Blood glucose uric acid electrochemical sensor and preparation and detection method thereof

Abstract

The invention discloses a blood glucose uric acid electrochemical sensor and a preparation and detection method, the electrochemical sensor comprises a lower substrate layer, a printed electrode layer, a reagent layer and an insulating layer which are sequentially arranged from bottom to top, and the printed electrode layer comprises a first electrode group consisting of a first working electrode and a first reference electrode; the second electrode group consists of a second working electrode and a second reference electrode, and each electrode is connected with a corresponding lead; the reagent layer and the first electrode group form an enzyme electrode which is used for measuring the blood glucose concentration of a blood sample, the second electrode group is used for measuring the hematocrit and the uric acid concentration of the blood sample, the second working electrode is subjected to surface hydrophilization treatment, and the blood glucose concentration and the uric acid concentration can be obtained according to signals measured by using the electrochemical sensor. According to the invention, mutual interference of two enzymes of blood glucose detection and uric acid detection can be completely avoided, the accuracy of a detection result is guaranteed, and blood glucose and uric acid detection and hematocrit correction are realized at the same time.

Description

Technical Field

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

[0002] Hyperuricemia and diabetes are both metabolic diseases caused by overnutrition. Research has found that insulin resistance is the common pathogenic basis for hyperuricemia and diabetes. Obese patients with typical insulin resistance characteristics are often the main source of hyperuricemia and type 2 diabetes. Hyperuricemia is also an independent risk factor for the occurrence of type 2 diabetes. The detection of uric acid and blood glucose biochemical indicators is of great significance for the prevention and diagnosis of hyperuricemia and diabetes.

[0003] When detecting uric acid and blood glucose biochemical indicators, it is necessary to go to a professional hospital and use professional measuring instruments for measurement. For sub-healthy people or patients with corresponding diseases, the above measurement methods are very inconvenient. Therefore, some household test strips have been invented on the market. The test strip is an electrochemical sensor with an enzyme-modified electrode. The electrode surface of the sensor is modified with a bio-enzyme. The commonly used enzymes for blood glucose testing are mainly glucose oxidase and glucose dehydrogenase, and the commonly used enzyme for uric acid testing is mainly uricase. The bio-enzyme of the sensor can react with glucose or uric acid in the blood to generate an electric current, and the glucose or uric acid concentration reading is converted according to the generated current signal. The common types of test strips include blood glucose test strips (single-function blood glucose), uric acid test strips (single-function uric acid), and blood glucose and uric acid test strips (dual-function blood glucose and uric acid). Among them, the single-function blood glucose test strips and uric acid test strips are very inconvenient to use. It is necessary to drop blood samples separately for the detection of blood glucose and uric acid. The operation is relatively cumbersome, which is not conducive to daily monitoring, and the detection cost is relatively high. For the dual-function test strip (blood glucose and uric acid test strip) that can simultaneously detect blood glucose and uric acid, the electrode surface needs to contain two enzymes. However, due to the different characteristics of different enzymes, they will affect each other, and the detection accuracy and anti-interference ability are different. In order to reduce the mutual influence, usually the two detection items of blood glucose and uric acid are set on the front and back sides of the test strip respectively, or the two detection item sample test chambers are divided into regions to avoid the interference of different types of enzyme reactions to achieve the simultaneous detection of blood glucose and uric acid. However, these methods result in a complex manufacturing process of the test strip, an increase in the number of electrodes, which is prone to electrode contamination and enzyme contamination. Since the test strips are all portable and small in size, the precision requirements for electrode production become harsh, leading to an increase in production costs, and it will also cause an increase in the amount of blood sample used, resulting in an increase in the blood collection volume of patients. Summary of the Invention

[0004] In view of the above 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 as follows:

[0006] The present invention provides a blood glucose and uric acid electrochemical sensor, which includes a lower substrate layer, a printed electrode layer, a reagent layer, and an insulating layer arranged in sequence from bottom to top. The printed electrode layer includes 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 form a first electrode group, the second working electrode and the second reference electrode form 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 for measuring the hematocrit and uric acid concentration of a blood sample, and the second working electrode is surface-hydrophilized.

[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, and conventional silver / silver chloride electrode preparation materials can be used without special limitations;

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

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

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

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

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

[0016] Preferably, the plasma cleaning is medium-frequency oxygen plasma cleaning, the intake of oxygen is 50 - 200 SCCM, the cleaning power is 300 - 1000 W, and the cleaning duration is 3 - 10 min.

[0017] The insulating layer is located above the reagent layer and the printed electrode layer and is made by printing a material with insulation properties. The insulating layer is used to prevent short circuits, while allowing electrochemical reactions to occur in specific areas, isolating current interference between electrodes, and ensuring the accuracy and stability of detection signals.

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

[0019] The insulating layer is provided with a first channel, the intermediate substrate is provided with a sample inlet and a second channel, the second channel corresponds to the first channel, and the second channel and the first channel form a fine reaction channel in combination. The blood sample is sucked into the reaction channel by the siphon effect through the sample inlet;

[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 the effect of the 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 in the printed electrode layer and the reagent layer. Each electrode is, in the order from near to far from the sample inlet, the second working electrode, the second reference electrode, the first working electrode, and the first reference electrode.

