An electrochemical sensor for multiple detection and its preparation method and application

Abstract

The present invention relates to an electrochemical sensor for multiple detection, a preparation method thereof, and an application thereof, and belongs to the field of biological detection technology. The present invention provides an electrochemical sensor for multiple detection, wherein the reference electrode, the working electrode, and the counter electrode of the sensor are sequentially modified with a GOD-HRP-CS-MB layer, a LOD-HRP-FMN layer, and a COD-CEH-HRP-β-CD layer from bottom to top, wherein the components of the GOD-HRP-CS-MB layer include horseradish peroxidase, methylene blue, glucose oxidase, and chitosan, the components of the LOD-HRP-FMN layer include lactate oxidase and flavin mononucleotide, and the components of the COD-CEH-HRP-β-CD layer include β-cyclodextrin, ferrocene, cholesterol oxidase, horseradish peroxidase, and cholesterol esterase. The sensor can simultaneously measure the concentrations of glucose, lactate, and cholesterol in a sample to be tested.

Description

Technical Field

[0001] The invention relates to an electrochemical sensor for multiple detection and a preparation method and application thereof, belonging to the technical field of biological detection. Background Art

[0002] Multiplex testing refers to a technology that can detect multiple targets in a single reaction. Its hallmarks are efficiency, systematicity, and cost-effectiveness. As the number of tests and indicators increases, the importance of multiplex testing is becoming increasingly prominent, particularly from a cost-effective perspective. Multiplex testing can reduce the cost of each target result. Furthermore, in the information age, multidimensional information enables more comprehensive analysis.

[0003] Blood is a special type of connective tissue, its main components being plasma, blood cells, and genetic material (chromosomes and genes). Plasma contains substances such as plasma proteins (albumin, globulin, and fibrinogen), lipoproteins, glucose, lactic acid, cholesterol, inorganic salts, oxygen, hormones, enzymes, antibodies, and cellular metabolites. Blood cells include red blood cells, white blood cells, and platelets. Physiological and pathological changes in the body often cause changes in blood composition. Therefore, blood stores information about human health, and the detection of blood components has important clinical significance for the diagnosis of diseases, including genetic disorders. If multiple detection targets in blood can be achieved, it will help significantly improve the efficiency of disease diagnosis.

[0004] Current multiplex detection technologies can be broadly categorized into three types: microfluidic structures for differential sample distribution, different enzymes and markers for sample identification, and a combination of detection methods for increased detection multiplexity. Microfluidic-based multiplex detection requires sophisticated manufacturing processes and skilled operators; multiplex detection based on different labeling methods requires demanding experimental conditions and specialized reagents; and multiplex detection instruments based on a combination of detection methods are bulky and expensive. Electrochemical methods, on the other hand, offer advantages such as high sensitivity, fast response time, low cost, and ease of integration and miniaturization. If electrochemical methods can achieve multiplex detection of multiple targets in blood, they could address the shortcomings of current multiplex detection technologies. Summary of the Invention

[0005] To solve the above problems, the present invention provides a three-electrode electrochemical sensor for multiple detection, comprising a substrate; a conductive track, a reference electrode, a working electrode, and a counter electrode are printed on the substrate; the conductive track comprises three groups, one end of each of the three groups of conductive tracks being connected to the reference electrode, the working electrode, and the counter electrode, respectively; the reference electrode, the working electrode, and the counter electrode are sequentially modified with a GOD-HRP-CS-MB layer, a LOD-HRP-FMN layer, and a COD-CEH-HRP-β-CD layer from bottom to top; the GOD-HRP-CS-MB layer comprises horseradish peroxidase, methylene blue, glucose oxidase, and chitosan; the LOD-HRP-FMN layer comprises lactate oxidase and flavin mononucleotide; and the COD-CEH-HRP-β-CD layer comprises β-cyclodextrin, ferrocene, cholesterol oxidase, horseradish peroxidase, and cholesterol esterase.

[0006] In one embodiment of the present invention, the three-electrode electrochemical sensor further comprises a hydrophilic layer; the hydrophilic layer is attached to one side of the substrate on which the conductive track, reference electrode, working electrode and counter electrode are printed, and an injection port is provided at a position of the hydrophilic layer corresponding to the working electrode.

[0007] In one embodiment of the present invention, the three-electrode electrochemical sensor further includes a blood cell filter membrane; the blood cell filter membrane is sandwiched between the substrate and the hydrophilic layer, and the blood cell filter membrane covers the sample inlet.

[0008] In one embodiment of the present invention, the GOD-HRP-CS-MB layer further comprises a bifunctional reagent; the bifunctional reagent is at least one of glutaraldehyde, thiol polyethylene glycol amino, carboxyl polyethylene glycol amino or phospholipid polyethylene glycol biotin.

[0009] In one embodiment of the present invention, the COD-CEH-HRP-β-CD layer further comprises NaOH.

[0010] The present invention also provides a method for preparing the above-mentioned three-electrode electrochemical sensor, the method comprising the following steps:

[0011] Preparation of electrodes: Using polyethylene terephthalate as a substrate, the substrate is washed with deionized water and ethanol in sequence and then dried to obtain a pretreated substrate; graphene and carbon ink are mixed to obtain GR-C ink; a defoamer and GR-C ink are mixed to obtain a mixture; after stirring the mixture, the stirred medium is separated from the ink to obtain a GR-C conductive paste; a conductive track is printed on the substrate using silver conductive paste, a working electrode is printed using GR-C conductive paste, a counter electrode is printed using GR-C conductive paste, and a reference electrode is printed using silver chloride conductive paste, and each printing is followed by drying; after the conductive track, working electrode, counter electrode, and reference electrode are printed, an insulating layer is printed on the conductive track, working electrode, counter electrode, and reference electrode of the substrate using insulating paste, and the insulating layer is dried after printing to obtain a three-electrode system electrochemical sensor to be processed;

[0012] GOD-HRP-CS-MB modification of the electrode: horseradish peroxidase is dissolved in a bifunctional reagent to obtain an HRP-bifunctional reagent solution; methylene blue is dissolved in a buffer solution to obtain an MB solution; glucose oxidase and chitosan are dissolved in a buffer solution to obtain a GOD-CS solution; the MB solution and the GOD-CS solution are mixed, incubated, and then ultrasonicated to obtain a GOD-HRP-CS-MB detection solution; the three-electrode system electrochemical sensor to be treated is immersed in the buffer solution, and the three-electrode system electrochemical sensor to be treated in the buffer solution is scanned by cyclic voltammetry until its reference electrode, working electrode, and counter electrode are stable, and the treated electrode is placed in the buffer solution. The three-electrode electrochemical sensor is taken out from the buffer solution to obtain the three-electrode electrochemical sensor A to be modified; first, a GOD-HRP-CS-MB detection solution is dropped onto the reference electrode, working electrode, and counter electrode surfaces of the three-electrode electrochemical sensor to be modified for incubation, and then an HRP-bifunctional reagent solution is dropped onto the reference electrode, working electrode, and counter electrode surfaces of the three-electrode electrochemical sensor A to be modified for drying, and finally, the reference electrode, working electrode, and counter electrode surfaces of the three-electrode electrochemical sensor A to be modified are rinsed with deionized water to achieve modification and immobilization, thereby obtaining a three-electrode electrochemical sensor modified with GOD-HRP-CS-MB;

[0013] LOD-HRP-FMN modification of the electrode: lactate oxidase and flavin mononucleotide were dissolved in a buffer solution and then sonicated to obtain a LOD-HRP-FMN detection solution; the GOD-HRP-CS-MB modified three-electrode system electrochemical sensor was immersed in the buffer solution, and the GOD-HRP-CS-MB modified three-electrode system electrochemical sensor in the buffer solution was scanned by cyclic voltammetry until its reference electrode, working electrode and counter electrode were stable, and the GOD-HRP-CS-MB modified three-electrode system electrochemical sensor was taken out from the buffer solution to obtain the electrode to be modified. Modifying a three-electrode electrochemical sensor B; first, dropping a LOD-HRP-FMN detection solution onto the reference electrode, working electrode, and counter electrode surfaces of the three-electrode electrochemical sensor B to be modified for incubation; then dropping a HRP-bifunctional reagent solution onto the reference electrode, working electrode, and counter electrode surfaces of the three-electrode electrochemical sensor B to be modified for drying; and finally, rinsing the reference electrode, working electrode, and counter electrode surfaces of the three-electrode electrochemical sensor B to be modified with deionized water to achieve modification and immobilization, thereby obtaining a three-electrode electrochemical sensor modified with LOD-HRP-FMN;

