0.70 mM, 0.90 mM, and 1.10 mM). The blood glucose concentrations in the samples were all within the normal range, with a low concentration (4.1 mM). The blood glucose and uric acid concentrations of the samples were measured using a Cobas c111 automatic biochemical analyzer after centrifugation of the plasma.

[0155] (2) The blood glucose and uric acid electrochemical sensor of Example 1 was used to test samples of different concentrations, and the blood glucose and uric acid data were recorded, as shown in Table 2.

[0156] (3) Add a high-concentration glucose solution to the blood sample from step 1 to increase the uric acid concentration in the blood sample to 25.0 mM. Use an automatic biochemical analyzer to measure blood glucose and uric acid concentrations.

[0157] (4) The blood glucose and uric acid electrochemical sensor of Example 1 was used to test samples of different concentrations, and the blood glucose and uric acid data were recorded, as shown in Table 2.

[0158] Table 2

[0159]

[0160] Table 1 shows the test performance of the blood glucose uric acid electrochemical sensor for blood glucose under the normal range of uric acid values ​​and at high concentrations. As can be seen from the results, the blood glucose measurement values ​​change little and the CV test performance is stable. Table 2 shows the test performance of the blood glucose uric acid electrochemical sensor for uric acid under the normal range of blood glucose values ​​and at high concentrations. As can be seen from the results, the uric acid measurement values ​​change little and the CV test performance is stable. This shows that under the electrochemical sensor structure and measurement method of the present invention, the concentrations of blood glucose and uric acid can be accurately tested.

[0161] Example 2

[0162] This embodiment differs from Example 1 in that the conductive carbon slurry is doped with 50% platinum-carbon catalyst powder. The slurries for the first working electrode 201, the second working electrode 202, and the first reference electrode 203 are prepared by adding 5 g of the 50% platinum-carbon catalyst powder to 500 g of the carbon slurry and mixing thoroughly. The remaining steps are the same as in Example 1.

[0163] The calculation equation between the initial uric acid concentration and the current signals I1 and I2 in Example 2 can be obtained by Cua=a*(I2-k1*I1)+b. The specific equation is Cua=1.112*(I2-0.85*I1)-0.1355, where I1 and I2 are in μA and Cua is in mmol / L.

[0164] The initial blood glucose concentration and current signal I3 can be obtained by Cglu=c*I3+d. The specific equation is Cglu=5.4986*I3-0.4990, where I3 is in μA and Cglu is in mmol / L.

[0165] The hematocrit (HCT) is determined by the electrical signal S1 obtained by the fourth voltage. The relevant equation is: HCT*100=k2+k3*S1. The specific equation is HCT*100=-11.07*S1+85.36, and the unit of S1 is μA.

[0166] The process of correcting the initial measured concentrations of uric acid and blood glucose by hematocrit (HCT) is the same as that in Example 1.

[0167] A comparative study was conducted using the blood glucose and uric acid electrochemical sensor of Example 2 and a fully automatic biochemical analyzer used by professionals, and the following evaluation results were obtained from 200 venous blood samples.

[0168] For blood glucose concentrations <5.55 mmol / L (100 mg / dL), 98% of cases were within ±0.83 mmol / L; for blood glucose concentrations ≥5.55 mmol / L (100 mg / dL), 100% were within ±15%. For uric acid concentrations <300 μmol / L, 99% were within ±60 μmol / L; for uric acid concentrations ≥300 μmol / L, 99% were within ±20%.

[0169] The test results of the biochemical analyzer are used as the X-axis and the test results of the electrochemical sensor are used as the Y-axis for regression analysis. Figure 5 shown.

[0170] The regression equation parameters of uric acid venous blood and its 95% CI and determination coefficient (R 2 )for:

[0171] Y=-20.013+1.003*X((R 2 )=0.9631).

[0172] The regression equation parameters of venous blood glucose and its 95% CI and determination coefficient (R 2 )for:

[0173] Y=0.022+0.997*X((R 2 )=0.9795).