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

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

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

[0025] Preferably, the upper substrate is made of a transparent or semi-transparent material, such as polyethylene terephthalate (PET), so as to facilitate the observation of the entry of the blood sample into 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. Fabricate the printed electrode layer

[0028] (1) Prepare the lower substrate as the lower substrate layer;

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

[0030] S2. Surface hydrophilic treatment

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

[0032] S3. Fabricate the reagent layer

[0033] Fabricate the reagent layer on the product obtained in step S2, and the reagent layer covers the first electrode group in the printed electrode layer;

[0034] S4. Fabricate the insulating layer

[0035] Fabricate the insulating layer 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 further includes:

[0037] S5. Assemble with the middle substrate and the upper substrate

[0038] Paste the middle substrate on the insulating layer, and assemble the upper substrate with the lower substrate layer in a matching manner.

[0039] Preferably, in step S2, the surface hydrophilic treatment is medium-frequency oxygen plasma cleaning, the intake of oxygen is 50-200 SCCM, the cleaning power is 300-1000 W, and the cleaning duration is 3-10 min.

[0040] Preferably, in step S2, use the perforated lower plate to press on the lower substrate, and only expose the second working electrode for oxygen plasma cleaning.

[0041] Preferably, in step S3, apply the reagent layer composition containing glucose reaction enzyme, electron mediator and buffer matrix on the first working electrode and the first reference electrode, and form the reagent layer by heating and drying, the temperature of heating and drying is 50-70 °C, and the time is 3-10 minutes.

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

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

[0044] The buffer matrix is phosphate buffer solution (PBS), citrate buffer solution, or a composite buffer solution of phosphate and citrate.

[0045] Preferably, in step S4, the mixture composed of insulating materials is printed on the lower substrate layer provided with a printed electrode layer and a reagent layer to form an insulating layer, covering part of the printed electrode layer and part of the reagent layer, and then heated and dried. The heating and drying temperature of the insulating layer should be selected as 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 includes the following steps:

[0047] (1) Connect the blood glucose and uric acid electrochemical sensor to an 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 a current is formed between the first working electrode and the first reference electrode, causing the electrochemical tester to start testing and start timing;

[0048] (2) Apply a first voltage between the second working electrode and the second reference electrode. The first voltage is a DC voltage, and the potential of this DC voltage is less than the uric acid oxidation potential. The second working electrode and the second reference electrode measure the current signal I of interfering substances such as ascorbic acid and paracetamol in the blood sample flowing through the reaction channel by using the oxidation-reduction characteristics of interfering substances such as ascorbic acid and paracetamol in the blood sample; 1 for measurement;

[0049] (3) Apply a second voltage between the second working electrode and the second reference electrode. The second voltage is a DC voltage. The second working electrode and the second reference electrode measure the current signal I of uric acid in the blood sample flowing through the reaction channel by using the oxidation-reduction characteristics of uric acid in the blood sample; 2 for measurement;

[0050] (4) Apply a third voltage between the first working electrode and the first reference electrode. The third voltage is a DC voltage. The enzyme electrode formed by the first working electrode, the first reference electrode, and the reagent layer measures the current signal I of blood glucose in the blood sample flowing through the reaction channel; 3 for measurement;

[0051] (5) Apply a fourth voltage to the second working electrode and the second reference electrode. The fourth voltage is an alternating current voltage. Measure the hematocrit (HCT) of the blood sample flowing through the reaction channel with the second working electrode and the second reference electrode, and the maximum value of the obtained alternating current signal is S. 1 ;

[0052] (6) First, calculate the initial measured concentration of uric acid according to the current signal I 1、 I 2 , calculate the initial measured concentration of blood glucose according to the current signal I 3 , and then correct the initial measured concentration of uric acid and the initial measured concentration of blood glucose to obtain the final concentration of uric acid and the final concentration of 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 sampling port, a current will be formed only when it reaches the first electrode group at the end of the reaction channel, which can avoid inaccurate measurement values caused by the reaction channel not being fully filled with blood.

[0054] Preferably, in step (1), the voltage range of the trigger voltage is 100 - 700 mV;

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

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

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

[0058] In step (5), the voltage range of the fourth voltage is 0.05 - 1 V, the frequency is 10 - 1000 Hz, and the duration of applying the voltage is 1 - 10 s.

