Electrode, test paper and preparation method thereof for detecting creatinine by electrochemical method

Abstract

The present invention provides an electrode for electrochemical detection of creatinine, including an electrode material base layer; the outer surface of the electrode material base layer is covered with an electron mediator enhancement layer; the outer surface of the electron mediator enhancement layer is fixed with an enzyme composition that can react with creatinine to generate hydrogen peroxide; the electrode material base layer is composed of carbon paste or graphite containing Prussian blue; the enzyme composition is composed of creatinase, creatinase and sarcosine oxidase in a ratio of 3.75-6.25:1.25-2:0.75-1.25. The present invention also provides a test paper and a biosensor based on the electrode. The present invention also provides a preparation method for the electrode and the test paper, and a method for detecting creatinine levels using the biosensor. The test paper of the present invention can be used in a variety of portable and fast small electrochemical detection equipment, and can achieve rapid and convenient clinical detection or home monitoring of blood and urine creatinine.

Description

Technical Field

[0001] The invention relates to a material for analyzing and testing clinical samples, and in particular to an electrode and a test paper for electrochemically detecting creatinine, and a preparation method thereof. Background Art

[0002] Currently, the number of people suffering from kidney diseases, especially chronic kidney disease (CKD), is increasing worldwide. In 2017, the Global Burden of Disease, Trauma and Risk Factors (GBD) study estimated that the global CKD prevalence was 9.1%. Between 1990 and 2017, the CKD mortality rate in all age groups increased by 41.5% worldwide. With high morbidity and mortality, CKD has brought a huge burden to the global economy. Therefore, it is very necessary to monitor renal function regularly to avoid renal damage to an irreversible degree.

[0003] Creatinine is the most commonly used renal function monitoring indicator in clinical work. Creatinine (2-amino-1-methyl-5H-imidazole-4-one) is a chemical waste metabolite of creatine phosphate produced by muscle and protein metabolism. In the human body, endogenous creatinine is produced by muscle tissue, and exogenous creatinine mainly comes from the metabolism of meat in the diet. Under normal circumstances, the amount of creatinine produced in our daily life is relatively stable, and almost all creatinine is filtered through the kidneys and released into the urine. Therefore, serum creatinine (SCr) levels will remain in a stable range of about 44-106μmol / L. When renal function is impaired and the ability to excrete creatinine is impaired, the creatinine concentration will exceed the upper limit of the physiological range. In the early stages, patients with kidney disease will not feel uncomfortable, but abnormal SCr levels will attract the attention of doctors. Therefore, SCr levels are generally considered to be an important biomarker for detecting renal insufficiency.

[0004] In the prior art, there are a series of methods for quantitative determination of creatinine. The traditional methods mainly include Jaffe reaction method and enzyme method. Jaffe reaction is fast and economical, but it is easily affected by interfering chromogens such as ketones and glucose in serum, making it sometimes inaccurate. Therefore, the enzyme method is considered to be a better method. The use of biological enzymes can indeed greatly improve the accuracy and efficiency of detection. Although the enzyme method imposes a heavier economic burden on patients than the Jaffe reaction, the higher specificity makes the use of the enzyme method to detect creatinine gradually become the mainstream means of clinical testing in hospitals. However, for patients who are inconvenient to go to the hospital but need to check their renal function regularly, the two existing creatinine detection methods are not easy to implement.

[0005] When it comes to daily monitoring of health conditions, POCT-based blood glucose meters have undoubtedly left the most profound impression. Unfortunately, no smart device like blood glucose meters has been widely accepted worldwide. Biosensors are an electrochemical detection technology that converts the concentration of biological materials into electrical signals. Compared with traditional measurement methods, biosensors have the advantages of short measurement time, simple operation, high sensitivity and good selectivity. Therefore, the use of biosensors to detect creatinine by electrochemical method is considered to be a promising alternative method for creatinine detection.

[0006] There are some disclosures in the prior art about the electrochemical detection method of creatinine and the materials used therein. For example, the I-SATA optical and electrical detection device of Abbott Care Point Company in the United States includes components for performing electrochemical detection of creatinine, but the device and components are complex in structure and expensive, and are not suitable for ordinary patients to use, and cannot meet the needs of fast and convenient home testing. In addition, some existing products have the disadvantages of poor anti-interference ability, and the accuracy and sensitivity of the test results are not high.

[0007] Due to the complex composition of blood, the electrochemical detection results of creatinine are easily interfered by other non-target substances in the blood. In order to reduce the detection error caused by this interference, many test materials in the prior art are equipped with blood filtration devices, the purpose of which is to filter out non-target substances in the blood that are prone to interference before the electrochemical reaction occurs. However, it is found in practice that the blood filtration device set in the prior art not only makes the sensor expensive and complicated in process, but also still cannot effectively avoid the interference of creatine in the blood on creatinine detection.

[0008] Therefore, it is necessary to provide an electrochemical electrode and test paper with a simple structure, low cost, and the ability to accurately and quantitatively detect blood creatinine, so that key indicators of kidney disease can also be self-monitored at home. Summary of the invention

[0009] In view of the above background, the primary purpose of the present invention is to provide an electrode for detecting creatinine by electrochemical method, which has the comprehensive advantages of high sensitivity, strong anti-interference ability, stable performance, simple structure, small size and low cost.

[0010] Another object of the present invention is to provide a test paper comprising the electrode of the present invention and capable of detecting creatinine by electrochemical method, which can be used for rapid and convenient detection in clinic or at home.

[0011] Another object of the present invention is to provide a method for preparing the electrode and the test paper, which has the comprehensive advantages of less material, high precision, low cost, etc. and is suitable for industrial manufacturing and promotion.

[0012] The above-mentioned object of the present invention is achieved by the following technical solutions:

[0013] In the first aspect, the present invention provides an electrode for electrochemical detection of creatinine, comprising an electrode material base layer; the outer surface of the electrode material base layer is covered with an electron mediator enhancement layer; the outer surface of the electron mediator enhancement layer is fixed with an enzyme composition that can react with creatinine to generate hydrogen peroxide.

[0014] In the electrode scheme described in the present invention, the material of the electrode material base layer can be various existing material compositions that can be used to make redox electrodes, including various compositions containing inert metal materials (such as platinum electrode materials, gold electrode materials or mercury electrode materials) or compositions containing carbon electrode materials, etc. The material composition may or may not contain an electron mediator component.

[0015] In a preferred embodiment of the present invention, the electrode material base layer is a composition mainly composed of carbon electrode materials, and the carbon electrode material can be selected from any one of graphite, carbon paste or glassy carbon; carbon paste or graphite is most preferred. In a further preferred embodiment of the present invention, the composition mainly composed of carbon electrode materials also contains an electron mediator, and the electron mediator can be selected from Prussian blue, methylene blue, ferrocene or its derivatives, or potassium ferrocyanide, etc.; Prussian blue is most preferred.