[0014] COD-CEH-HRP-β-CD modification of the electrode: β-cyclodextrin is dissolved in NaOH solution to obtain a β-CD-Fc electron mediator solution; ferrocene is added to the β-CD-Fc electron mediator solution, stirred, and then the filtrate is filtered to obtain a β-CD-Fc solution; cholesterol oxidase, horseradish peroxidase, and cholesterol esterase are dissolved in a buffer solution to obtain a COD-CEH-HRP solution; the COD-CEH-HRP solution and the β-CD-Fc solution are mixed and ultrasonicated to obtain a COD-CEH-HRP-β-CD detection solution; the LOD-HRP-FMN modified three-electrode system electrochemical sensor is immersed in a buffer solution, and the LOD-HRP-FMN modified three-electrode system electrochemical sensor in the buffer solution is scanned by cyclic voltammetry. After the reference electrode, working electrode and counter electrode of the three-electrode electrochemical sensor are stable, the LOD-HRP-FMN modified three-electrode system electrochemical sensor is removed from the buffer solution to obtain the three-electrode system electrochemical sensor C to be modified; first, the COD-CEH-HRP-β-CD detection solution is dropped onto the reference electrode, working electrode and counter electrode surfaces of the three-electrode system electrochemical sensor C to be modified for incubation, and then the HRP-bifunctional reagent solution is dropped onto the reference electrode, working electrode and counter electrode surfaces of the three-electrode system electrochemical sensor C to be modified for drying, and finally, the reference electrode, working electrode and counter electrode surfaces of the three-electrode system electrochemical sensor C to be modified are rinsed with deionized water to achieve modification and immobilization, thereby obtaining the COD-CEH-HRP-β-CD modified three-electrode system electrochemical sensor;

[0015] Preparation of a three-electrode electrochemical sensor: a hydrophilic material, a blood cell filtration membrane and a COD-CEH-HRP-β-CD modified three-electrode electrochemical sensor are bonded with an adhesive material and packaged to obtain a three-electrode electrochemical sensor.

[0016] In one embodiment of the present invention, the incubation temperature is 37° C., the pH is 7, and the incubation time is 4 hours.

[0017] The present invention also provides a method for synchronously and quantitatively detecting glucose, lactic acid and cholesterol in a sample, wherein the method uses the three-electrode system electrochemical sensor to detect the sample to be tested.

[0018] In one embodiment of the present invention, the method is: three clips of the electrochemical workstation are respectively clamped on the working electrode, reference electrode and counter electrode of the three-electrode system electrochemical sensor to achieve connection between the electrochemical workstation and the three-electrode system electrochemical sensor; after the connection is completed, the sample to be tested is first dripped into the liquid inlet of the three-electrode system electrochemical sensor, and then the current signal value generated when the glucose, lactic acid and / or cholesterol contained in the sample to be tested undergoes redox reaction is detected by differential pulse voltammetry of the electrochemical workstation; the measured current signal value is substituted into the standard curve to calculate the concentration of glucose, lactic acid and / or cholesterol in the sample to be tested.

[0019] In one embodiment of the present invention, the sample to be tested is blood.

[0020] The present invention also provides the use of the three-electrode electrochemical sensor or the method in the simultaneous quantitative detection of glucose, lactic acid and cholesterol.

[0021] The technical solution of the present invention has the following advantages:

[0022] The present invention provides a three-electrode electrochemical sensor capable of realizing multiple detections on one electrode while containing multiple enzymes and electron mediators. The three-electrode electrochemical sensor comprises a substrate; a conductive track, a reference electrode, a working electrode, and a counter electrode are printed on the substrate; the conductive track comprises three groups, one end of each of the three groups of conductive tracks being connected to the reference electrode, the working electrode, and the counter electrode, respectively; the reference electrode, the working electrode, and the counter electrode are sequentially modified with a GOD-HRP-CS-MB layer, a LOD-HRP-FMN layer, and a COD-CEH-HRP-β-CD layer from bottom to top; the GOD-HRP-CS-MB layer comprises horseradish peroxidase, methylene blue, glucose oxidase, and chitosan; the LOD-HRP-FMN layer comprises lactate oxidase and flavin mononucleotide; and the COD-CEH-HRP-β-CD layer comprises β-cyclodextrin, ferrocene, cholesterol oxidase, horseradish peroxidase, and cholesterol esterase. Electron mediators and specific oxidases can increase specificity and amplify current signals in the detection based on electrochemical methods, thereby improving detection sensitivity. However, due to the immobilization of the multi-enzyme system, mutual interference is easily generated. Therefore, when using the multi-enzyme system for multiple detection based on electrochemical methods, it is easily cross-influenced and thus causes the detection sensitivity to decrease. The accuracy is insufficient, which is not conducive to clinical diagnosis and evaluation. The three-electrode system electrochemical sensor provided by the present invention is cross-linked in different ways by electron mediators and specific oxidases of different properties, mediating dielectric pathways under different potentials, achieving the separation of the three oxidation peaks on the same detection curve, and the peak current is related to the concentration of the detected substance. Therefore, the three-electrode system electrochemical sensor provided by the present invention can simultaneously measure the concentrations of glucose (Glu), lactic acid (Lac) and cholesterol (Chol) in the sample to be tested by differential pulse voltammetry, and has high sensitivity and accuracy, which is conducive to clinical diagnosis and evaluation. In addition, the three-electrode system electrochemical sensor provided by the present invention detects the sample to be tested within 5 μL, and the result can be detected within 2 min, and the detection repeatability is good.

[0023] Specifically, the three-electrode system electrochemical sensor provided by the present invention uses a specific recognition layer containing glucose oxidase (GOD), lactate oxidase (LOD) and cholesterol oxidase (COD) to respectively recognize glucose, lactate and cholesterol, and produce hydrogen peroxide (H2O2) through redox reaction. These components contain the same flavin adenine dinucleotide (FAD) active center and are wrapped by different amino acids to form different structures with different but related properties; the three-electrode system electrochemical sensor provided by the present invention uses a multiple amplification layer containing horseradish peroxidase (HRP). Horseradish peroxidase is an oxidoreductase, and horseradish peroxidase is relatively stable, shows a wide range of substrate specificity, is easy to couple with other molecules and can be fixed on the electrode. The multiple amplification layer containing horseradish peroxidase mainly uses iron porphyrin as a prosthetic group, which is a common feature of all pathways. Horseradish peroxidase decomposes the hydrogen peroxide produced by the redox reaction into water and oxygen, which is used to reduce test interference, protect electrodes and enzymes, and amplify current signals; the present invention provides Three-electrode electrochemical sensors use a dielectric pathway layer composed of an electron mediator and a coenzyme. Methylene blue (MB), a phenothiazine dye, has an intermediate sulfur atom and a high affinity for hydroxyl groups, making it an electron mediator in biosensors. Electropolymerization improves electrochemical electrode performance, and it can be highly selectively adsorbed by chitosan (CS). Flavin mononucleotide (FMN) catalyzes redox reactions in biological systems, reacting with a suitable substrate in a two-electron process along with the free coenzyme. Following the reaction, flavin mononucleotide is reduced and the substrate is oxidized, accelerating the reaction and increasing the reaction rate. Ferrocene (Fc) has excellent redox activity and can be introduced as a ligand to increase the solubility of the catalyst flavin mononucleotide in organic solvents, significantly enhancing its catalytic activity. β-cyclodextrin has a unique cavity structure (hydrophilic on the outside and hydrophobic on the inside) that can accommodate a variety of suitable guest molecules. The combination of β-cyclodextrin and ferrocene has great potential for application in biosensors.