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

[0060] a. Substitute the I 2 and I 1 of the test blood sample into the equation I u = I 2 - k 1 * I 1 to obtain the uric acid response current intensity I u , where the range of k 1 is 0.8 - 1.2;

[0061] b. Then substitute the I obtained in step a u into Cua = a * I u + b to obtain the initial measured concentration Cua of the corresponding uric acid. Substitute I 3 into Cglu = c * I 3 + d to obtain the initial measured concentration Cglu of the corresponding blood glucose;

[0062] c. Substitute the electrical signal S obtained through the fourth voltage 1 into the linear equation HCT * 100 = k 2 + k 3 * S 1 to obtain the hematocrit HCT, where the range of k 2 is 50 to 150, the range of k 3 is -50 to 0, and the unit of S 1 is μA;

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

[0064] Cuat = Cua / (k 4 * HCT + k 5 ), where the range of k 4 is -5 to 0, and the range of k 5 is 0 to +5;

[0065] Cglut = Cglu / (k 6 * HCT + k 7 ), where the range of k 6 is -5 to 0, and the range of k 7 is 0 to +5.

[0066] Among them, k 1 , a, and b are obtained by the method of multiple linear regression, and c, d, and k 2 ~k 7 are obtained by the method of simple linear regression.

[0067] The linear equation I u = I 2 - k 1 * I 1 is obtained by the following method: Configure a series of blood samples with different concentrations of interferents (for example, a series of blood samples with different ascorbic acid concentrations), and measure the corresponding I 1 and I 2 according to the methods in steps (2) and (3). Analyze that the current I 1 has a linear relationship with the interferent concentration, and the current I 2 has a linear relationship with the uric acid and interferent concentrations. After deducting proportionally, the relevant relationship I u = I2 -k 1 *I 1 , finally substitute this relationship into the uric acid and interferent mixed solution for verification;

[0068] Taking the initial measured concentration of uric acid as an example, prepare a series of blood samples with different uric acid concentrations, and measure the corresponding I according to the methods in steps (2) and (3) 1 and I 2 , substitute into I u =I 2 -k 1 *I 1 to obtain the corresponding I u , perform linear fitting on multiple groups of uric acid concentrations and multiple groups of I u to obtain the corresponding standard curve Cua = a*I u +b = a*(I 2 -k 1 *I 1 );

[0069] Taking the initial measured concentration of blood glucose as an example, prepare a series of blood samples with different blood glucose concentrations, and obtain the corresponding current signal I of blood glucose according to the operation in step (4) 3 , perform linear fitting on multiple groups of blood glucose concentrations and multiple groups of current signals I 3 to obtain the corresponding standard curve Cglu = c*I 3 +d;

[0070] HCT*100 = k 2 +k 3 *S 1 Obtained by the following method: Prepare a series of blood samples with different hematocrits (HCT), measure S according to the method in step (5) 1 , perform linear fitting on multiple groups of hematocrits and multiple groups of S 1 to obtain the linear equation HCT*100 = k 2 +k 3 *S 1 ;

[0071] The two calibration equations are obtained by the following method: Prepare a series of blood samples with different hematocrits (HCT), measure the uric acid and blood glucose concentrations of the samples using a professional instrument (denoted as the final uric acid concentration Cuat and the final blood glucose concentration Cglut), then measure the initial measured concentrations of different samples (denoted as the initial measured uric acid concentration Cua and the initial measured blood glucose concentration Cglu) according to the method in step b, perform linear fitting on multiple groups of Cua / Cuat values and multiple groups of HCT to obtain the equation Cuat = Cua / (k 4 *HCT + k 5), linearly fit multiple sets of Cglu / Cuat values with multiple sets of HCT, Cglut = Cglu / (k 6 *HCT + k 7 ).

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

[0073] (1) Without introducing additional reaction channels and electrodes, the uric acid detection electrode and the blood glucose detection electrode of the present invention share a reaction channel to achieve combined measurement, requiring less blood sample volume, reducing the blood collection amount of patients, reducing pain, and also having the advantages of simple manufacturing process and avoiding a substantial increase in production costs;

[0074] (2) The present invention can detect uric acid and correct hematocrit on the same electrode, completely avoiding the mutual interference of the two enzymes for blood glucose detection and uric acid detection, ensuring the accuracy of the detection results, and at the same time realizing blood glucose and uric acid detection and hematocrit correction, improving the detection efficiency of the test strip. Description of the Drawings

[0075] The present invention will be further described below in conjunction with the drawings and embodiments:

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

[0077] Figure 2 is a partial structural schematic diagram of the blood glucose and uric acid electrochemical sensor of Embodiment 1 of the present invention;

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

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

[0080] Figure 5 is a linear graph of the electrochemical sensor for detecting blood glucose and uric acid according to Embodiment 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 inlet; 502, second channel; 6, upper substrate; 601, air outlet. Detailed Embodiments

[0082] To make the objectives, technical solutions, and advantages of the present invention more clear and understandable, 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 unnecessarily confusing the concepts of the present invention.

[0083] As Figure 1 and 2 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 that are sequentially distributed from bottom to top.

[0084] The lower substrate layer 1 is selected from 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 composed of a first working electrode 201 and a first reference electrode 203, and a second electrode group composed 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 for measuring the hematocrit (HCT) and uric acid concentration of a blood sample.

[0086] The electrodes of the printed electrode layer 2 are, in the order from near to far from the sampling 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 subjected to surface hydrophilization treatment. The second reference electrode 204 is a silver / silver chloride electrode. The wire 205 is a silver wire. The surface hydrophilization treatment process of the manufacturing 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 to start an electrochemical tester.