[0016] In the electrode of the present invention, the enzyme composition capable of reacting with creatinine to generate hydrogen peroxide is preferably a composition of creatinase (CA), creatinase (CI) and sarcosine oxidase (SO); in a further preferred embodiment, the ratio of creatinase, creatinase and sarcosine oxidase is: 3.75-6.25:1.25-2:0.75-1.25; a more preferred ratio is 4-5.5:1.75-2: 1.0-1.25; and the most preferred ratio is 4-5:2:1.25.

[0017] In a further preferred embodiment, the creatinine loading on the electrode surface is 0.5-0.85 U / mm 2 , creatinase loading is 0.15-0.28U / mm 2 , and the sarcosine oxidase loading is 0.1-0.15U / mm 2 .

[0018] In a second aspect, the present invention provides a test paper that can detect creatinine using an electrochemical method, the test paper comprising an insulating base plate, an electrode group and a circuit being provided on the surface of the insulating base plate; the electrode group is composed of at least a working electrode, a counter electrode and a reference electrode, the working electrode being the electrode for detecting creatinine using an electrochemical method as described in the first aspect of the present invention; the circuit comprising a loop formed between the working electrode and the counter electrode, and between the working electrode and the reference electrode.

[0019] The working principle of the test paper of the present invention as an electrochemical biosensor is: after adding the sample according to the conventional method during detection, the creatinine in the sample will react with the enzyme composition fixed on the working electrode in a series of reactions, the creatinine is decomposed into creatine by creatinase, the creatine generates hydrogen peroxide under the action of creatinase and sarcosine oxidase, the hydrogen peroxide is catalytically reduced by the electron mediator of the electron mediator enhancement layer, the electrons are transferred to the electrode material base layer of the working electrode, and a reduction current is generated. According to the relationship between the current response value and the concentration of creatine in the sample, the creatine content of the sample can be known. When the test paper of the present invention is used to detect creatinine, the test sample can be in various forms such as whole blood, plasma, serum, urine, etc.

[0020] In the test paper described in the present invention, there is no particular restriction on the specific material, specification and shape of the insulating base plate. The insulating base plate material is preferably insulating paper, plastic, rubber and other materials with a hydrophobic surface; the plastic is further preferably any one of PET, PVC, PE or PP. The specifications of the insulating base plate can be manufactured into different thicknesses and sizes as needed. The shape of the insulating base plate can be any shape acceptable to the application scenario during actual measurement.

[0021] In the test paper described in the present invention, there is no particular restriction on the specific shape and specification of each electrode in the electrode group. Under the premise of achieving effective electrochemical detection, each electrode can be made into various acceptable shapes, for example, the electrode cross-section can be rectangular, circular or other arbitrary shapes.

[0022] In the test paper of the present invention, the material of the counter electrode is preferably the same as the electrode material base layer of the working electrode; the reference electrode can be made of a variety of existing materials suitable for use as a reference electrode, and the preferred reference electrode is a silver-silver chloride electrode.

[0023] In one embodiment of the present invention, the electrode group may consist of only one working electrode, one counter electrode and one reference electrode, and the one working electrode is the electrode for electrochemical detection of creatinine according to the present invention. The test paper can be used as an electrochemical sensor for detecting the creatinine content in a single sample, and can be used in a simple and portable creatinine detection device, which is suitable for home self-monitoring of patients with kidney disease.

[0024] In a third aspect, the present invention provides a biosensor for electrochemical detection of creatinine, comprising an insulating base plate, wherein an electrode group, a wire and a sample loading chamber are fixedly connected to the surface of the insulating base plate; the sample loading chamber is composed of insulating material walls on all sides and a hollow area in the middle; the main body of the electrode group is within the hollow area, and the electrode group is composed of at least a working electrode, a counter electrode and a reference electrode, and the working electrode is the electrode for electrochemical detection of creatinine as described in the first aspect of the present invention; the wire comprises metal wires respectively connected to the working electrode, the counter electrode and the reference electrode, and is used to electrically connect the electrodes to an external potential detection device.

[0025] When the biosensor described in the present invention is used to detect the blood creatinine level, the wire of the biosensor is connected to the external potential detection device, and the collected blood sample is dripped into the hollow area of ​​the sample addition chamber at an appropriate dose. The insulating material wall around it can limit the sample droplet to the hollow area and fully contact the electrode. The creatinine in the sample reacts with the enzyme composition on the working electrode to generate hydrogen peroxide, and the hydrogen peroxide is catalytically reduced by the electron mediator of the electron mediator enhancement layer on the working electrode, thereby transferring the electrons in the active center of the enzyme composition to the electrode material base layer, generating a reduction current on the working electrode, and the potential detection device measures the potential through the reference electrode. The current response value is related to the blood creatinine level within a certain range, so that the corresponding creatinine level is calculated based on the current detection result.

[0026] In the biosensor described in the present invention, there is no particular restriction on the specific material, specification and shape of the insulating base plate. The insulating base plate material is preferably a material with a hydrophobic surface such as insulating paper, plastic, rubber, etc.; the plastic is further preferably any one of PET, PVC, PE or PP. The specifications of the insulating base plate can be manufactured into different thicknesses and sizes as needed. The shape of the insulating base plate can be any shape acceptable to the application scenario during actual measurement.

[0027] In the biosensor described in the present invention, the specific shape and specifications of each electrode in the electrode group are not particularly limited. Under the premise of achieving effective electrochemical detection, each electrode can be made into various acceptable shapes, for example, the electrode cross section can be rectangular, circular or other arbitrary shapes. The material of the counter electrode is preferably the same as the electrode material base layer of the working electrode; the reference electrode can be made of a variety of existing materials suitable for use as a reference electrode, and the preferred reference electrode is a silver-silver chloride electrode.

[0028] In the biosensor of the present invention, the wire can be any metal conductive material, preferably a copper wire.

[0029] In the biosensor of the present invention, the insulating material wall of the sample loading chamber can be made of a variety of existing insulating materials, preferably hot melt adhesive.

[0030] In a fourth aspect, the present invention also provides a method for preparing the electrode and test paper of the present invention.

[0031] A method for preparing an electrode according to the first aspect of the present invention comprises:

[0032] 1) forming an electrode material base layer with a carbon slurry containing an electron mediator and drying the layer;

[0033] 2) Arranging an electron mediator enhancement layer on the upper surface of the electrode material base layer obtained in 1) and drying the layer;

[0034] 3) Immobilizing an enzyme composition on the surface of the electron mediator enhanced layer obtained in 2); the enzyme composition is a composition of creatinase, creatinase and sarcosine oxidase; in a further preferred enzyme composition, the ratio of creatinase, creatinase and sarcosine oxidase is: 3.75-6.25:1.25-2:0.75-1.25; the preferred ratio is 4-5.5:1.75-2:1.0-1.25; the most preferred ratio is 4-5:2:1.25; and an enzyme-modified electrode is obtained, i.e., the electrode for electrochemical detection of creatinine.