[0024] When the three-electrode system electrochemical sensor provided by the present invention is used to detect a test sample, when the sample to be tested enters the three-electrode system electrochemical sensor, the glucose, lactate and cholesterol in the sample to be tested are specifically recognized by glucose oxidase, lactate oxidase and cholesterol oxidase, respectively. The current signal is amplified by horseradish peroxidase, and electron transfer is promoted under the redox potential induced by the electron mediator and the coenzyme. The higher the concentration of glucose, lactate and cholesterol in the sample to be tested, the more electron transfer occurs. Under different redox potentials mediated by the electron mediator and the coenzyme, the distinguished peak current is related to the concentration of glucose, lactate and cholesterol in the sample to be tested. Therefore, the concentrations of glucose, lactate and cholesterol in the sample to be tested can be simultaneously measured by differential pulse voltammetry with high sensitivity and accuracy.

[0025] Furthermore, the three-electrode electrochemical sensor provided by the present invention is produced by printing a composite material of graphene (GR), conductive carbon paste, conductive silver paste, etc., which significantly improves the current detection signal and detection sensitivity.

[0026] Furthermore, the three-electrode electrochemical sensor provided by the present invention uses a bifunctional reagent during preparation, which makes the cross-linking more stable and the immobilization degree better.

[0027] Furthermore, the three-electrode electrochemical sensor provided by the present invention uses NaOH solution during preparation. NaOH solution helps to increase the solubility of the catalyst flavin mononucleotide in organic solvents and can cooperate with ferrocene to further enhance the catalytic effect of the catalyst flavin mononucleotide. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : Schematic diagram of the structure of the electrochemical sensor. Figure 1 In the figure, substrate 1, hydrophilic layer 2, blood cell filter membrane 3, conductive track 4, reference electrode 5, working electrode 6, counter electrode 7, injection port 8.

[0029] Figure 2 : Flow chart of the preparation of electrochemical sensors.

[0030] Figure 3 : Detection principle diagram of electrochemical sensor.

[0031] Figure 4 : Using glucose dilution as the test sample, the current signals measured by different groups of electrochemical sensors.

[0032] Figure 5 : Using lactic acid dilution as the test sample, the current signals measured by different groups of electrochemical sensors.

[0033] Figure 6 : Using cholesterol dilution as the test sample, the current signals measured by different groups of electrochemical sensors.

[0034] Figure 7 : Current signals measured by the three-electrode electrochemical sensor 1 using standard glucose solutions with concentrations of 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 15, 20, and 25 mM as test samples.

[0035] Figure 8 : A standard curve is plotted with the concentration of glucose in the standard glucose solution as the abscissa and the peak current signal measured by the three-electrode electrochemical sensor 1 as the ordinate.

[0036] Figure 9 : Current signals measured by the three-electrode electrochemical sensor 1 using standard lactic acid solutions with concentrations of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8, and 10 mM as test samples.

[0037] Figure 10 : A standard curve is plotted with the concentration of lactic acid in the standard lactic acid solution as the abscissa and the peak current signal measured by the three-electrode electrochemical sensor 1 as the ordinate.

[0038] Figure 11 : Current signals measured by the three-electrode electrochemical sensor 1 using standard cholesterol solutions with concentrations of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8, and 10 mM as test samples.

[0039] Figure 12 : A standard curve is plotted with the concentration of cholesterol in the standard cholesterol solution as the abscissa and the peak current signal measured by the three-electrode electrochemical sensor 1 as the ordinate.

[0040] Figure 13 : Current signals measured by the three-electrode electrochemical sensor 4 using standard glucose solutions with concentrations of 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 15, 20, and 25 mM as test samples.

[0041] Figure 14 : A standard curve is plotted with the concentration of glucose in the standard glucose solution as the abscissa and the peak current signal measured by the three-electrode electrochemical sensor 4 as the ordinate.

[0042] Figure 15 : Current signals measured by the three-electrode electrochemical sensor 4 using standard lactic acid solutions with concentrations of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8, and 10 mM as test samples.

[0043] Figure 16 : A standard curve is plotted with the concentration of lactic acid in the standard lactic acid solution as the abscissa and the peak current signal measured by the three-electrode electrochemical sensor 4 as the ordinate.

[0044] Figure 17 : Current signals measured by the three-electrode electrochemical sensor 4 using standard cholesterol solutions with concentrations of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8 and 10 mM as test samples.

[0045] Figure 18 : A standard curve is plotted with the concentration of cholesterol in the standard cholesterol solution as the abscissa and the peak current signal measured by the three-electrode electrochemical sensor 4 as the ordinate.

[0046] Figure 19 : Current signals measured by the three-electrode electrochemical sensor 1 using different mixed solutions as test samples (PBS buffer system + plasma system).

[0047] Figure 20 : Peak current signals measured by three-electrode electrochemical sensors 6-13.

[0048] Figure 21 : Peak current signals measured by the three-electrode electrochemical sensors 14-20.

[0049] Figure 22 : Peak current signals measured by three-electrode electrochemical sensors 21-25.

[0050] Figure 23 : Peak current signals measured by the three-electrode electrochemical sensors 26-32.

[0051] Figure 24 : Clinical sample 1 is used as the test sample and the current signal is measured by the three-electrode electrochemical sensor 1.

[0052] Figure 25 : The current signal is measured by the three-electrode electrochemical sensor 1 using the clinical sample 2 as the test sample.

[0053] Figure 26 : The current signal is measured by the three-electrode electrochemical sensor 1 using the clinical sample 3 as the test sample.

[0054] Figure 27 : Linear fitting results between glucose concentrations measured by the three-electrode electrochemical sensor 1 and the actual results using 243 clinical samples as test samples.

[0055] Figure 28 : Linear fitting results between lactate concentrations measured by the three-electrode electrochemical sensor 1 and the actual results using 243 clinical samples as test samples.

[0056] Figure 29 : Linear fitting results between cholesterol concentrations measured by three-electrode electrochemical sensor 1 and actual results using 243 clinical samples as test samples.

[0057] Figure 30: Data processing process of the current signal measured by the three-electrode electrochemical sensor 1 using different mixed solutions as test samples.

[0058] Figure 31 : A standard curve is plotted with the concentration of glucose in different mixed solutions as the abscissa and the peak current signal measured by the three-electrode electrochemical sensor 1 as the ordinate.

[0059] Figure 32 : A standard curve was plotted with the concentration of lactic acid in different mixed solutions as the abscissa and the peak current signal measured by the three-electrode electrochemical sensor 1 as the ordinate.

[0060] Figure 33 : A standard curve was plotted with the cholesterol concentration in different mixed solutions as the abscissa and the peak current signal measured by the three-electrode electrochemical sensor 1 as the ordinate. DETAILED DESCRIPTION

[0061] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0062] Example 1: A three-electrode electrochemical sensor for multiple detection and its preparation method

[0063] like Figure 1 As shown, this embodiment provides a three-electrode electrochemical sensor for multiple detection, the three-electrode electrochemical sensor comprising a substrate 1, a hydrophilic layer 2 and a blood cell filtration membrane 3; the substrate 1 is printed with a conductive track 4, a reference electrode 5, a working electrode 6 and a counter electrode 7; the conductive track 4 has three groups, one end of the three groups of conductive tracks 4 is connected to the reference electrode 5, the working electrode 6 and the counter electrode 7 respectively, and the other end is used to connect to three clips of the electrochemical workstation respectively; the reference electrode 5, the working electrode 6 and the counter electrode 7 are modified with a GOD-HRP-CS-MB layer, a LOD-HRP-FMN layer and a COD-CEH-HRP-β-CD layer from bottom to top; the G The components of the OD-HRP-CS-MB layer include horseradish peroxidase, methylene blue, glucose oxidase and chitosan; the components of the LOD-HRP-FMN layer include lactate oxidase and flavin mononucleotide; the components of the COD-CEH-HRP-β-CD layer include β-cyclodextrin, ferrocene, cholesterol oxidase, horseradish peroxidase and cholesterol esterase; the hydrophilic layer 2 is attached to the side of the substrate 1 printed with a conductive track 4, a reference electrode 5, a working electrode 6 and a counter electrode 7, and an injection port 8 is opened at a position corresponding to the working electrode 6 of the hydrophilic layer 2; the blood cell filter membrane 3 is sandwiched between the substrate 1 and the hydrophilic layer 2, and the blood cell filter membrane 3 covers the injection port 8.