[0087] The reagent layer 3 covers the first electrode group in the printed electrode layer 2. The reagent layer 3 contains a glucose reaction enzyme and an electron mediator. The reagent layer 3 and the first electrode group form an enzyme electrode, which is the region where an electrochemical reaction of blood glucose occurs under the action of the enzyme and is used for measuring the blood glucose concentration (i.e., the glucose concentration) of a blood sample.

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

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

[0090] The insulating layer 4 is located above the reagent layer 3 and the printed electrode layer 2. A first channel 401 is provided on the insulating layer 4 and is made by printing a stirring material with insulating properties. Specifically, a stirring material composed of insulating materials is printed on the lower substrate layer provided with the printed electrode layer and the reagent layer to form the insulating layer, covering part of the printed electrode layer and part of the reagent layer, and then heated and dried. The heating and drying temperature of the insulating layer should be selected as an appropriate temperature that does not damage the glucose reaction enzyme in the reagent layer.

[0091] The insulating layer 4 is used to prevent short circuits, while allowing electrochemical reactions to occur in specific areas, isolating the current interference between the electrodes, and ensuring the accuracy and stability of the detection signal. The insulating layer 4 does not cover corresponding parts of the first working electrode 201, part of the first reference electrode 203, part of the second working electrode 202, and part of the second reference electrode 204.

[0092] The intermediate substrate 5 can be a double-sided adhesive tape with adhesiveness on both sides, used to paste the lower substrate layer 1 and the upper substrate 6. A sample suction port 501 and a second channel 502 are provided on the intermediate substrate 5. 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 in the printed electrode layer 2 and the reagent layer 3.

[0093] The upper substrate 6 covers the reaction channel and part of the printed electrode layer 2. The upper substrate 6 is provided with an air outlet 601. The end of the reaction channel is connected to the air outlet 601 to achieve the effect of siphon. When the blood sample flows into the reaction channel, the air in the reaction channel can be discharged through the air outlet 601. The upper substrate 6 is made of a transparent or semi-transparent material, such as polyethylene terephthalate (PET), so as to facilitate observing the situation of the blood sample entering the reaction channel.

[0094] Using the above blood glucose and uric acid electrochemical sensor can 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, and the voltage range of the trigger voltage is 100 - 700 mV. Blood enters the reaction channel, and a current is formed between the first working electrode and the first reference electrode, causing the electrochemical tester to start testing and start timing;

[0096] (2) Apply a first voltage between the second working electrode and the second reference electrode. The first voltage is a DC voltage, and the potential of this DC voltage is less than the uric acid oxidation potential. The voltage range of the first voltage is 0.05 - 0.25 V, and the duration of applying the voltage is 1 - 3 s. The second working electrode 202 and the second reference electrode 204 measure the current signal I of interfering substances such as ascorbic acid and paracetamol in the blood sample flowing through the reaction channel by using the oxidation-reduction characteristics of the interfering substances; 1 for measurement;

[0097] (3) Apply a second voltage between the second working electrode and the second reference electrode. The second voltage is a DC voltage, and the voltage range of the second voltage is 0.3 - 0.6 V. The duration of applying the voltage is 1 - 4 s. The second working electrode and the second reference electrode measure the current signal I of uric acid in the blood sample flowing through the reaction channel by using the oxidation-reduction characteristics of uric acid in the blood sample; 2 for measurement;

[0098] (4) Apply a third voltage between the first working electrode and the first reference electrode. The third voltage is a DC voltage, and the voltage range of the third voltage is 0.2 - 0.6 V. The duration of applying the voltage is 5 - 6 s. The enzyme electrode formed by the first working electrode, the first reference electrode, and the reagent layer measures the current signal I of blood glucose in the blood sample flowing through the reaction channel; 3 for measurement;

[0099] (5) Apply a fourth voltage between the second working electrode and the second reference electrode. The fourth voltage is an AC voltage, and the voltage range of the fourth voltage is 0.05 - 1 V, and the frequency is 10 - 1000 Hz. The duration of applying the voltage is 1 - 10 s. The second working electrode and the second reference electrode measure the hematocrit (HCT) of the blood sample flowing through the reaction channel, and the maximum value of the obtained AC current electrical signal is S; 1 ;

[0100] (6) a. Substitute the I 2 and I 1 of the test blood sample into the linear equation I u = I 2 - k 1 * I 1 to obtain the uric acid response current intensity I u, where k 1 ranges from 0.8 to 1.2;

[0101] Linear equation I u = I 2 - k 1 * I 1 is obtained by the following method: Configure a series of blood samples with different ascorbic acid concentrations, and measure the corresponding I 1 and I 2 using an electrochemical sensor according to the methods in steps (2) and (3). Analyze that the current I 1 has a linear relationship with the interferent concentration, and the current I 2 has a linear relationship with the uric acid and ascorbic acid concentrations. After proportional deduction, the relevant relationship can be obtained: I u = I 2 - k 1 * I 1 , and finally substitute this relationship into the mixed solution of uric acid and ascorbic acid for verification;