[0035] In the preferred method for preparing the electrode of the present invention, 1) forming the electrode material base layer with the carbon paste containing the electron mediator is to print the carbon paste on an acceptable surface by screen printing based on a pre-designed screen to form the electrode material base layer.

[0036] In a further preferred method for preparing the electrode of the present invention, 1) the carbon paste containing the electron mediator is a carbon paste doped with Prussian blue.

[0037] In the preferred method for preparing the electrode of the present invention, 2) the electron mediator enhancement layer is provided by modifying the electron mediator on the surface of the electrode material base layer by an electrodeposition method to form the electron mediator enhancement layer.

[0038] In a more preferred embodiment, the electron mediator is Prussian blue, and the electrodeposition method specifically comprises:

[0039] The electrode material base layer described in 1) is immersed in FeCl 3 and K 3 [Fe(CN) 6] in a mixture of KCl and HCl, the scanning speed is controlled at 10-30 mV / s, the scanning voltage is controlled at -0.15 V-0.3 V, and the scanning is continued for 4-20 cycles, preferably 8-16 cycles, and most preferably 8-10 cycles; then in a mixed solution of KCl and HCl, the scanning is further continued for 4-20 cycles, preferably 8-16 cycles, and most preferably 8-10 cycles at a scanning speed of 40-60 mV / s and in a voltage range of -0.2-0.5 V.

[0040] In the preferred method for preparing the electrode of the present invention, the fixation of the enzyme composition described in 3) is completed by glutaraldehyde cross-linking; further preferably, it is completed by the following method:

[0041] 3.1) dissolving creatinase, creatinase and sarcosine oxidase in a neutral buffer to obtain a mixed enzyme solution; controlling the concentrations of creatinase, creatinase and sarcosine oxidase in the mixed enzyme solution to be 5-8.5 / μL, 1.5-2.8 / μL and 1-1.8 / μL, respectively; further preferred concentrations are 5.3-7.3 / μL, 2.3-2.7 / μL and 1.3-1.7 / μL, respectively; the most preferred concentrations of the three enzymes are 5.3-6.7 / μL, 2.5-2.7 / μL and 1.6 / μL, respectively;

[0042] 3.2) preparing a glutaraldehyde solution with a concentration of 0.5-1%;

[0043] 3.3) mixing the mixed enzyme solution obtained in 3.1) and the glutaraldehyde solution obtained in 3.2) in an equal volume ratio to obtain an enzyme modification solution;

[0044] 3.4) The enzyme modification solution obtained in 3.3) is added dropwise to the surface of the electron mediator enhancement layer obtained in 2), and then air-dried at 0-4° C., thereby completing the fixation and obtaining an enzyme-modified electrode.

[0045] The method for preparing the test paper capable of detecting creatinine by electrochemical method according to the second aspect of the present invention comprises:

[0046] Using carbon slurry containing electron mediator as working electrode and counter electrode materials and silver-silver chloride slurry as reference electrode material, a working electrode, a counter electrode, a reference electrode and a matching circuit are prepared on the surface of an insulating and hydrophobic bottom plate; an electron mediator enhancement layer is applied on the surface of the prepared working electrode; then an enzyme composition consisting of creatinase, creatinase and sarcosine oxidase is fixed on the surface of the electron mediator enhancement layer; and a test paper that can detect creatinine by an electrochemical method is obtained.

[0047] In the method for preparing the test paper described in the present invention, the carbon slurry containing the electron mediator is preferably a carbon paste doped with Prussian blue; the preparation of the working electrode, the counter electrode, the reference electrode and the matching circuit on the surface of the insulating and hydrophobic bottom plate is preferably completed by screen printing; the application of the electron mediator enhancement layer on the surface of the prepared working electrode is preferably performed by modifying the Prussian blue on the surface of the prepared working electrode by electrodeposition; the ratio of creatinase, creatinase and sarcosine oxidase in the enzyme composition is preferably 3.75-6.25:1.25-2:0.75-1.25; the more preferred ratio is 4-5.5: 1.75-2:1.0-1.25; the most preferred ratio is 4-5:2:1.25; the enzyme composition is fixed on the surface of the electron mediator enhancement layer, preferably by dripping a mixed solution of the enzyme composition and glutaraldehyde on the surface of the electron mediator enhancement layer and drying it at 0-4°C.

[0048] In a fifth aspect, the present invention also provides a method for detecting creatinine by electrochemical method, comprising:

[0049] Using the biosensor for electrochemical detection of creatinine of the present invention, the wire of the biosensor is connected to an external potential detection device; a buffer solution with a pH value of 7-9.5 is dripped into the sample addition chamber of the biosensor, and then a plasma or whole blood sample to be tested is dripped into the buffer solution, and the potential detection device performs detection at a working voltage of -0.4 to -0.1V to obtain the intensity of the response current, and finally the creatinine concentration in the sample is calculated based on a relationship curve between the creatinine concentration and the current signal.

[0050] In the preferred electrochemical method for detecting creatinine of the present invention, the pH value of the buffer solution is 7.4.

[0051] In the preferred electrochemical method for detecting creatinine of the present invention, the working voltage is -0.2 to -0.1V, and -0.1V is most preferred.

[0052] Compared with the prior art, the electrode of the present invention is provided with an electron mediator enhancement layer between the three-enzyme composition and the electrode electrode material base layer for detecting creatinine by optimizing the internal composition structure, thereby significantly enhancing the electron transfer efficiency, reducing the working potential during detection to -0.1V, thereby effectively eliminating the interference caused by non-target substances such as creatine in creatinine detection, and significantly improving the anti-interference ability of the electrode and test paper of the present invention. At the same time, the electrode of the present invention also has higher sensitivity and repeated measurement stability. In addition, the test paper of the present invention can be used in a variety of portable and fast small electrochemical detection equipment, so the test paper of the present invention can realize fast and convenient clinical detection or home monitoring of blood creatinine or urine creatinine. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 It is a schematic diagram of the preparation process of the biosensor for electrochemical detection of creatinine described in Example 1 of the present invention.

[0054] Figure 2 It is a schematic diagram of the structure of the biosensor prepared by the method described in Example 1 of the present invention.

[0055] Figure 3 a shows the cyclic voltammetry curves of the biosensor of Example 3 at different scan rates in 1 mM creatinine solution.

[0056] Figure 3 b shows the concentration of 1 mM H 2 O 2 Cyclic voltammetry curves of the biosensor of Example 3 in solution at different scan rates.