[0064] The preparation method of the three-electrode electrochemical sensor comprises the following steps (the preparation process of the three-electrode electrochemical sensor is shown in Figure 2 ):

[0065] Preparation of the electrode: Polyethylene terephthalate (PET, purchased from Shanghai Julong Electronic Technology Co., Ltd.) was selected as the substrate, and the substrate was washed with deionized (DI) water and ethanol in sequence, and then dried in nitrogen to obtain a pretreated substrate; graphene and carbon ink were mixed in a volume ratio of 5:1 to obtain GR-C ink; a defoamer (the defoamer was HT-630 oil-based ink) and GR-C ink were mixed in a volume ratio of 0.06:20 to obtain a mixture; the mixture was stirred at 1200 rpm for 10 hours using a stirred medium grinder, and the stirred medium was separated from the ink using a steel sieve to obtain GR-C conductive paste; a conductive track with a length of 15 mm was printed on the substrate using a silver conductive paste (purchased from Shenzhen Seya Electronic Paste Co., Ltd.) using a fully automatic screen printing machine. A working electrode (WE) with a width of 2.5 mm and a length of 5 mm was printed using GR-C conductive paste, a counter electrode (CE) with a width of 1.5 mm and a length of 5 mm was printed using GR-C conductive paste, and a reference electrode (RE) with a width of 1.5 mm and a length of 5 mm was printed using silver chloride conductive paste (purchased from Shanghai Julong Electronic Technology Co., Ltd.). After each printing, the substrate was dried in a vacuum oven at 120°C for 1 hour. After the conductive track, working electrode, counter electrode, and reference electrode were printed, an insulating layer was printed on the conductive track, working electrode, counter electrode, and reference electrode of the substrate using an insulating paste (purchased from Shenzhen Jieyongcheng Technology Co., Ltd.) using a fully automatic screen printer. After the insulating layer was printed, the substrate was dried in a vacuum oven at 90°C for 6 hours to obtain a three-electrode electrochemical sensor to be processed.

[0066] GOD-HRP-CS-MB modification of the electrode: 5 mg of horseradish peroxidase (HRP) powder (purchased from Shanghai Lianshuo Biotechnology Co., Ltd.) was dissolved in 0.5 mL of 2.5% (m / v, g / 100 mL) glutaraldehyde (GA) aqueous solution and incubated in a water bath at 37°C for 2 h to obtain an HRP-GA solution; 3.2 mg of methylene blue (MB) powder (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) was dissolved in 10 mL of PBS buffer (purchased from Shanghai Lianshuo Biotechnology Co., Ltd.) to obtain an MB solution; 2 mg of glucose oxidase ( GOD) powder (purchased from Sigma, USA) and 0.5 mg chitosan (CS) powder (purchased from Shanghai Lianshuo Biotechnology Co., Ltd.) were dissolved in 10 mL PBS buffer (purchased from Shanghai Lianshuo Biotechnology Co., Ltd.) to obtain GOD-CS solution; MB solution and GOD-CS solution were mixed in a volume ratio of 1:1, first incubated at 37°C for 30 min, and then ultrasonicated at 37°C for 30 min to obtain GOD-HRP-CS-MB detection solution; the three-electrode system electrochemical sensor to be treated was immersed in PBS buffer and the mixture was stirred at pH 8. At 7.0, the three-electrode electrochemical sensor to be treated in the PBS buffer was scanned by cyclic voltammetry in the range of 0.6+0.2V at a scan rate of 50mV / s until its reference electrode, working electrode and counter electrode were stable, and the treated three-electrode electrochemical sensor was removed from the PBS buffer to obtain the three-electrode electrochemical sensor A to be modified; first, 2μL GOD-HRP-CS-MB detection solution was dropped onto the reference electrode, working electrode and counter electrode surfaces of the three-electrode electrochemical sensor to be modified, and incubated at 37°C for 4h, and then 1μL HRP-GA solution was dropped onto the reference electrode, working electrode and counter electrode surfaces of the three-electrode electrochemical sensor A to be modified, and dried at 25°C for 10min. Finally, the reference electrode, working electrode and counter electrode surfaces of the three-electrode electrochemical sensor A to be modified were rinsed with deionized water to achieve modification and immobilization, thereby obtaining a three-electrode electrochemical sensor modified with GOD-HRP-CS-MB;

[0067] LOD-HRP-FMN modification of the electrode: 2 mg lactate oxidase (LOD) powder (purchased from Sigma, USA) and 0.5 mg flavin mononucleotide (FMN) powder (purchased from Sigma, USA) were dissolved in 10 mL PBS buffer and ultrasonicated at 37°C for 30 min to obtain LOD-HRP-FMN detection solution; the GOD-HRP-CS-MB modified three-electrode system electrochemical sensor was immersed in PBS buffer, and the GOD-HRP-CS-MB modified three-electrode system electrochemical sensor in the PBS buffer was scanned by cyclic voltammetry at a scan rate of 50 mV / s in the range of 0.6+0.2 V at pH 7.0 until its reference electrode, working electrode and counter electrode were stable. The GOD-HRP-CS-MB modified three-electrode system electrochemical sensor was removed from the PBS buffer to obtain the three-electrode system electrochemical sensor B to be modified; first, 2 μL The LOD-HRP-FMN detection solution was dropped onto the reference electrode, working electrode, and counter electrode surfaces of the three-electrode system electrochemical sensor B to be modified, and incubated at 37°C for 4 hours. Then, 1 μL of HRP-GA solution was dropped onto the reference electrode, working electrode, and counter electrode surfaces of the three-electrode system electrochemical sensor B to be modified, and dried at 25°C for 20 minutes. Finally, the reference electrode, working electrode, and counter electrode surfaces of the three-electrode system electrochemical sensor B to be modified were rinsed with deionized water to achieve modification and immobilization, thereby obtaining a three-electrode system electrochemical sensor modified with LOD-HRP-FMN.

[0068] COD-CEH-HRP-β-CD modification of the electrode: 18.5 mg of β-cyclodextrin (β-CD) powder (purchased from Shanghai Lianshuo Biotechnology Co., Ltd.) was dissolved in 50 μL of 0.1 M NaOH (50 mg / mL) aqueous solution, first diluted to 1 mL with pure water, and then stirred at 60°C for 24 h to obtain a β-CD-Fc electron mediator solution; 3.75 mg of ferrocene (Fc) powder (purchased from Shanghai Lianshuo Biotechnology Co., Ltd.) was weighed and divided into three equal parts, and the first part of 1.25 mg of ferrocene powder was added to the β-CD-Fc electron mediator solution, stirred at 60°C for 8 h, and then the second part of 1.25 mg of ferrocene powder was added, stirred at 60°C for 8 h, and then the third part of 1.25 mg of ferrocene powder was added, stirred at 60°C for 8 h, and finally the filtrate was filtered to obtain a β-CD-Fc solution; 2 mg of cholesterol oxide was added. 10mg of COD powder (purchased from Sigma, USA), 3mg of horseradish peroxidase (HRP) powder (purchased from Shanghai Lianshuo Biotechnology Co., Ltd.) and 2mg of cholesterol esterase (CEH) powder (purchased from Sigma, USA) were dissolved in 10mL of PBS buffer to obtain COD-CEH-HRP solution; COD-CEH-HRP solution and β-CD-Fc solution were mixed in a volume ratio of 10:1, and ultrasonicated at 37°C for 30min to obtain COD-CEH-HRP-β-CD detection solution; the three-electrode electrochemical sensor modified with LOD-HRP-FMN was immersed in PBS buffer and the mixture was stirred at pH 8. At 7.0, the three-electrode electrochemical sensor modified with LOD-HRP-FMN in PBS buffer was scanned by cyclic voltammetry at a scan rate of 50 mV / s in the range of 0.6+0.2 V until its reference electrode, working electrode and counter electrode were stable. The three-electrode electrochemical sensor modified with LOD-HRP-FMN was taken out from the PBS buffer to obtain the three-electrode electrochemical sensor C to be modified. First, 2 μL The COD-CEH-HRP-β-CD detection solution was dropped onto the reference electrode, working electrode, and counter electrode surfaces of the three-electrode system electrochemical sensor C to be modified, and incubated at 37°C for 4 hours. Then, 1 μL of HRP-GA solution was dropped onto the reference electrode, working electrode, and counter electrode surfaces of the three-electrode system electrochemical sensor C to be modified, and dried at 25°C for 30 minutes. Finally, the reference electrode, working electrode, and counter electrode surfaces of the three-electrode system electrochemical sensor C to be modified were rinsed with deionized water to achieve modification and immobilization, thereby obtaining a COD-CEH-HRP-β-CD modified three-electrode system electrochemical sensor;

[0069] Preparation of a three-electrode electrochemical sensor: A hydrophilic material with an injection port (the hydrophilic material is polyethylene terephthalate treated with polar groups, purchased from Shenzhen Dimas Technology Co., Ltd.), a blood cell filter membrane with a pore size of 3 μm (the blood cell filter membrane is a square with the injection port as the inscribed circle, purchased from Shanghai Merck Company) and a COD-CEH-HRP-β-CD modified three-electrode electrochemical sensor adhesive material (the adhesive material is double-sided tape, purchased from Wuxi Smeida Technology Co., Ltd.) are bonded and packaged to obtain a three-electrode electrochemical sensor 1 (the detection principle of the three-electrode electrochemical sensor is shown in FIG1 ). Figure 3 ).