[0102] b. Then substitute the I u obtained in step a into Cua = a * I u + b to obtain the initial measured concentration Cua of the corresponding uric acid. Substitute the I 3 into Cglu = c * I 3 + d to obtain the initial measured concentration Cglu of the corresponding blood glucose;

[0103] a, b, c, and d are coefficients determined by conducting a standard curve method experiment. Taking the initial measured concentration of blood glucose as an example, configure a series of blood samples with different blood glucose concentrations, and use an electrochemical sensor to obtain the corresponding current signal I 3 of blood glucose according to the operation in step (4). Perform linear fitting on multiple groups of blood glucose concentrations and multiple groups of current signals I 3 to obtain the corresponding standard curve Cglu = c * I 3 + d;

[0104] Taking the initial measured concentration of uric acid as an example, configure a series of blood samples with different uric acid concentrations, and use an electrochemical sensor to measure the corresponding I 1 and I 2 , substitute into I u = I 2 - k 1 * I 1 to obtain the corresponding I u , perform linear fitting on multiple groups of uric acid concentrations and multiple groups of I u to obtain the corresponding standard curve Cua = a * I u + b = a * (I 2 - k 1 * I1 ) + b;

[0105] c. Substitute the electrical signal S obtained from the fourth voltage 1 into the linear equation HCT*100 = k 2 + k 3 *S 1 to obtain the hematocrit HCT, where k 2 ranges from 50 to 150, and k 3 ranges from -50 to 0, and S 1 is in the unit of μA;

[0106] HCT*100 = k 2 + k 3 *S 1 is obtained by the following method: Prepare a series of blood samples with different hematocrits (HCT), measure S using an electrochemical sensor according to the method in step (5) 1 , and perform linear fitting on multiple groups of hematocrits and multiple groups of S 1 to obtain the linear equation HCT*100 = k 2 + k 3 *S 1 ;

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

[0108] Cuat = Cua / (k 4 *HCT + k 5 ), where k 4 ranges from -5 to 0, and k 5 ranges from 0 to +5;

[0109] Cglut = Cglu / (k 6 *HCT + k 7 ), where k 6 ranges from -5 to 0, and k 7 ranges from 0 to +5.

[0110] The two calibration equations are obtained by the following method: Prepare a series of blood samples with different hematocrits (HCT), measure the uric acid and blood glucose concentrations of the samples using a professional instrument (denoted as the final uric acid concentration Cuat and the final blood glucose concentration Cglut), then use an electrochemical sensor to obtain the initial measured concentrations of different samples according to the method in step b (denoted as the initial measured concentration Cua of uric acid and the initial measured concentration Cglu of blood glucose), perform linear fitting on multiple groups of Cua / Cuat values and multiple groups of HCT to obtain the equation Cuat = Cua / (k 4 *HCT + k 5), linearly fit multiple sets of Cglu / Cuat values with multiple sets of HCT, Cglut = Cglu / (k 6 *HCT + k 7 ).

[0111] Unless otherwise specified, the test methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

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

[0113] Example 1

[0114] A preparation method of a blood glucose and uric acid electrochemical sensor includes the following steps:

[0115] S1. Fabricate a printed electrode layer

[0116] (1) Prepare a lower substrate;

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

[0118] (2) Use the method of screen printing and then curing to prepare 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;

[0119] The preparation method of the slurry for preparing 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 paste and mix well.

[0120] The second reference electrode 204 is a silver / silver chloride electrode, and conventional silver / silver chloride electrode preparation materials can be used without special limitations;

[0121] The wire 205 is a silver wire, and conventional silver electrode preparation materials can be used without special limitations.

[0122] S2. Oxygen plasma cleaning

[0123] Put the product obtained in step S1 into an oxygen plasma machine for medium-frequency oxygen plasma cleaning. When in use, press the lower plate with holes on the lower substrate to expose the corresponding second working electrode 202 for oxygen plasma cleaning. The intake of oxygen is 100 SCCM, the cleaning power is 600 W, and the cleaning duration is 5 min.

[0124] S3. Fabricate the reagent layer

[0125] Apply the reagent layer composition containing glucose reaction enzyme, electron mediator, and buffer matrix onto the first working electrode 201 and the first reference electrode 203, and form the reagent layer 3 by heating and drying.

[0126] The glucose reaction enzyme is glucose dehydrogenase; the electron mediator is hexammine ruthenium(III) chloride, benzidine, and Prussian blue; the buffer matrix is phosphate buffer solution (PBS).

[0127] The temperature for heating and drying is 70 °C, and the time is 3 minutes.

[0128] S4. Fabricate the insulating layer

[0129] Print the mixture composed of insulating materials on the lower substrate layer 1 provided with the printed electrode layer 2 and the reagent layer 3 to form the insulating layer 4, covering 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 a constant area of the exposed (uncovered) reagent layer 3. Heat and dry the insulating layer 4. The temperature for heating and drying should be selected as an appropriate temperature that does not damage the glucose reaction enzyme in the reagent layer. Specifically, the drying temperature is 70 °C, and the time is 4 minutes.