[0057] Figure 4 a shows the results of the enzyme loading study of CA in Experimental Example 2.

[0058] Figure 4 b shows the enzyme loading study results of CI in Experimental Example 2.

[0059] Figure 4 c shows the results of the enzyme loading study of SO in Experimental Example 2.

[0060] Figure 5 The detection performance of the sensor of Example 3 under different pH conditions is shown.

[0061] Figure 6 The detection performance of the sensor of Example 3 is shown at different potentials of the working electrode relative to the reference electrode.

[0062] Figure 7 The sensitivity study results of sensors prepared with different electrode materials are shown.

[0063] Fig. 8A Example 3 shows the biosensor detection of H 2 O 2 The standard curve of

[0064] Figure 8B The standard curve of the biosensor in Example 3 for detecting creatinine solution is shown.

[0065] Fig. 9 The measurement repeatability of the biosensor in Example 3 for detecting creatinine solution is shown.

[0066] Fig. 10A The standard curve for detecting plasma creatinine concentration using the biosensor of Example 3 is shown.

[0067] Fig. 10BThe results show the correlation between the results of plasma creatinine concentration detected by the biosensor of Example 3 and the results of plasma creatinine concentration detected by the traditional Jaffe method. DETAILED DESCRIPTION

[0068] The technical solution of the present invention will be described in detail below by enumerating embodiments, but the scope of the present invention is not limited to the enumerated embodiments.

[0069] Example 1

[0070] An enzyme electrode structure that can be used for electrochemical detection of creatinine includes an electrode material base layer; the outer surface of the electrode material base layer is covered with an electron mediator enhancement layer; the outer surface of the electron mediator enhancement layer is fixed with an enzyme composition that can react with creatinine to generate hydrogen peroxide. The electrode material base layer is composed of a carbon paste doped with Prussian blue; the electron mediator enhancement layer is a Prussian blue layer modified on the electrode material base layer by electrochemical deposition; the enzyme composition is composed of creatinase, creatinase and sarcosine oxidase in a ratio of 5:2:1.25.

[0071] The method for preparing the enzyme electrode is as follows:

[0072] 1) Applying a carbon paste slurry doped with Prussian blue on a PET substrate to form a strip with a thickness of 0.2 mm and drying the strip to obtain an electrode material base layer;

[0073] 2) First, the electrode material base layer described in 1) is placed in 0.1MH 2 SO solution at a scanning speed of 1000 mV / s and a potential range of 0.2-1.2 V for 20 cycles to clean the electrode material base layer; then the cleaned electrode material base layer was immersed in 2.5 mM FeCl 3 and 2.5 mM K 3 [Fe(CN) 6 ] in a mixture of 0.1M KCl and 0.1M HCl as supporting electrolytes, with the scanning speed controlled at 20mV / s and the scanning voltage controlled at -0.15V-0.3V, and the scanning was continued for 8 cycles; then the electrode material base layer was further scanned for 8 cycles in a mixed solution of 0.1M KCl and 0.1M HCl at a scanning speed of 50mV / s and within a voltage range of -0.2-0.5V to stabilize the Prussian blue on the electrode; finally, after drying, an electrode with the surface of the electrode material base layer modified with Prussian blue was obtained, and the Prussian blue modified on the surface of the electrode material base layer was used as an electron mediator enhancement layer;

[0074] 3) Immobilizing an enzyme composition on the surface of the electron mediator enhanced layer obtained in 2):

[0075] 3.1) taking out powdered creatinase, creatinase and sarcosine oxidase stored at -20°C and preparing a mixed enzyme solution; controlling the ratio of creatinase, creatinase and sarcosine oxidase in the mixed enzyme solution to be 5:2:1.25;

[0076] 3.2) preparing a 0.8% glutaraldehyde solution; mixing the mixed enzyme solution and the glutaraldehyde solution in equal volumes to obtain an enzyme modification solution;

[0077] 3.3) The enzyme modified solution obtained in 3.2) is added dropwise to the surface of the electron mediator enhanced layer obtained in 2), and then refrigerated in a 4° C. refrigerator overnight to complete the fixation of the enzyme composition and obtain the enzyme electrode.

[0078] Example 2

[0079] A test paper capable of detecting creatinine by an electrochemical method comprises a rectangular PET base plate, wherein an electrode group and a circuit are arranged on the surface of the PET base plate; the electrode group is composed of a long strip working electrode, a long strip counter electrode and a long strip reference electrode arranged in parallel, and the structure of the working electrode is the same as that of the electrode described in Example 1; the material of the counter electrode is composed of a carbon paste doped with Prussian blue; and the reference electrode is a silver-silver chloride electrode.

[0080] Example 3

[0081] Prepare a biosensor that can detect the creatinine level of a sample by electrochemical method, the process is as follows Figure 1 As shown, including:

[0082] (1) Select a rectangular PET material of 25 mm × 30 mm as the insulating base plate, and preset the positions of the working electrode, counter electrode, and reference electrode on the insulating base plate;

[0083] (2) Electrodes were fabricated at predetermined positions on the insulating substrate described in (1), i.e., the working electrode and the counter electrode were prepared by brushing with carbon paste doped with Prussian blue, and the reference electrode was prepared by brushing with Ag / AgCl ink. The length, width and thickness of each electrode were 20 mm, 2 mm and 0.2 mm, respectively. Figure 1 (2) As shown, the middle is the working electrode, the left is the counter electrode, and the right is the reference electrode; the insulating base plate coated with electrodes is placed in a drying oven at 75°C for 30 minutes and cooled to room temperature;

[0084] (3) On the cooled insulating base plate obtained in (2), copper wires are connected to the ends of the three electrodes on the same side using silver paste; the connection parts are then sealed with glue to ensure the stability of the connection. Then, an insulating material wall is built around the ends of the other side of the three electrodes that are not connected to the copper wires using hot melt glue to form a sample loading cavity with a 2 mm × 8 mm hollow area in the middle and a raised area around it.

[0085] (4) modifying the surface of the working electrode in the sample loading chamber obtained in (3) by electrodeposition to form a Prussian blue layer as an electron mediator enhancement layer;

[0086] (5) Immobilizing the complex enzyme on the working electrode surface treated in (4) in the following manner:

[0087] The powdered creatinase, creatinase and sarcosine oxidase stored at -20°C are taken out to prepare a composite enzyme solution; the ratio of creatinase, creatinase and sarcosine oxidase in the mixed enzyme solution is controlled to be 4:1.6:1;

[0088] preparing a 0.8% glutaraldehyde solution; mixing the complex enzyme solution and the glutaraldehyde solution in equal volume ratio to obtain an enzyme modification solution;

[0089] The enzyme modification solution is added dropwise to the surface of the electron mediator enhancement layer obtained by modification (4) on the surface of the working electrode, and then refrigerated overnight at 4°C to complete the fixation of the complex enzyme and obtain the biosensor.