[0174] Example 3

[0175] The difference between this embodiment and embodiment 1 lies in the oxygen plasma cleaning process S2. The specific differences in this process are: the oxygen intake volume is 100 SCCM, the cleaning power is 600 W, and the cleaning time is 3 minutes. The remaining steps are the same as those in embodiment 1.

[0176] The blood glucose and uric acid electrochemical sensor of Example 3 is used to detect blood glucose and uric acid, and the specific detection process is the same as the process of detecting blood glucose and uric acid using the blood glucose and uric acid electrochemical sensor of Example 1.

[0177] Example 4

[0178] The difference between this embodiment and embodiment 1 lies in the oxygen plasma cleaning process S2. The specific differences in this process are: the oxygen intake volume is 100 SCCM, the cleaning power is 600 W, and the cleaning time is 10 minutes. The remaining steps are the same as those in embodiment 1.

[0179] The blood glucose and uric acid electrochemical sensor of Example 4 is used to detect blood glucose and uric acid, and the specific detection process is the same as the process of detecting blood glucose and uric acid using the blood glucose and uric acid electrochemical sensor of Example 1.

[0180] Comparative Example 1

[0181] The difference between Comparative Example 1 and Example 1 is that the S2 and oxygen plasma cleaning processes are not performed, and the remaining steps are the same as those in Example 1.

[0182] The electrochemical sensor of Comparative Example 1 was used to detect blood glucose and uric acid, and the specific detection process was the same as the process of detecting blood glucose and uric acid using the blood glucose and uric acid electrochemical sensor of Example 1.

[0183] The results obtained by the electrochemical sensor test of Comparative Example 1 are as follows:

[0184] The electrochemical tester failed to obtain the current signal related to uric acid of the comparative electrochemical sensor within the accuracy range, that is, the detected current signal related to uric acid was extremely weak and could not be used to measure the uric acid concentration.

[0185] The initial blood glucose concentration and current signal I3 can be obtained by Cglu=c*I3+d. The specific equation is Cglu=5.163*I3-0.3294, where I3 is in μA and Cua is in mmol / L.

[0186] The hematocrit (HCT) is determined by the electrical signal S1 obtained by the fourth voltage. The relevant equation is: HCT*100=k2+k3*S1. The specific equation is HCT*100=-8.67*S1+73.54, and the unit of S1 is μA.

[0187] The final blood glucose concentration (Cglut) at room temperature was calculated by correcting the initial blood glucose concentration with the hematocrit (HCT) to obtain the equation of Cglut = Cglu / (-0.0217*HCT+1.1254).

[0188] Comparative Example 2

[0189] The difference between Comparative Example 2 and Example 1 lies in the oxygen plasma cleaning process S2. The specific differences in this process are: the oxygen intake volume is 100 SCCM, the cleaning power is 600 W, and the cleaning time is 1 minute. The remaining steps are the same as in Example 1.

[0190] The blood glucose and uric acid electrochemical sensor of Comparative Example 2 was used to detect blood glucose and uric acid, and the specific detection process was the same as the process of detecting blood glucose and uric acid using the blood glucose and uric acid electrochemical sensor of Example 1.

[0191] The electrochemical sensors prepared in the above examples and comparative examples were tested for their sensitivity in uric acid and blood glucose detection, and the R of the fitting equation was obtained. 2 , intercept / slope, coefficient of variation, and specific test results are shown in Table 3.

[0192] Table 3

[0193]

[0194] According to the comparison between Example 1 and Comparative Example 1, if S2 and oxygen plasma cleaning are not performed during the production process of the electrochemical sensor, the uric acid concentration cannot be measured, and the measurement accuracy of the blood glucose concentration is slightly affected.

[0195] According to the comparison between Examples 1, 3, and 4 and Comparative Example 2, the duration of the S2 and oxygen plasma cleaning process in the production process of the electrochemical sensor is too short. Although a certain uric acid current signal can be sensed, the sensitivity of detecting uric acid is seriously insufficient, and the R of the fitting equation is 2 , intercept / slope, and coefficient of variation all reflect the low repeatability and accuracy of uric acid concentration test results.