[0130] S5. Assemble with the middle substrate 5 and the upper substrate 6

[0131] Select double-sided tape for the middle substrate 5, paste the upper substrate 6 on the middle substrate 5, and assemble the upper substrate 6 with the lower substrate layer to form a blood glucose and uric acid electrochemical sensor.

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

[0133] Connect the blood glucose and uric acid electrochemical sensor to an 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 from the sampling port 501 by siphon effect. A current is formed between the first working electrode 201 and the first reference electrode 203, causing the electrochemical tester to start testing and start timing (the trigger voltage is a DC voltage, and the voltage is 500 mV).

[0134] First, apply a first voltage between the second working electrode 202 and the second reference electrode 204. Specifically, the first voltage is a DC voltage, the potential of this DC voltage is less than the uric acid oxidation potential, this DC voltage is 200 mV, and the duration of applying the voltage is 1.0 s. The second working electrode 202 and the second reference electrode 204 utilize the oxidation-reduction characteristics of interferents such as ascorbic acid and paracetamol in the blood sample to measure the current signal I of interferents such as ascorbic acid and paracetamol in the blood sample flowing through the reaction channel 1Perform measurement;

[0135] Next, apply a second voltage to the second working electrode 202 and the second reference electrode 204. Specifically, the second voltage is a DC voltage with a value of 400 mV, and the duration of voltage application is 1.0 s. The second working electrode 202 and the second reference electrode 204 measure the current signal I of uric acid in the blood sample flowing through the reaction channel using the oxidation-reduction characteristics of uric acid in the blood sample 2 Perform measurement;

[0136] Then, apply a third voltage to the first working electrode 201 and the first reference electrode 203. Specifically, the third voltage is a DC voltage with a value of 200 mV, and the duration of voltage application is 5.0 s. The enzyme electrode formed by the first working electrode 201, the first reference electrode 203, and the reagent layer 3 measures the current signal I of blood glucose in the blood sample flowing through the reaction channel 3 Perform measurement;

[0137] Finally, apply a fourth voltage to the second working electrode 202 and the second reference electrode 204. Specifically, the fourth voltage is an AC voltage with a value of 200 mV and a frequency of 200 Hz, and the duration of voltage application is 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, and the maximum value of the obtained AC current signal is S 1 ;

[0138] During the above detection process, the electrochemical tester first calculates the initial measured concentration of uric acid based on the current signal I 1、 I 2 calculates the initial measured concentration of blood glucose based on the current signal I 3 Then, using the measured value of hematocrit (HCT), correct the initial measured concentration of uric acid and the initial measured concentration of blood glucose through hematocrit (HCT) to obtain a more accurate final concentration of uric acid and a more accurate final concentration of blood glucose.

[0139] The calculation equation between the initial measured concentration of uric acid and the current signal I 1、 I 2 can be obtained from Cua = a * (I 2 - k 1 * I 1 ) + b. The specific equation is Cua = 1.414 * (I 2 - 0.82 * I 1 ) - 0.0771, where I 1 , I 2 are in μA, and Cua is in mmol / L.

[0140] The calculation equation between the initial measured concentration of blood glucose and the current signal I 3 can be Cglu = c * I3 It is obtained by +d, and the specific equation is Cglu = 5.863 * I 3 - 0.4514 where I 3 The unit of I is μA, and the unit of Cua is mmol / L.

[0141] The hematocrit (HCT) is determined by the electrical signal S obtained from the fourth voltage 1 The relevant equation is: HCT * 100 = k 2 + k 3 * S 1 The specific equation is HCT * 100 = - 12.12 * S 1 + 88.49, and the unit of S 1 is μA.

[0142] The equation for calculating the final concentration values of the analytes (final uric acid concentration Cuat and final glucose concentration Cglut) at room temperature through the correction calculation of the hematocrit (HCT) for the initial measured concentration of uric acid and the initial measured concentration of glucose can be:

[0143] Cuat = Cua / (k 4 * HCT + k 5 ), specifically Cuat = Cua / (- 0.0116 * HCT + 0.934);

[0144] Cglut = Cglu / (k 6 * HCT + k 7 ), 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) Using the fresh venous blood of normal people as the matrix, with the hematocrit range of 35% - 50%, blood samples with different blood glucose concentrations (concentrations are 2.2 mM, 5.6 mM, 11.1 mM, 16.7 mM, and 25.0 mM) are prepared; at this time, the uric acid concentration in the blood samples is within the normal range value and is low (0.30 mM). The blood glucose and uric acid concentrations of the samples are obtained from the centrifuged plasma tested by the fully automatic biochemical analyzer Cobas c111.

[0148] (2) Use the blood glucose and uric acid electrochemical sensor of this Example 1 to test blood samples with different concentrations, and record the blood glucose and uric acid concentration data, as shown in Table 1.