[0090] The structure of the biosensor obtained in this example is as follows Figure 2 As shown, it includes a rectangular PET insulating base plate 1, and the surface of the insulating base plate 1 is fixedly connected with an electrode group, a wire and a sample loading chamber; the sample loading chamber 2 is composed of hot melt adhesive walls on all sides and a hollow area in the middle; the main body of the electrode group is within the hollow area, and the electrode group is composed of a working electrode 31, a counter electrode 32 and a reference electrode 33, and the working electrode 31 is the electrode described in Example 1; the wire includes a copper wire 4 connected to the working electrode 31, the counter electrode 32 and the reference electrode 33 respectively, and is used to electrically connect each electrode to an external potential detection device.

[0091] Before use in detection, the biosensor prepared in this example was taken out of the refrigerator and incubated in a buffer at room temperature for 1 hour. When the biosensor described in the present invention is used to detect blood creatinine levels, Figure 2 As shown, the wire 4 of the biosensor is connected to the external potential detection device, and the buffer solution and the collected blood sample are dripped into the hollow area of ​​the sample addition chamber 2 in appropriate doses, and the insulating material wall around it can limit the droplets in the hollow area to fully contact the electrode. The creatinine 0 in the sample reacts with the complex enzyme on the working electrode 31 to generate hydrogen peroxide, and the hydrogen peroxide is catalytically reduced by the electron mediator of the electron mediator enhancement layer on the working electrode 31, thereby transferring the electrons in the active center of the complex enzyme to the electrode material base layer, generating a reduction current on the working electrode 31, and the potential detection device measures the potential through the reference electrode 33. The current response value is related to the blood creatinine level within a certain range, so that the corresponding creatinine level is calculated based on the current detection result.

[0092] Example 4

[0093] A biosensor for detecting creatinine level in a sample by electrochemical method is prepared, comprising:

[0094] The working electrode, the counter electrode and the reference electrode are printed on appropriate positions on the surface of the PET material by screen printing based on a pre-designed screen, wherein the working electrode and the counter electrode use carbon paste doped with Prussian blue as printing ink, and the reference electrode uses Ag / AgCl ink as printing ink;

[0095] Further modifying the surface of the working electrode with Prussian blue by an electrodeposition method to form the electron mediator enhancement layer;

[0096] Immobilizing the complex enzyme on the surface of the electron mediator enhancement layer on the working electrode according to the method of step 3) of Example 1 to obtain an enzyme-modified working electrode;

[0097] Using silver paste to connect copper wires to the ends of the same side of the three electrodes respectively; then using glue to seal the connection parts to ensure the stability of the connection;

[0098] Then, a wall of pure raw material is built around the ends of the other side of the three electrodes that are not connected to the copper wires with hot melt adhesive to form a sample adding cavity with surrounding protrusions and a hollow area in the middle, thus obtaining the biosensor.

[0099] Example 5

[0100] Method for detecting blood creatinine by electrochemical method using the biosensor described in Example 3:

[0101] The copper wire of the biosensor is connected to an external potential detection device; a buffer solution with a pH value of 7.4 is dripped into the sample chamber of the biosensor, and then the plasma or whole blood sample to be tested is dripped into the buffer solution. The potential detection device performs detection at a working voltage of -0.1V to obtain the intensity of the response current, and finally the creatinine concentration in the sample is calculated based on the relationship curve between the creatinine concentration and the current signal.

[0102] Comparative Example 1

[0103] A biosensor for electrochemical detection of creatinine was prepared in a method substantially the same as that of Example 3, except that the working electrode was not modified with a Prussian blue layer.

[0104] Comparative Example 2

[0105] A biosensor for electrochemical detection of creatinine was prepared in a method substantially the same as that of Example 3, except that carbon paste without Prussian blue doping was used as printing ink for the working electrode and the counter electrode.

[0106] Comparative Example 3

[0107] A control biosensor was prepared in substantially the same manner as in Example 3, except that the working electrode and the counter electrode used carbon paste not doped with Prussian blue as printing ink, and no Prussian blue-modified electron mediator enhancement layer was added to the working electrode.

[0108] Experimental Example 1

[0109] In order to explore the effect of the creatinine sensor of Example 3 on H 2 O 2 or creatinine detection performance in the presence of 1 mM H 2 O 2 The cyclic voltammetry curves at different scan rates were studied in the buffer containing 1 mM creatinine or 1 mM creatinine. Figure 3 a shows the cyclic voltammetry curves of 1mM creatinine solution at scan rates of 30, 50, 70 and 100mV / s. Although the scan rates are different, the shape and change trend of the curves are similar. As the scan rate increases, the redox peak current gradually increases. As the scan rate increases, the oxidation peak potential shows an upward trend, and the reduction peak potential shows a downward trend. The results show that the curve presents a pair of obvious redox peaks, with potentials of -0.034V (a) and -0.325V (d), respectively. At a potential of -0.034V, creatinine seems to undergo a strong oxidation reaction on the working electrode. There are also two oxidation peaks on the curve, with corresponding potentials of -0.169v (b) and 0.254v (c), respectively. Therefore, the optimal potential needs further research, exploration and verification. Figure 3 b shows 1 mM H 2 O 2 Cyclic voltammetry curves of the solution at different scan rates. 2 O 2 The curve of H is similar to that of creatinine solution, which is mainly due to the properties of the printed electrode material. In addition, by comparing the current response at the same potential, H 2 O 2 Produces a greater response than creatinine due to H 2 O 2 The redox reaction occurs directly on the electrode, and creatinine is converted to H 2 O 2 Requires an enzymatic reaction, then H 2 O 2 Oxidized / reduced at the electrode.

[0110] Experimental Example 2

[0111] The effect of the concentration of three enzymes CA, CI, and SO on the sensitivity of the creatinine biosensor was verified. Theoretically, the current signal of the sensor depends largely on the amount of enzyme, but too much enzyme will reduce the current signal. Therefore, considering the measurement performance and cost of the sensor, it is necessary to study the optimal enzyme concentration of these three enzymes in order to obtain better detection results at a lower cost. CA, CI, and SO enzyme solutions were prepared at 100U / μl, 8U / μl, and 5U / μl, respectively, and the three enzyme solutions were mixed at a volume ratio of 1:1:1. Then 2μL of the mixed enzyme solution was mixed with 2μL of 0.8% glutaraldehyde solution to fix the enzyme composition on the working electrode. Therefore, the actual concentration of each enzyme on the working electrode is one-sixth of its single enzyme solution. Figure 4 The enzyme loading study of the three enzymes is shown. The concentration of each enzyme solution is the horizontal axis, and the vertical axis is the slope of the standard curve, which is the current response caused by the unit creatinine concentration and represents the sensitivity of the sensor. The higher the slope, the better the sensitivity of the sensor.