[0196] It should be understood that the above-described specific embodiments of the present invention are merely illustrative or illustrative of the principles of the present invention and do not constitute limitations of the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included within the scope of protection of the present invention. In addition, the appended claims are intended to cover all variations and modifications that fall within the scope and metes and bounds of the appended claims, or equivalents thereof.

Claims

1. A blood glucose and uric acid electrochemical sensor, comprising, arranged from bottom to top, a lower substrate layer, a printed electrode layer, a reagent layer, and an insulating layer, wherein the printed electrode layer comprises a first working electrode, a first reference electrode, a second working electrode, and a second reference electrode, the first working electrode, the first reference electrode, the second working electrode, and the second reference electrode being connected to corresponding wires; The first working electrode and the first reference electrode constitute a first electrode group, the second working electrode and the second reference electrode constitute a second electrode group, the reagent layer covers the first electrode group in the printed electrode layer, and the insulating layer covers part of the printed electrode layer and part of the reagent layer; Its characteristics are: The first working electrode, the second working electrode and the first reference electrode are carbon electrodes, and the second reference electrode is a silver / silver chloride electrode; a reagent layer composition consisting of a glucose responsive enzyme, an electron mediator and a buffer matrix is ​​applied to the first working electrode and the first reference electrode, and a reagent layer is formed by heating and drying; The reagent layer and the first electrode group form an enzyme electrode for measuring the blood glucose concentration of the blood sample, and the second electrode group is used to measure the hematocrit and uric acid concentration of the blood sample, and the second working electrode is subjected to a surface hydrophilization treatment; The hydrophilic treatment is medium frequency oxygen plasma cleaning, the oxygen intake volume is 50-200 SCCM, the cleaning power is 300-1000 W, and the cleaning time is 3-10 minutes.

2. The blood glucose and uric acid electrochemical sensor according to claim 1, characterized in that: The carbon electrodes used in 0-3 of the first working electrode, the second working electrode, and the first reference electrode are carbon electrodes containing a doping material, wherein the doping material is a mixture of one or more of single-walled carbon nanotubes, multi-walled carbon nanotubes, platinum-carbon nanoparticles, and micron-sized platinum-carbon particles; The slurry for preparing the carbon electrode containing the doping material is a mixture of the doping material and the conductive carbon slurry used for screen printing, and the mass ratio of the conductive carbon slurry to the doping material is 1:0.01-0.

06.

3. The blood glucose and uric acid electrochemical sensor according to claim 1, characterized in that: Also included is an intermediate substrate and an upper substrate, wherein the intermediate substrate is sandwiched between the upper substrate and the lower substrate layer; The insulating layer is provided with a first channel, the intermediate substrate is provided with a sample suction port and a second channel, the second channel corresponds to the first channel, and the second channel and the first channel are combined to form a fine reaction channel, the upper substrate is provided with an air outlet, and the end of the reaction channel is connected to the air outlet; The bottom of the reaction channel is the first electrode group, the second electrode group and the reagent layer in the printed electrode layer. The electrodes are the second working electrode, the second reference electrode, the first working electrode and the first reference electrode in order from near to far from the sample suction port.

4. The blood glucose and uric acid electrochemical sensor according to claim 1, characterized in that: The first reference electrode and the second reference electrode are connected to each other.

5. A method for preparing the blood glucose and uric acid electrochemical sensor according to any one of claims 1 to 4, characterized in that: The steps include: S1. Making printed electrode layer (1) Preparing a lower substrate as a lower substrate layer; (2) preparing a first working electrode, a first reference electrode, a second working electrode, a second reference electrode and a wire on the lower substrate; S2. Surface hydrophilic treatment Performing surface hydrophilization treatment on the second working electrode of the product obtained in step S1; S3. Make reagent layer forming a reagent layer on the product obtained in step S2, wherein the reagent layer covers the first electrode group in the printed electrode layer; S4. Make insulation layer An insulating layer is formed on the product obtained in step S3, and the insulating layer covers part of the printed electrode layer and part of the reagent layer.