[0070] Comparative Example 1: A three-electrode electrochemical sensor for multiple detection and its preparation method

[0071] This comparative example provides a three-electrode electrochemical sensor for multiple detection. The preparation method of the three-electrode electrochemical sensor is as follows: based on Example 1, the GOD-HRP-CS-MB modification step of the electrode is retained, and the LOD-HRP-FMN modification step and the COD-CEH-HRP-β-CD modification step of the electrode are omitted to obtain a three-electrode electrochemical sensor 2.

[0072] Comparative Example 2: A three-electrode electrochemical sensor for multiple detection and its preparation method

[0073] This comparative example provides a three-electrode electrochemical sensor for multiple detection. The preparation method of the three-electrode electrochemical sensor is as follows: based on Example 1, the LOD-HRP-FMN modification step of the electrode is retained, and the GOD-HRP-CS-MB modification step and the COD-CEH-HRP-β-CD modification step of the electrode are omitted to obtain a three-electrode electrochemical sensor 3.

[0074] Comparative Example 3: A three-electrode electrochemical sensor for multiple detection and its preparation method

[0075] This comparative example provides a three-electrode electrochemical sensor for multiple detection. The preparation method of the three-electrode electrochemical sensor is as follows: based on Example 1, the COD-CEH-HRP-β-CD modification step of the electrode is retained, and the GOD-HRP-CS-MB modification step and the LOD-HRP-FMN modification step of the electrode are omitted, and the dropwise addition and drying processes of the HRP-GA solution in the COD-CEH-HRP-β-CD modification step of the electrode are removed to obtain a three-electrode electrochemical sensor 4.

[0076] Example 2: A method for simultaneous quantitative detection of glucose, lactate, and cholesterol in a sample

[0077] This embodiment provides a method for simultaneous quantitative detection of glucose, lactate, and cholesterol in a sample, using the three-electrode electrochemical sensor described in Example 1, comprising the following steps:

[0078] Step 1: First, place the three-electrode electrochemical sensor on a stable experimental table, and then clamp the three clips of the CHI660E electrochemical workstation (purchased from Shanghai Chenhua Technology Co., Ltd.) on the working electrode, reference electrode, and counter electrode of the three-electrode electrochemical sensor to achieve the connection between the electrochemical workstation and the three-electrode electrochemical sensor;

[0079] Step 2: After the connection is completed, the sample to be tested is added to the liquid inlet of the three-electrode system electrochemical sensor. After the sample to be tested enters the liquid inlet, the blood cells are first removed by the blood cell filter membrane, and the remaining plasma enters the reaction area and fully contacts with the GOD-HRP-CS-MB layer, LOD-HRP-FMN layer and / or COD-CEH-HRP-β-CD layer modified on the working electrode, reference electrode and counter electrode. If the sample to be tested contains glucose, lactic acid and / or cholesterol, the glucose, lactic acid and / or cholesterol contained in the sample to be tested will react with the GOD -Specific enzymes (i.e., glucose oxidase, lactate oxidase, and / or cholesterol oxidase) contained in the HRP-CS-MB layer, the LOD-HRP-FMN layer, and / or the COD-CEH-HRP-β-CD layer recognize each other and undergo redox reactions. At this time, differential pulse voltammetry can be used on an electrochemical workstation to detect the current signal values generated when the redox reactions of glucose, lactate, and / or cholesterol contained in the test sample occur at different potentials (glucose: -0.41 V, lactate: -0.19 V, cholesterol: 0.21 V);

[0080] Step 3: Substitute the measured current signal values (glucose i1, lactate i2, cholesterol i3) into the standard curve to calculate the concentration of glucose, lactate and / or cholesterol in the sample to be tested; wherein the standard curve is as follows:

[0081] (Glu, Lac, Chol):

[0082] In the standard curve, n is a unit vector and has nothing to do with the detection.

[0083] Experimental Example 1: Performance Experiment of Three-Electrode Electrochemical Sensor

[0084] This experimental example provides a performance test of a three-electrode electrochemical sensor. The experimental process is as follows:

[0085] Experiment 1: Glucose was diluted to a concentration of 5 mM using PBS buffer to obtain a glucose dilution solution; lactic acid was diluted to a concentration of 1 mM using PBS buffer to obtain a lactic acid dilution solution; cholesterol was diluted to a concentration of 4 mM using PBS buffer to obtain a cholesterol dilution solution; the three-electrode electrochemical sensor to be treated (Unmodified), the three-electrode electrochemical sensor modified only with GOD solution (2 mg glucose oxidase dissolved in 10 mL PBS buffer) (GODmodified), the three-electrode electrochemical sensor modified only with GOD solution (2 mg glucose oxidase dissolved in 10 mL PBS buffer) and HRP-GA solution (GOD+HRP modified), and the three-electrode electrochemical sensor modified only with GOD-HRP-CS-MB detection solution (GOD+CS_MB modified) were used as controls, and the three-electrode electrochemical sensor 2 of Comparative Example 1 (GOD+HRP+CS_MB The method of Example 2 was modified. Glucose was quantitatively detected using glucose dilution as the test sample. Differential pulse voltammetry (DPV) was used on a CHI660E electrochemical workstation (purchased from Shanghai Chenhua Technology Co., Ltd.) at -0.29 V to collect test data. The peak current signals measured by the three-electrode electrochemical sensors of different groups were compared. The comparison results are shown in FIG. Figure 4 ; The three-electrode electrochemical sensor to be treated (Unmodified), the three-electrode electrochemical sensor modified only with LOD solution (2 mg lactate oxidase dissolved in 10 mL PBS buffer) (LOD modified), the three-electrode electrochemical sensor modified only with LOD solution (2 mg lactate oxidase dissolved in 10 mL PBS buffer) and HRP-GA solution (LOD+HRPmodified), and the three-electrode electrochemical sensor modified only with LOD-HRP-FMN detection solution (LOD+FMN modified) were used as controls. The three-electrode electrochemical sensor 3 (LOD+HRP+FMN modified) of Comparative Example 2 was used. Referring to the method of Example 2, quantitative detection of lactic acid was performed using lactic acid dilution as the sample to be tested. The test data was collected at 0.09 V using differential pulse voltammetry (DPV) of a CHI660E electrochemical workstation (purchased from Shanghai Chenhua Technology Co., Ltd.). The peak current signals measured by the three-electrode electrochemical sensors of different groups were compared. The comparison results are shown in FIG. Figure 5The three-electrode electrochemical sensor to be treated (Unmodified), the three-electrode electrochemical sensor modified with COD-CEH solution (2 mg cholesterol oxidase and 2 mg cholesterol esterase dissolved in 10 mL PBS buffer) ((COD_CEH)modified), the three-electrode electrochemical sensor modified with COD-CEH-HRP solution ((COD_CEH)+HRP modified), and the three-electrode electrochemical sensor modified with COD-CEH-β-CD solution (COD-CEH solution and β-CD-Fc solution mixed in a volume ratio of 10:1) (GOD+CS_MB The three-electrode electrochemical sensor 4 ((COD_CEH)+HRP+(β-CD-Fc)modified) of Comparative Example 3 was used as a control. Referring to steps 1 and 2 of Example 2, cholesterol dilution was used as the test sample for detection. The differential pulse voltammetry (DPV) of the CHI660E electrochemical workstation (purchased from Shanghai Chenhua Technology Co., Ltd.) was used to collect test data at 0.30 V. The peak current signal sizes measured by the three-electrode electrochemical sensors of different groups were compared. The comparison results are shown in FIG. Figure 6 .