[0112] In this experiment, the concentrations of CI enzyme solution and SO enzyme solution prepared separately were 8U / μL and 5U / μL, respectively. In order to observe the change of sensor slope with the increase of CA concentration, a series of CA enzyme solution concentrations were designed, including 0.1, 1, 5, 10, 20, 50, and 100U / μl. Figure 4 As can be seen in a, when the CA concentration is between 0.1-20U / μL, the sensor slope increases significantly from -0.4255μA / mM to -2.3413μA / mM. When the CA concentration is 50 and 100U / μL, the slope does not change much compared with the CA concentration of 20U / μL. Therefore, 20u / μL is considered to be a more ideal CA concentration.

[0113] The concentrations of CI enzyme solution used in CI enzyme loading experiments were 0.5, 1, 2, 4, and 8 U / μL, respectively, and the concentrations of the other two enzymes CA and SO were 20u / μL and 5u / μL, respectively. Figure 4 As shown in b, unlike CA, the slope is proportional to the CI concentration under unsaturated conditions.

[0114] Figure 4 c shows the study of SO enzyme loading, where SO concentrations were 0.05, 0.2, 0.5, 1, 2, 4, and 5 U / μL. Figure 4 As shown in Figure c, with the increase of SO concentration, the change trend of the slope is similar to that of CA. When the SO concentration is 5 U / μL, the slope of the sensor is the largest.

[0115] The results showed that 20U / μL, 8U / μL and 5U / μL were the optimal enzyme concentrations for CA, CI and SO, respectively.

[0116] like Figure 4As shown in Figures 4a and 4b, the slope tends to saturate as the enzyme concentration increases. This is expected because the reaction rate is positively correlated with the enzyme concentration when the amount of substrate is not considered. When the substrate dosage is constant, the reaction rate can reach a limit at a certain enzyme concentration. Increasing the enzyme concentration at this time does not speed up the reaction rate, and excess enzyme may hinder the H produced by the decomposition of creatinine. 2 O 2 It is also worth mentioning that the optimal ratio of CA:CI:SO is 5:2:1.25, which suggests that in order to improve the detection performance of the sensor, the conversion of creatinine to creatine catalyzed by CA needs to be sufficient, and the conversion of creatine to H 2 O 2 Only small amounts of SO enzyme appear to be required.

[0117] Experimental Example 3

[0118] During the creatinine determination process, 200 μL NaH 2 PO 4 ·2H 2 O buffer soaked electrode and enzyme fixed on the working electrode. It was found that the enzyme activity would change under the above different buffer pH conditions, thus affecting the detection performance of the sensor. Therefore, we prepared a series of buffers with pH values ​​of 6.0-9.5 to explore the suitable acid-base environment for the sensor to work. The results showed that the sensor maintained good detection performance in the pH range of 7.5-9.5. The worst detection effect occurred at pH 6, and the best detection effect occurred at pH 8.5. Figure 5 shown.

[0119] Since the pH value of the actual blood sample ranges from 7.35 to 7.45, a buffer with a pH value of 7.4 was selected for further study. Through this study, we believe that the creatinine biosensor of the present invention can accurately detect human blood samples without any acid-base pretreatment.

[0120] Experimental Example 4

[0121] As described in Experimental Example 1, 1 mM creatinine and 1 mM H 2 O 2 In the cyclic voltammetry curve study of the solution, the redox potential curve of the sensor of Example 3 has multiple peaks, and it is necessary to study the optimal detection potential of the sensor. Figure 6As shown in the figure, the sensor exhibits high sensitivity at -0.4, -0.2, -0.1, -0.05 and 0V potentials, and obtains the best detection effect at -0.1V, while the sensor signal is significantly reduced at 0.05, 0.1 and 0.2V potentials, especially at 0.1V and 0.2V. In short, the performance of the sensor at -0.1V potential is the best, which is also extremely important and critical for clinical human blood sample measurement. Because the detection principle of many existing sensors is based on H 2 O 2 The main problem of this sensor in practical application is that H 2 O 2 The oxidation potential of the biosensor is relatively high. In the actual blood sample being tested, some reducing substances, such as ascorbic acid, uric acid and bilirubin, will also be oxidized at high potential. Once these substances are oxidized, the interference current generated will hinder the accurate detection of the target substance (creatinine). In this case, the biosensor of the present invention can exert superior detection performance at a low potential of -0.1V, which can significantly improve the anti-interference ability of the sensor in actual blood sample detection, thereby making the detection results more accurate and reliable.

[0122] Experimental Example 5

[0123] This experiment studies the detection sensitivity of four biosensors with different electrode materials. The four biosensors are biosensors prepared by the methods of Example 3, Comparative Example 1, Comparative Example 2 and Comparative Example 3. The research results are as follows Figure 7 As shown, the biosensor of comparative example 3 does not generate a current signal. The biosensors of comparative example 2 and comparative example 1 have similar detection performances, with a sensitivity of about 1.4 μA / mM. Compared with comparative examples 1 and 2, the sensor of Example 3 has a significantly improved sensitivity to about 2.2 μA / mM after the PB carbon paste electrode is modified with a Prussian blue electrodeposition layer. The results show that the electron mediator enhancement layer of the present invention can enable the sensor to exhibit good detection performance at a potential of -0.1 V.

[0124] Experimental Example 6

[0125] Referring to the detection method described in Example 5, the biosensor of Example 3 was used as the detection sensor, a buffer solution with a pH value of 7.4 was dripped into the sample chamber of the biosensor of Example 3, and when the current baseline of the buffer solution was stable, a series of H2O2 concentrations from low to high were added to the buffer solution. 2 O 2 Solution. 2 O 2 After diffusing to the surface of the working electrode, it is oxidized and generates a current signal through electron transfer. The results show that after adding H 2 O 2When the working electrode surface is locally H 2 O 2 The instantaneous increase in concentration causes the current to rise immediately; 2 O 2 diffusion, the current peak gradually decreases; when H 2 O 2 When the diffusion and oxidation process reaches equilibrium, the current signal reaches a new platform; this current change process takes only about 100 seconds, indicating that the biosensor of the present invention has a fast sensing measurement speed and high efficiency. Fig. 8A As shown, when H 2 O 2 When the concentration is in the range of 0.05-1.4 mM, the current response is similar to H 2 O 2 The slope of the standard curve is -5.5739 μA / mM, and the correlation coefficient is 0.999, showing a nearly perfect ratio. The experimental results show that the sensor can accurately and sensitively achieve H 2 O 2 Detection of H 2 O 2 The successful detection showed that the sensor has the potential to detect creatinine.