[0149] (3) Add a high-concentration uric acid solution to the blood sample in step 1 to make the uric acid concentration in the blood sample reach 0.80 mM. Use an automatic biochemical analyzer to measure the blood glucose and uric acid concentrations.

[0150] (4) Use the blood glucose and uric acid electrochemical sensor of Example 1 to test samples with different concentrations, and record the blood glucose and uric acid data, as shown in Table 1.

[0151] Table 1

[0152]

[0153] 2. Uric acid monitoring accuracy

[0154] (1) Using fresh venous blood from normal people as the matrix, with the hematocrit range of 35% - 50%, prepare blood samples with different uric acid concentrations (concentrations are 0.30 mM, 0.50 mM, 0.70 mM, 0.90 mM, and 1.10 mM respectively); at this time, the blood glucose concentration in the blood sample is within the normal range value and is low (4.1 mM). The blood glucose and uric acid concentrations of the samples are obtained by centrifuging the plasma and measuring with an automatic biochemical analyzer Cobas c111.

[0155] (2) Use the blood glucose and uric acid electrochemical sensor of Example 1 to test samples with different concentrations, and record the blood glucose and uric acid data, as shown in Table 2.

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

[0157] (4) Use the blood glucose and uric acid electrochemical sensor of Example 1 to test samples with different concentrations, and record the blood glucose and uric acid data, as shown in Table 2.

[0158] Table 2

[0159]

[0160] Table 1 shows the test performance of the blood glucose and uric acid electrochemical sensor for blood glucose under normal and high-concentration uric acid range values. From the results, it can be seen that the change in the blood glucose measurement value is small and the CV test performance is stable. Table 2 shows the test performance of the blood glucose and uric acid electrochemical sensor for uric acid under normal and high-concentration blood glucose range values. From the results, it can be seen that the change in the uric acid measurement value is small and the CV test performance is stable. This shows that under the structure and measurement method of the electrochemical sensor of the present invention, the concentrations of blood glucose and uric acid can be accurately measured.

[0161] Example 2

[0162] The difference between this embodiment and Embodiment 1 is that the material doped with conductive carbon paste is 50% platinum-carbon catalyst powder, and the preparation method of the slurry for preparing the first working electrode 201, the second working electrode 202 and the first reference electrode 203 is as follows: Add 5 g of 50% platinum-carbon catalyst powder to 500 g of carbon paste and mix well. The remaining steps are the same as those in Embodiment 1.

[0163] The initial measured concentration of uric acid and the current signal I 1 、I 2 in Embodiment 2, the calculation equation between them can be obtained from Cua = a*(I 2 -k 1 *I 1 ) + b, and the specific equation is Cua = 1.112*(I 2 -0.85*I 1 ) - 0.1355, where I 1 , I 2 is in μA and Cua is in mmol / L.

[0164] The initial measured concentration of blood glucose and the current signal I 3 can be obtained from Cglu = c*I 3 +d, and the specific equation is Cglu = 5.4986*I 3 -0.4990 where I 3 is in μA and Cglu is in mmol / L.

[0165] The hematocrit (HCT) is determined by the electrical signal S 1 obtained through the fourth voltage, and the relevant equation is: HCT*100 = k 2 +k 3 *S 1 , and the specific equation is HCT*100 = -11.07*S 1 +85.36, and the unit of S 1 is μA.

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

[0167] Using the blood glucose and uric acid electrochemical sensor of Embodiment 2 for a comparative study with the method of the fully automatic biochemical analyzer used by professionals, the following evaluation results were obtained from 200 venous blood samples.

[0168] When the blood glucose concentration < 5.55 mmol / L (100 mg / dL), 98% is within the range of ±0.83 mmol / L; when the blood glucose concentration ≥ 5.55 mmol / L (100 mg / dL), 100% is within the range of ±15%. When the uric acid concentration < 300 μmol / L, 99% is within the range of ±60 μmol / L; when the uric acid concentration ≥ 300 μmol / L, 99% is within the range of ±20%.

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

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

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

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

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

[0174] Example 3

[0175] The difference between this example and Example 1 lies in S2, the oxygen plasma cleaning process. The specific difference in this process is that the inlet gas volume of oxygen is 100 SCCM, the cleaning power is 600 W, and the cleaning duration is 3 min. The remaining steps are the same as those in Example 1.

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

[0177] Example 4

[0178] The difference between this example and Example 1 lies in S2, the oxygen plasma cleaning process. The specific difference in this process is that the inlet gas volume of oxygen is 100 SCCM, the cleaning power is 600 W, and the cleaning duration is 10 min. The remaining steps are the same as those in Example 1.

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

[0180] Comparative Example 1

[0181] The difference between Comparative Example 1 and Example 1 is that the process of S2, oxygen plasma cleaning was not carried out, and the remaining steps are the same as those in Example 1.

[0182] The blood glucose and uric acid were detected using the electrochemical sensor of Comparative Example 1, and the specific detection process was the same as that of using the blood glucose and uric acid electrochemical sensor of Example 1.