[0086] Experiment 2: Glucose was diluted to a standard glucose solution with a concentration of 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 15, 20 and 25 mM using PBS buffer; lactic acid was diluted to a standard lactic acid solution with a concentration of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8 and 10 mM using PBS buffer; cholesterol was diluted to a standard cholesterol solution with a concentration of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8 and 10 mM using PBS buffer; the three-electrode electrochemical sensors 1 to 4 of Example 1 and Comparative Examples 1 to 3 were used, with reference to steps 1 and 2 of Example 2, and the concentrations of 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8 and 10 mM were used. Standard glucose solutions at concentrations of 5, 0.5, 1, 2.5, 5, 10, 15, 20, and 25 mM, standard lactic acid solutions at concentrations of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8, and 10 mM, and standard cholesterol solutions at concentrations of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8, and 10 mM were used as test samples for detection, and differential pulse voltammetry (DPV) was used on a CHI660E electrochemical workstation (purchased from Shanghai Chenhua Technology Co., Ltd.) at different potentials (glucose: -0.29 V, lactic acid: 0.09 V, cholesterol: 0.30 V) for test data acquisition. Origin2019 software was used for data analysis and processing and standard curves were drawn. The detection and drawing results are shown in Figure 4. Figures 7 to 18 .

[0087] Experiment 3: Lactic acid and cholesterol were added to standard glucose solutions with concentrations of 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 15, 20 and 25 mM to a final concentration of 0.01 mM and 0.01 mM, respectively, to obtain a low-low concentration three-target mixed solution with a gradient glucose concentration; Lactic acid and cholesterol were added to standard glucose solutions with concentrations of 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 15, 20 and 25 mM to a final concentration of 0.01 mM and 10 mM, respectively, to obtain a low-high concentration three-target mixed solution with a gradient glucose concentration; Lactic acid and cholesterol were added to the standard glucose solutions with concentrations of 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 15, 20 and 25 mM to a final concentration of 10 mM and 0.01 mM, respectively, to obtain a high-low concentration three-target mixed solution with gradient glucose concentrations; lactic acid and cholesterol were added to the standard glucose solutions with concentrations of 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 15, 20 and 25 mM to a final concentration of 10 mM and 10 mM, respectively, to obtain a high-high concentration three-target mixed solution with gradient glucose concentrations; lactic acid was added to the standard glucose solution with concentrations of 15 mM to a final concentration of 0 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8 and 10 mM, and cholesterol was added to the final concentrations of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8 and 10 mM, respectively, to obtain a three-target mixed solution with a fixed glucose concentration; glucose and cholesterol were added to the standard lactic acid solutions with concentrations of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8 and 10 mM, respectively, to obtain a three-target mixed solution with a gradient lactic acid concentration; at a concentration of 0. Glucose and cholesterol were added to the standard lactic acid solutions with the concentrations of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8 and 10 mM, respectively, to reach the final concentrations of 0.01 mM and 10 mM, respectively, to obtain the low and high concentration three-target mixed solutions with gradient lactic acid concentrations; glucose and cholesterol were added to the standard lactic acid solutions with the concentrations of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8 and 10 mM, respectively, to reach the final concentrations of 25 mM and 0.01 mM, respectively, to obtain the high and low concentration three-target mixed solutions with gradient lactic acid concentrations;Glucose and cholesterol were added to the standard lactic acid solutions with a concentration of 5, 1, 2, 4, 6, 8 and 10 mM, respectively, to a final concentration of 25 mM and 10 mM, respectively, to obtain a high-high concentration three-target mixed solution with a gradient lactic acid concentration; glucose was added to the standard lactic acid solution with a concentration of 2 mM, respectively, to a final concentration of 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 15, 20 and 25 mM, respectively, and cholesterol was added to a final concentration of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1 , 2, 4, 6, 8 and 10mM to obtain a three-target mixed solution with a fixed lactic acid concentration; glucose and lactic acid were added to the standard cholesterol solutions with concentrations of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8 and 10mM to obtain a three-target mixed solution with a gradient cholesterol concentration; in the standard cholesterol solutions with concentrations of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8 and 10mM, glucose and lactic acid were added to the standard cholesterol solutions with concentrations of 0.01mM and 0.01 ... Glucose and lactic acid were added to the standard cholesterol solution to the final concentrations of 0.01 mM and 10 mM, respectively, to obtain a three-target mixed solution with a gradient lactic acid concentration of low and high concentrations; glucose and lactic acid were added to the standard cholesterol solution with the concentrations of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8 and 10 mM, respectively, to obtain a three-target mixed solution with a gradient lactic acid concentration of high and low concentrations; Glucose and lactic acid were added to the standard cholesterol solution with a concentration of 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 15, 20 and 25 mM, respectively, and lactic acid was added to the standard cholesterol solution with a concentration of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 15, 20 and 25 mM, respectively, to obtain a high-high concentration three-target mixed solution with a gradient lactic acid concentration; glucose was added to the standard cholesterol solution with a concentration of 4 mM, to obtain a final concentration of 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 15, 20 and 25 mM, respectively, and lactic acid was added to the standard cholesterol solution with a concentration of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8 and 10 mM to obtain a three-target mixed solution with a fixed cholesterol concentration; using the three-electrode system electrochemical sensor 1 of Example 1, referring to steps 1 and 2 of Example 2, respectively, a low-low concentration three-target mixed solution of gradient glucose concentration, a low-high concentration three-target mixed solution of gradient glucose concentration, a high-low concentration three-target mixed solution of gradient glucose concentration, a high-high concentration three-target mixed solution of gradient glucose concentration, a variable concentration three-target mixed solution of fixed glucose concentration, a low-low concentration three-target mixed solution of gradient lactic acid concentration, a low-high concentration three-target mixed solution of gradient lactic acid concentration, a high-low concentration three-target mixed solution of gradient lactic acid concentration, a variable concentration three-target mixed solution of fixed lactic acid concentration Mixed solutions, three-target mixed solutions with gradient cholesterol concentrations of low and low concentrations, three-target mixed solutions with gradient cholesterol concentrations of low and high concentrations, three-target mixed solutions with gradient cholesterol concentrations of high and low concentrations, three-target mixed solutions with gradient cholesterol concentrations of high and high concentrations, three-target mixed solutions with gradient cholesterol concentrations of high and high concentrations, and three-target mixed solutions with fixed cholesterol concentrations of varying concentrations were used as test samples. Differential pulse voltammetry (DPV) was used on a CHI660E electrochemical workstation (purchased from Shanghai Chenhua Technology Co., Ltd.) to collect test data at different potentials (glucose: -0.41 V, lactate: -0.19 V, cholesterol: 0.21 V). Data analysis and processing were performed using Origin 2019 software. The test results in the PBS buffer system are shown in [ ]. Figure 19 ; On the basis of the above experiments, the solvents of the low-low concentration three-target mixed solution of gradient glucose concentration, the low-high concentration three-target mixed solution of gradient glucose concentration, the high-low concentration three-target mixed solution of gradient glucose concentration, the high-high concentration three-target mixed solution of gradient glucose concentration, the variable concentration three-target mixed solution of fixed glucose concentration, the low-low concentration three-target mixed solution of gradient lactic acid concentration, the low-high concentration three-target mixed solution of gradient lactic acid concentration, the high-low concentration three-target mixed solution of gradient lactic acid concentration, the high-high concentration three-target mixed solution of gradient lactic acid concentration, the variable concentration three-target mixed solution of fixed lactic acid concentration, the low-low concentration three-target mixed solution of gradient cholesterol concentration, the low-high concentration three-target mixed solution of gradient cholesterol concentration, the high-low concentration three-target mixed solution of gradient cholesterol concentration, the high-high concentration three-target mixed solution of gradient cholesterol concentration, and the variable concentration three-target mixed solution of fixed cholesterol concentration were replaced by PBS buffer with plasma (sourced from Suzhou Science and Technology City Hospital Affiliated to Nanjing Medical University), and the above experiments were repeated to obtain the test results under the plasma system. The test results under the plasma system are shown in FIG. Figure 19 .