[0126] Referring to the detection method described in Example 5, multiple parallel test groups were set up. Each group first added 200 μl of a buffer solution with a pH value of 7.4 into the sample chamber of the biosensor prepared in Example 3 to immerse the electrode group. When the current baseline of the buffer solution was stable, each group added creatinine solutions of different concentrations into the buffer solution. It can be observed that the current signal of each group increased within 1 second after the addition of creatinine, and reached a stable state in about 100 seconds, indicating that the sensor responded quickly.

[0127] In order to explore the detection range of the creatinine sensor, this experiment also measured several high-concentration creatinine solutions. Figure 8B As can be seen in the figure, the current signal is proportional to the creatinine concentration in the range of 0.05-1.4 mM. However, as the creatinine concentration continues to increase, the current signal tends to be flat. Therefore, the linear detection range of the biosensor for creatinine in the present invention is 0.05-1.4 mM, which is sufficient to measure actual human blood samples in clinical practice.

[0128] Figure 8B The standard curve between the current response and creatinine concentration of the biosensor for electrochemical detection of creatinine prepared in Example 3 is shown. Figure 8B As shown, with H 2 O 2Consistently, the current response is proportional to the creatinine concentration. On the standard curve, we can intuitively see the linear detection range of 0.05-1.4mM, the slope is -1.8918μa / mM, and the correlation coefficient is 0.9957. These results show that the biosensor successfully achieves rapid and sensitive detection of creatinine.

[0129] Experimental Example 7

[0130] In order to study the measurement stability of the biosensor prepared by the method of Example 3, the same creatinine solution was measured continuously 74 times using the method described in Example 5. First, 200 μL of buffer solution was dropped on the electrode surface to immerse the electrode group. When the current baseline of the buffer solution tended to be stable, 5 μL of 41 mM creatinine solution was added to the buffer solution, so that the final concentration of creatinine was 1 mM. Then the current response caused by the 1 mM creatinine solution was recorded. The current response of the first measurement was 100%, and the current responses of the subsequent 73 measurements were quantified based on the response of the first measurement and expressed as a percentage.

[0131] 74 test results Fig. 9 As shown, the horizontal axis is the number of measurements, and the vertical axis is the normalized response ratio. It can be observed that, taking the current response of the first detection as the standard, the current response of the subsequent 73 detections tends to be stable, and the current response varies within a range of 20% (80-120%). It is worth noting that after 74 measurements, the sensor still maintains excellent performance, and compared with the initial signal, the current response has not decreased, but increased, which may be due to the increase in enzyme activity. The results show that the sensor has good measurement stability when continuously measuring creatinine.

[0132] Experimental Example 8

[0133] The method of Example 5 of the present invention was used to detect the creatinine levels in 7 groups of plasma samples. Each group of plasma samples was also tested using the traditional Jaffe method as a control. The two test results and their error rates are shown in Table 1 below:

[0134] Table 1

[0135]

[0136] The standard curve of plasma creatinine concentration detected by the biosensor in Example 3 is as follows: Fig. 10A As shown in FIG. 1 , it can be seen that the response current intensity detected by the biosensor of the present invention is highly correlated with the creatinine concentration. Fig. 10B As shown, there is also a high correlation between the detection results of the biosensor of the present invention and the detection results of the traditional Jaffe method, which proves that the biosensor of the present invention has good accuracy in detecting plasma creatinine levels.

[0137] Experimental Example 9

[0138] The storage stability of the sensor determines the shelf life of the commercial creatinine biosensor. This experiment studied the storage stability of the creatinine sensor. On the first day, a sufficient number of biosensors prepared in Example 3 were prepared and stored in a refrigerator at 4 degrees Celsius. Creatinine was measured on the 2nd, 5th, 8th, 11th, 14th, 17th, and 20th days, using 4 biosensors each time. Subsequently, measurements were performed on the 30th and 60th days. The changes in the sensor detection performance over time were observed. The results showed that the detection performance of the biosensor for electrochemical detection of creatinine prepared in Example 3 did not decrease over time compared with the first detection. Among them, the slopes of the standard curves for creatinine determination on the 2nd, 30th, and 60th days after storage were -1.9475μA / mM, -2.5791μA / mM, and -2.3389μA / mM, respectively, with good consistency. These results show that the creatinine biosensor of the present invention still maintains good detection performance after being stored at 4°C for 2 months, providing the possibility of long-term effectiveness.

Claims

1. An electrode for electrochemical detection of creatinine, comprising an electrode material base layer; the outer surface of the electrode material base layer is modified with Prussian blue as an electron mediator enhancement layer by electrodeposition; an enzyme composition that can react with creatinine to generate hydrogen peroxide is fixed on the outer surface of the electron mediator enhancement layer; the electrode material base layer is composed of carbon paste or graphite containing Prussian blue; the enzyme composition is composed of creatinase (CA), creatinase (CI) and sarcosine oxidase (SO) in a ratio of 5:2:1.25; on the surface of the electrode, the creatinase loading is 0.5-0.85U / mm 2 The creatinase loading is 0.15-0.28U / mm 2 , and the sarcosine oxidase loading is 0.1-0.15U / mm 2 ; The electrode material base layer is formed by printing carbon paste or graphite containing Prussian blue on an acceptable surface through screen printing; The electrodeposition method for modifying Prussian blue is carried out as follows: the electrode material base layer is immersed in a mixture of FeCl3 and K3[Fe(CN)6], the scanning speed is controlled at 10-30mV / s, the scanning voltage is controlled at -0.15 V-0.3V, and the scanning is continued for 4-20 cycles; then, in a mixed solution of KCl and HCl, the scanning speed is further scanned at 40-60mV / s and within a voltage range of -0.2-0.5V for 4-20 cycles.

2. The electrode according to claim 1, characterized in that The electrodeposition method for modifying Prussian blue is carried out as follows: the electrode material base layer is immersed in a mixture of FeCl3 and K3[Fe(CN)6], the scanning speed is controlled at 10-30mV / s, the scanning voltage is controlled at -0.15 V-0.3V, and the scanning is continued for 8-16 cycles; then, in a mixed solution of KCl and HCl, the scanning speed is further scanned at 40-60mV / s and in the voltage range of -0.2-0.5V for 8-16 cycles.

3. The electrode according to claim 1, characterized in that The electrodeposition method for modifying Prussian blue is carried out as follows: the electrode material base layer is immersed in a mixture of FeCl3 and K3[Fe(CN)6], the scanning speed is controlled at 10-30mV / s, the scanning voltage is controlled at -0.15 V-0.3V, and the scanning is continued for 8-10 cycles; then, in a mixed solution of KCl and HCl, the scanning speed is further scanned at 40-60mV / s and in the voltage range of -0.2-0.5V for 8-10 cycles.