6. The preparation method according to claim 5, characterized in that Also includes: S5, assemble with the middle substrate and upper substrate The middle substrate is pasted on the insulating layer, and the upper substrate and the lower substrate are assembled together.

7. A method for detecting blood sugar and uric acid, characterized in that: Using the blood glucose and uric acid electrochemical sensor according to any one of claims 1 to 4, the detection method comprises the following steps: (1) Connect the blood glucose and uric acid electrochemical sensor to the electrochemical tester, turn on the electrochemical tester, and then apply a trigger voltage between the first working electrode and the first reference electrode. The trigger voltage is a DC voltage. Blood enters the reaction channel, and the first working electrode and the first reference electrode form a current, causing the electrochemical tester to start testing and start timing. (2) applying a first voltage to the second working electrode and the second reference electrode, wherein the first voltage is a DC voltage, the potential of which is less than the oxidation potential of uric acid, and measuring the current signal I1 of the interfering substance, wherein the voltage range of the first voltage is 0.05 to 0.25 V; (3) applying a second voltage to the second working electrode and the second reference electrode, where the second voltage is a DC voltage, and measuring the current signal I2 of uric acid; (4) applying a third voltage to the first working electrode and the first reference electrode, where the third voltage is a DC voltage, and measuring the current signal I3 of the blood glucose; (5) applying a fourth voltage to the second working electrode and the second reference electrode, the fourth voltage being an AC voltage, and measuring the hematocrit, where the maximum value of the AC current electrical signal obtained is S1; (6) First, according to the current signal I 1、 I2 calculates the initial concentration of uric acid, and calculates the initial concentration of blood glucose based on the current signal I3. The initial concentrations of uric acid and blood glucose are then corrected to obtain the final concentrations of uric acid and blood glucose.

8. The detection method according to claim 7, characterized in that In step (1), the trigger voltage range is 100 to 700 mV; In step (2), the voltage is applied for a duration of 1 to 3 seconds; In step (3), the second voltage has a voltage range of 0.3 to 0.6 V, and the duration of applying the voltage is 1 to 4 seconds; In step (4), the voltage range of the third voltage is 0.2 to 0.6 V, and the duration of applying the voltage is 5 to 6 seconds; In step (5), the fourth voltage has a voltage range of 0.05 to 1 V, a frequency of 10 to 1000 Hz, and a duration of applying the voltage of 1 to 10 seconds.

9. The detection method according to claim 7, characterized in that The final concentration of uric acid and the final concentration of blood glucose in step (6) are specifically obtained by the following method: a. Substitute I2 and I1 of the test blood sample into the linear equation I u =I2-k1*I1, and the uric acid response current intensity I is obtained. u , where k1 ranges from 0.8 to 1.2; b. Then, the I obtained in step a is u Substitute Cua=a*I u +b, obtain the corresponding initial concentration of uric acid Cua, substitute I3 into Cglu=c*I3+d, and obtain the corresponding initial concentration of blood glucose Cglu; c. Substituting the electrical signal S1 obtained by the fourth voltage into the linear equation HCT*100=k2+k3*S1 to obtain the hematocrit HCT, where k2 ranges from 50 to 150, k3 ranges from -50 to 0, and the unit of S1 is μA; d. Obtain the final concentration of uric acid Cuat and blood glucose Cglut: Cuat=Cua / (k4*HCT+k5), where k4 ranges from -5 to 0 and k5 ranges from 0 to +5; Cglut=Cglu / (k6*HCT+k7), where k6 ranges from -5 to 0 and k7 ranges from 0 to +5; Among them, k1, a and b are obtained by the multivariate linear regression method, and c, d and k2~k7 are obtained by the unit linear regression method.

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