[0088] Experiment 4: Based on Example 1, the pH of PBS buffer (pH 7.0) was replaced with 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, and 7.8, respectively, to obtain three-electrode electrochemical sensors 6 to 13; Based on Example 1, the incubation temperature (37°C) was replaced with 4°C, 25°C, 30°C, 35°C, 37°C, 40°C, and 45°C, respectively, to obtain three-electrode electrochemical sensors 14 to 20; Based on Example 1, the GOD-HRP-CS- The modified dosages (2 μL) of MB detection solution, LOD-HRP-FMN detection solution and COD-CEH-HRP-β-CD detection solution were replaced with 0.5 μL, 1 μL, 1.5 μL, 2 μL and 3 μL, respectively, to obtain three-electrode electrochemical sensors 21 to 25; based on Example 1, the incubation time (4 h) was replaced with 0.5 h, 1 h, 2 h, 4 h, 6 h, 12 h and 24 h, respectively, to obtain three-electrode electrochemical sensors 26 to 32; using three Electrode system electrochemical sensors 6 to 32, referring to steps 1 and 2 of Example 2, are prepared with standard glucose solutions having concentrations of 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 15, 20, and 25 mM, standard lactic acid solutions having concentrations of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8, and 10 mM, and standard glucose solutions having concentrations of 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8 and 10 mM standard cholesterol solutions were used as test samples, and differential pulse voltammetry (DPV) was used on a CHI660E electrochemical workstation (purchased from Shanghai Chenhua Technology Co., Ltd.) to collect test data at different potentials (glucose: -0.41 V, lactate: -0.19 V, cholesterol: 0.21 V). The peak current signal sizes measured by the three-electrode system electrochemical sensors 6 to 32 were compared. The comparison results are shown in Figure 5. Figures 20-23 .

[0089] Experiment 5: Clinical samples 1 to 3 (from Suzhou Science and Technology City Hospital affiliated to Nanjing Medical University) were tested using the method of Example 2. The test results are shown in Figures 24-26 .

[0090] Experiment 6: 243 clinical samples (from Suzhou Science and Technology City Hospital affiliated to Nanjing Medical University) were tested using the method of Example 2, and a linear fit was performed between the test results and the actual results of the clinical samples (the actual results of the clinical samples were measured by Siemens automatic biochemical analyzer). The fitting results are shown in Figures 27-29 .

[0091] Depend on Figures 4-6It can be seen that in the single modification systems of the GOD-HRP-CS-MB layer, the LOD-HRP-FMN layer, and the COD-CEH-HRP-β-CD layer (i.e., Comparative Examples 1, 2, and 3), Glu, Lac, and Chol are specifically recognized by the oxidase, respectively, and the current signal is amplified by HRP, promoting electron transfer under the redox potential mediated by the electron mediator and the coenzyme. Moreover, the higher the sample concentration, the more electron transfer occurs in the redox reaction. Under the redox potential mediated by the electron mediator and the coenzyme, the peak current is related to the sample concentration.

[0092] Depend on Figures 7 to 18 It can be seen that in the mixed modification system of GOD-HRP-CS-MB layer, LOD-HRP-FMN layer and COD-CEH-HRP-β-CD layer (ie, Example 1), Glu, Lac and Chol can be detected simultaneously and the detection results are almost the same as those of the single modification system of GOD-HRP-CS-MB layer, LOD-HRP-FMN layer and COD-CEH-HRP-β-CD layer (ie, Comparative Examples 1, 2, 3), indicating that it overcomes the problem of reduced detection sensitivity and insufficient accuracy caused by cross-influence when using a multi-enzyme system for multiple detection based on electrochemical methods. In addition, the peak current at different potentials is related to the corresponding sample concentration.

[0093] Depend on Figure 19 It can be seen that in the detection of a single substance, the peak current is linearly correlated with the substance concentration. Therefore, the multivariate linear regression method was used to analyze the metabolite peak current detection data under PBS and plasma backgrounds, and the linear regression coefficient matrix [P1] (150 groups of observations) and the linear regression coefficient matrix [P2] (1884 groups of observations) of all gradient tests under PBS background were obtained, as well as the linear regression coefficient matrix [P1'] (150 groups of observations) of some gradient tests under plasma background. Among them, the concentrations of [P1] and [P1'] correspond one to one (see the data processing process for details). Figure 30 ). Use the processed data to draw a standard curve (see the results of the drawing). Figures 31-33 ). According to the standard curve, in each gradient test, the three metabolite concentrations have different weight relationships with the three peak currents. Among them, in the PBS background, the correlation coefficient between Glu and the peak current value i1 at a potential of -0.41V is 4.202, which is the largest correlation, with a standard error of 0.013 and a fitting correlation coefficient of R 2 =0.999; the correlation coefficient between Lac and the peak current value i2 is 2.012 at the potential of -0.19V, which is the largest correlation, with a standard error of 0.035 and a fitting correlation coefficient of R 2=0.979; the correlation coefficient between Chol and the peak current value i3 at a potential of 0.21 V is 1.673, which is the largest correlation, with a standard error of 0.019 and a fitting correlation coefficient of R 2 =0.990. In summary, when the sample contains Glu, Lac, and Chol simultaneously, the multilayer modification system of the GOD-HRP-CS-MB layer, the LOD-HRP-FMN layer, and the COD-CEH-HRP-β-CD layer (i.e., Example 1) can achieve accurate detection under combined gradients of high and low concentrations of different substances, exhibiting good sensitivity and anti-interference properties, and can be used for the simultaneous detection of the three metabolites of Glu, Lac, and Chol. The resulting standard curve is as follows:

[0094] (Glu, Lac, Chol):

[0095] In the standard curve, n is a unit vector and has nothing to do with the detection.

[0096] Depend on Figures 20-23 It can be seen that the peak current signal measured by the three-electrode electrochemical sensor is the largest when incubated at 37°C and pH 7 for 4 hours. It can be seen that incubating at 37°C and pH 7 for 4 hours is the optimal modification condition for the three-electrode electrochemical sensor.

[0097] Depend on Figures 24-29 It can be seen that the linear correlation between the multilayer modification system (i.e., embodiment 1) of GOD-HRP-CS-MB layer, LOD-HRP-FMN layer and COD-CEH-HRP-β-CD layer and Siemens automatic biochemical analyzer for detecting Glu, Chol and Lac is 0.985, 0.980 and 0.984 respectively, which shows that the system has good consistency with clinical instruments. The linear range of Glu, Chol and Lac measured based on DPV electrochemical method using the multilayer modification system (i.e., embodiment 1) of GOD-HRP-CS-MB layer, LOD-HRP-FMN layer and COD-CEH-HRP-β-CD layer is 0.01~25mM, 0.01~10mM and 0.01~10mM respectively, which shows that the system has higher sensitivity and wider detection range. Moreover, the multilayer modification system using a GOD-HRP-CS-MB layer, a LOD-HRP-FMN layer, and a COD-CEH-HRP-β-CD layer (i.e., Example 1) was used to determine the concentrations of Glu, Chol, and Lac based on the DPV electrochemical method, which was calculated from three peak current values in one test and has high clinical application value.

[0098] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A three-electrode electrochemical sensor for multiple detection, characterized in that: The three-electrode electrochemical sensor includes a substrate; a conductive track, a reference electrode, a working electrode, and a counter electrode are printed on the substrate; the conductive track has three groups, and one end of the three groups of conductive tracks is connected to the reference electrode, the working electrode, and the counter electrode, respectively; the reference electrode, the working electrode, and the counter electrode are sequentially modified with a GOD-HRP-CS-MB layer, a LOD-HRP-FMN layer, and a COD-CEH-HRP-β-CD-Fc layer from bottom to top; the GOD-HRP-CS-MB layer contains horseradish peroxidase, methylene blue, glucose oxidase, and chitosan; the LOD-HRP-FMN layer contains horseradish peroxidase, lactate oxidase, and flavin mononucleotide; and the COD-CEH-HRP-β-CD-Fc layer contains β-cyclodextrin, ferrocene, cholesterol oxidase, horseradish peroxidase, and cholesterol esterase.