4. A test paper that can detect creatinine using an electrochemical method, the test paper comprising an insulating base plate, an electrode group and a circuit are provided on the surface of the insulating base plate; the electrode group is composed of at least a working electrode, a counter electrode and a reference electrode, the working electrode is the electrode described in any one of claims 1-3; the circuit comprises a loop formed between the working electrode and the counter electrode, and between the working electrode and the reference electrode.

5. A biosensor for electrochemical detection of creatinine, comprising an insulating base plate, wherein an electrode group, a wire and a sample loading chamber are fixedly connected to the surface of the insulating base plate; the sample loading chamber is composed of insulating material walls on all sides and a hollow area in the middle; the main body of the electrode group is within the hollow area, and the electrode group is composed of at least a working electrode, a counter electrode and a reference electrode, and the working electrode is the electrode according to any one of claims 1 to 3; the wire comprises metal wires respectively connected to the working electrode, the counter electrode and the reference electrode, and is used to electrically connect each electrode to an external potential detection device.

6. A method for preparing the electrode according to claim 1, comprising: 1) Forming a base layer of electrode material by screen printing with carbon paste or graphite containing Prussian blue on an acceptable surface and drying; 2) A layer of Prussian blue is modified on the surface of the electrode material base layer obtained in 1) as an electron mediator enhancement layer and dried; the modification is performed in the following manner: the electrode material base layer is immersed in a mixture of FeCl3 and K3[Fe(CN)6], the scanning speed is controlled at 10-30mV / s, the scanning voltage is controlled at -0.15 V-0.3V, and the scanning is continued for 4-20 cycles; then, in a mixed solution of KCl and HCl, the scanning speed is further scanned at 40-60mV / s and the voltage range is -0.2-0.5V for 4-20 cycles; 3) Fixing a layer of enzyme composition on the surface of the electron mediator enhancement layer obtained in 2) to obtain an enzyme-modified electrode; the enzyme composition is composed of creatinase, creatinase and sarcosine oxidase mixed in a ratio of 5:2:1.25; after fixation, the creatinase loading on the electrode surface is 0.5-0.85U / mm 2 The creatinase loading is 0.15-0.28U / mm 2 , and the sarcosine oxidase loading is 0.1-0.15U / mm 2 .

7. The method according to claim 6, characterized in that 2) The modification is carried out in the following manner: the electrode material base layer is immersed in a mixture of FeCl3 and K3[Fe(CN)6], the scanning speed is controlled at 10-30mV / s, the scanning voltage is controlled at -0.15 V-0.3V, and the scanning is continued for 8-16 cycles; then in a mixed solution of KCl and HCl, the scanning speed is further scanned at 40-60mV / s and the voltage range is -0.2-0.5V for 8-16 cycles.

8. The method according to claim 6, characterized in that 2) The modification is carried out in the following manner: the electrode material base layer is immersed in a mixture of FeCl3 and K3[Fe(CN)6], the scanning speed is controlled at 10-30mV / s, the scanning voltage is controlled at -0.15 V-0.3V, and the scanning is continued for 8-10 cycles; then in a mixed solution of KCl and HCl, the scanning is further carried out at a scanning speed of 40-60mV / s and in a voltage range of -0.2-0.5V for 8-10 cycles.

9. The method according to any one of claims 6 to 8, characterized in that 3) The fixation is accomplished by: 3.1) Dissolving creatinase, creatinase and sarcosine oxidase in a neutral buffer to obtain a mixed enzyme solution; controlling the concentrations of creatinase, creatinase and sarcosine oxidase in the mixed enzyme solution to be 5.3-6.7 / μL, 2.5-2.7 / μL and 1.6 / μL, respectively; 3.2) Prepare a 0.5-1% glutaraldehyde solution; 3.3) mixing the mixed enzyme solution obtained in 3.1) and the glutaraldehyde solution obtained in 3.2) in an equal volume ratio to obtain an enzyme modification solution; 3.4) The enzyme modification solution obtained in 3.3) is added dropwise to the surface of the electron mediator enhancement layer obtained in 2), and then air-dried at 0-4°C to complete the fixation and obtain the enzyme-modified electrode.

10. A method for preparing the test paper capable of detecting creatinine by electrochemical method according to claim 4, comprising: Using carbon slurry containing Prussian blue as working electrode and counter electrode materials, and silver-silver chloride slurry as reference electrode material, a working electrode, a counter electrode, a reference electrode and a matching circuit are prepared on the surface of an insulating and hydrophobic bottom plate; an electron mediator enhancement layer is applied on the surface of the prepared working electrode; then an enzyme composition consisting of creatinase, creatinase and sarcosine oxidase is fixed on the surface of the electron mediator enhancement layer; a test paper that can detect creatinine by electrochemical method is obtained; the preparation of the working electrode, the counter electrode, the reference electrode and the matching circuit on the surface of the insulating and hydrophobic bottom plate is completed by screen printing; the application of the electron mediator enhancement layer on the surface of the prepared working electrode is carried out by modifying the surface of the prepared working electrode with Prussian blue by electrodeposition; the modification of Prussian blue by electrodeposition is carried out in the following manner: the electrode material base layer is immersed in a mixture of FeCl3 and K3[Fe(CN)6], the scanning speed is controlled at 10-30mV / s, the scanning voltage is controlled at -0.15 V-0.3V, continue scanning for 4-20 cycles; then in a mixed solution of KCl and HCl, further scan at a scanning speed of 40-60mV / s in a voltage range of -0.2-0.5V for 4-20 cycles; the ratio of creatinase, creatinase and sarcosine oxidase in the enzyme composition is 5:2:1.25; on the electrode surface, the creatinase loading is 0.5-0.85U / mm 2 The creatinase loading is 0.15-0.28U / mm 2 , and the sarcosine oxidase loading is 0.1-0.15U / mm 2 .

11. The method according to claim 10, characterized in that: The enzyme composition is fixed on the surface of the electron mediator enhancement layer by dripping a mixture of the enzyme composition and glutaraldehyde on the surface of the electron mediator enhancement layer and drying it at 0-4°C.

12. A method for detecting creatinine by electrochemical method, comprising: Using the biosensor for electrochemical detection of creatinine as claimed in claim 5, connecting the wire of the biosensor to an external potential detection device; A buffer solution with a pH value of 7-9.5 is dripped into the sample loading chamber of the biosensor, and then a plasma or whole blood sample to be tested is dripped into the buffer solution. The potential detection device performs detection at an operating voltage of -0.2~0V to obtain the intensity of the response current, and finally the creatinine concentration in the sample is calculated based on the relationship curve between the creatinine concentration and the current signal.

13. The method according to claim 12, characterized in that: The pH value of the buffer solution is 7.

4.

14. The method of claim 12, wherein: The working voltage is -0.2~-0.1V.

15. The method of claim 12, wherein: The working voltage is -0.1V.

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