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

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

[0185] Initial blood glucose concentration and current signal I 3 can be obtained from Cglu = c*I 3 +d, and the specific equation is Cglu = 5.163*I 3 -0.3294 where I 3 is in μA and Cua is in mmol / L.

[0186] The hematocrit (HCT) is determined by the electrical signal S obtained from the fourth voltage 1 and the related equation is: HCT * 100 = k 2 +k 3 *S 1 , and the specific equation is HCT * 100 = -8.67*S 1 +73.54, where S 1 is in μA.

[0187] The equation for calculating the final blood glucose concentration value (Cglut) at room temperature after correcting the initial blood glucose concentration by the hematocrit (HCT) is Cglut = Cglu / (-0.0217 * HCT + 1.1254).

[0188] Comparative Example 2

[0189] The difference between Comparative Example 2 and Example 1 is the process of S2, oxygen plasma cleaning. The specific difference in this process is that the intake of oxygen is 100 SCCM, the cleaning power is 600 W, and the cleaning duration is 1 min. The remaining steps are the same as those in Example 1.

[0190] The blood glucose and uric acid were detected using the blood glucose and uric acid electrochemical sensor of Comparative Example 2, and the specific detection process was the same as that of using the blood glucose and uric acid electrochemical sensor of Example 1.

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

[0192] Table 3

[0193]

[0194] From the comparison between Example 1 and Comparative Example 1, it can be seen that if the oxygen plasma cleaning in step S2 is not carried out during the manufacturing process of the electrochemical sensor, the measurement of uric acid concentration cannot be performed, and the measurement accuracy of glucose concentration is slightly affected.

[0195] From the comparison between Examples 1, 3, 4 and Comparative Example 2, it can be seen that if the duration of the oxygen plasma cleaning process in step S2 during the manufacturing process of the electrochemical sensor is too short, although a certain uric acid current signal can be sensed, the sensitivity for detecting uric acid is seriously insufficient, and the R of the fitting equation 2 , intercept / slope, and coefficient of variation all reflect the low repeatability and accuracy of the uric acid concentration detection results.

[0196] It should be understood that the above specific embodiments of the present invention are only used for exemplary illustration or explanation of the principle of the present invention, and do not constitute a limitation to the present invention. Therefore, any modifications, equivalent replacements, improvements, etc. made without departing from the spirit and scope of the present invention should be included within the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundary of the appended claims, or equivalent forms of such scope and boundary.

Claims

1. 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; The first working electrode and the first reference electrode form a first electrode group, the second working electrode and the second reference electrode form 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; Features: 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 for measuring the hematocrit and uric acid concentration of a blood sample, and the second working electrode is subjected to a surface hydrophilization treatment.

2. The blood glucose and uric acid electrochemical sensor according to claim 1, characterized in that: The first working electrode, the second working electrode and the first reference electrode are carbon electrodes; The second reference electrode is a silver / silver chloride electrode; The reagent layer contains glucose responsive enzyme and electron mediator.

3. The blood glucose and uric acid electrochemical sensor according to claim 2, 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 doping materials, and the doping materials are 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.

4. The blood glucose and uric acid electrochemical sensor according to claim 1, characterized in that: The surface hydrophilization treatment is plasma cleaning or laser etching.

5. The blood glucose and uric acid electrochemical sensor according to claim 4, characterized in that: The plasma cleaning 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.

6. 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, 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.

7. 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.

8. A method for preparing a blood glucose and uric acid electrochemical sensor according to any one of claims 1 to 7, 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 a lower substrate; S2. Surface hydrophilization treatment The second working electrode of the product obtained in step S1 is subjected to surface hydrophilization treatment; S3. Making reagent layer A reagent layer is formed on the product obtained in step S2, and 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.

9. The preparation method according to claim 8, 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 is assembled with the lower substrate layer.

10. 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 7, 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, so that the electrochemical tester starts testing and starts 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 the DC voltage is less than the oxidation potential of uric acid, and measuring the current signal I1 of the interferent; (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 blood glucose; (5) applying a fourth voltage to the second working electrode and the second reference electrode, the fourth voltage being an alternating current voltage, measuring the hematocrit, and obtaining a maximum value of the alternating current electrical signal as 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 according to the current signal I3. The initial concentration of uric acid and the initial concentration of blood glucose are then corrected to obtain the final concentration of uric acid and the final concentration of blood glucose.

11. The detection method according to claim 10, characterized in that: In step (1), the trigger voltage ranges from 100 to 700 mV; 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; In step (3), 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; 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.

12. The detection method according to claim 10, characterized in that: The final concentration of uric acid and the final concentration of blood glucose in step (6) are obtained by the following method: a. Substitute I2 and I1 of the test blood sample into the linear equation I u =I2-k1*I1, get the uric acid response current intensity I u , where k1 ranges from 0.8 to 1.2; b. Then, 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 the range of k2 is 50 to 150, the range of k3 is -50 to 0, and the unit of S1 is μA; d. Get the final concentration of uric acid Cuat and the final concentration of 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 method of multiple linear regression, and c, d and k2~k7 are obtained by the method of unit linear regression.

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