2. The three-electrode electrochemical sensor according to claim 1, wherein: The three-electrode electrochemical sensor further comprises a hydrophilic layer, which is attached to the side of the substrate printed with the conductive track, reference electrode, working electrode and counter electrode, and an injection port is provided at a position of the hydrophilic layer corresponding to the working electrode.

3. The three-electrode electrochemical sensor according to claim 2, wherein: The three-electrode electrochemical sensor further comprises a blood cell filter membrane; the blood cell filter membrane is sandwiched between the substrate and the hydrophilic layer, and the blood cell filter membrane covers the sample inlet.

4. The three-electrode electrochemical sensor according to claim 1, wherein: The GOD-HRP-CS-MB layer further comprises a bifunctional reagent; the bifunctional reagent is at least one of glutaraldehyde, thiol polyethylene glycol amino, carboxyl polyethylene glycol amino or phospholipid polyethylene glycol biotin.

5. The three-electrode electrochemical sensor according to any one of claims 1 to 4, characterized in that: The composition of the COD-CEH-HRP-β-CD-Fc layer further includes NaOH.

6. A method for preparing the three-electrode electrochemical sensor according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: Preparation of electrodes: Using polyethylene terephthalate as a substrate, the substrate is washed with deionized water and ethanol in sequence and then dried to obtain a pretreated substrate; graphene and carbon ink are mixed to obtain GR-C ink; a defoamer and GR-C ink are mixed to obtain a mixture; after stirring the mixture, the stirred medium is separated from the ink to obtain a GR-C conductive paste; a conductive track is printed on the substrate using silver conductive paste, a working electrode is printed using GR-C conductive paste, a counter electrode is printed using GR-C conductive paste, and a reference electrode is printed using silver chloride conductive paste, and each printing is followed by drying; after the conductive track, working electrode, counter electrode, and reference electrode are printed, an insulating layer is printed on the conductive track, working electrode, counter electrode, and reference electrode of the substrate using insulating paste, and the insulating layer is dried after printing to obtain a three-electrode system electrochemical sensor to be processed; GOD-HRP-CS-MB modification of the electrode: horseradish peroxidase was dissolved in a bifunctional reagent to obtain an HRP-bifunctional reagent solution; methylene blue was dissolved in a buffer solution to obtain an MB solution; Glucose oxidase and chitosan are dissolved in a buffer solution to obtain a GOD-CS solution; the MB solution and the GOD-CS solution are mixed, incubated, and then ultrasonicated to obtain a GOD-CS-MB detection solution; The three-electrode electrochemical sensor to be treated is immersed in a buffer solution, and the three-electrode electrochemical sensor to be treated in the buffer solution is scanned by cyclic voltammetry until its reference electrode, working electrode and counter electrode are stable, and the treated three-electrode electrochemical sensor is taken out from the buffer solution to obtain a three-electrode electrochemical sensor A to be modified; first, a GOD-CS-MB detection solution is dropped onto the reference electrode, working electrode and counter electrode surfaces of the three-electrode electrochemical sensor to be modified for incubation, and then an HRP-bifunctional reagent solution is dropped onto the reference electrode, working electrode and counter electrode surfaces of the three-electrode electrochemical sensor A to be modified for drying, and finally, the reference electrode, working electrode and counter electrode surfaces of the three-electrode electrochemical sensor A to be modified are rinsed with deionized water to achieve modification and immobilization, thereby obtaining a three-electrode electrochemical sensor modified with GOD-HRP-CS-MB; LOD-HRP-FMN modification of the electrode: lactate oxidase and flavin mononucleotide were dissolved in a buffer solution and then sonicated to obtain a LOD-FMN detection solution; the GOD-HRP-CS-MB modified three-electrode system electrochemical sensor was immersed in the buffer solution, and the GOD-HRP-CS-MB modified three-electrode system electrochemical sensor in the buffer solution was scanned by cyclic voltammetry until its reference electrode, working electrode and counter electrode were stable, and the GOD-HRP-CS-MB modified three-electrode system electrochemical sensor was taken out from the buffer solution to obtain the electrode to be modified. Modifying a three-electrode electrochemical sensor B; first, dropping a LOD-FMN detection solution onto the reference electrode, working electrode, and counter electrode surfaces of the three-electrode electrochemical sensor B to be modified for incubation; then dropping a HRP-bifunctional reagent solution onto the reference electrode, working electrode, and counter electrode surfaces of the three-electrode electrochemical sensor B to be modified for drying; and finally, rinsing the reference electrode, working electrode, and counter electrode surfaces of the three-electrode electrochemical sensor B to be modified with deionized water to achieve modification and immobilization, thereby obtaining a three-electrode electrochemical sensor modified with LOD-HRP-FMN; COD-CEH-HRP-β-CD-Fc modification of the electrode: β-cyclodextrin is dissolved in NaOH solution to obtain a β-CD electron mediator solution; ferrocene is added to the β-CD electron mediator solution, stirred, and then the filtrate is filtered to obtain a β-CD-Fc solution; cholesterol oxidase, horseradish peroxidase, and cholesterol esterase are dissolved in a buffer solution to obtain a COD-CEH-HRP solution; the COD-CEH-HRP solution and the β-CD-Fc solution are mixed and ultrasonicated to obtain a COD-CEH-HRP-β-CD-Fc detection solution; the LOD-HRP-FMN modified three-electrode system electrochemical sensor is immersed in the buffer solution, and the LOD-HRP-FMN modified three-electrode system electrochemical sensor in the buffer solution is scanned by cyclic voltammetry. After the reference electrode, working electrode and counter electrode are stabilized, the LOD-HRP-FMN modified three-electrode system electrochemical sensor is removed from the buffer solution to obtain the three-electrode system electrochemical sensor C to be modified; first, a COD-CEH-HRP-β-CD-Fc detection solution is dropped onto the reference electrode, working electrode and counter electrode surfaces of the three-electrode system electrochemical sensor C to be modified for incubation, and then an HRP-bifunctional reagent solution is dropped onto the reference electrode, working electrode and counter electrode surfaces of the three-electrode system electrochemical sensor C to be modified for drying, and finally, the reference electrode, working electrode and counter electrode surfaces of the three-electrode system electrochemical sensor C to be modified are rinsed with deionized water to achieve modification and immobilization, thereby obtaining the COD-CEH-HRP-β-CD-Fc modified three-electrode system electrochemical sensor; Preparation of a three-electrode electrochemical sensor: a hydrophilic material, a blood cell filtration membrane, and a COD-CEH-HRP-β-CD-Fc modified three-electrode electrochemical sensor are bonded and encapsulated with an adhesive material to obtain a three-electrode electrochemical sensor; The bifunctional reagent is at least one of glutaraldehyde, thiol polyethylene glycol amino, carboxyl polyethylene glycol amino or phospholipid polyethylene glycol biotin.

7. The method according to claim 6, wherein The incubation temperature is 37° C., the pH is 7, and the incubation time is 4 hours.

8. A method for simultaneous quantitative detection of glucose, lactate and cholesterol in a sample, characterized in that: The method uses the three-electrode electrochemical sensor according to any one of claims 1 to 5 to detect the sample to be tested.

9. The method according to claim 8, wherein The method comprises: respectively clamping three clips of the electrochemical workstation on the working electrode, the reference electrode and the counter electrode of the three-electrode electrochemical sensor to achieve connection between the electrochemical workstation and the three-electrode electrochemical sensor; After the connection is completed, the sample to be tested is first added to the liquid inlet of the three-electrode system electrochemical sensor, and then the current signal value generated by the redox reaction of glucose, lactic acid and cholesterol contained in the sample to be tested is detected by differential pulse voltammetry of the electrochemical workstation; the measured current signal value is substituted into the standard curve to calculate the concentration of glucose, lactic acid and cholesterol in the sample to be tested.

10. Use of the three-electrode electrochemical sensor according to any one of claims 1 to 5 for simultaneous quantitative detection of glucose, lactate and cholesterol in a sample.

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