Working electrode, preparation method thereof, electrochemical wearable biosensor, detection kit and miniature electrochemical workstation

Abstract

The invention relates to a working electrode, a preparation method thereof, an electrochemical wearable biosensor, a detection kit and a miniature electrochemical workstation, the working electrode comprises a substrate electrode, and a metal nano-enzyme and glucose oxidase located on the substrate electrode, and the metal nano-enzyme has peroxidase-like activity. Palladium is used as a main alloy, boron and phosphorus elements are added through a hydrothermal method, the multi-element three-dimensional palladium / boron / phosphorus metal wire net-shaped nano composite material which is simple in structure and has the electron transfer advantage is synthesized, gaps between palladium atoms are reduced through introduction of boron and phosphorus, the efficient electron transfer effect is achieved, the electro-catalytic performance is optimized, and the electro-catalytic performance is improved. The prepared nano composite material has peroxidase-like catalytic activity, and the characteristics of peroxidase-like catalytic activity, higher catalytic activity and stability and the like of the material are utilized, and the electrochemical sensing technology, the screen-printed electrode and the introduction of wearable equipment capable of promoting perspiration are combined; therefore, high-sensitivity detection of the glucose content in sweat is realized.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a working electrode, a preparation method thereof, an electrochemical biosensor, a detection kit and a micro electrochemical workstation. Background Art

[0002] Diabetes is a chronic disease characterized by hyperglycemia, which is caused by absolute or relative insufficient secretion of insulin and impaired utilization. Effective treatment and management of diabetes requires regular monitoring of blood sugar, especially for patients with type 2 diabetes and gestational diabetes who can have a good prognosis by controlling blood sugar through self-management. Continuous blood glucose monitoring can well guide diabetic patients to strengthen exercise and adjust their diet and other self-management of blood sugar. At present, the more popular blood sugar test is mainly through blood testing. Blood testing of blood sugar levels is an invasive test, which brings certain distress to patients, and it is also impossible to perform continuous real-time monitoring. In addition, during the self-management process of diabetic patients, they lack the affirmation and sense of autonomy of their own health because they cannot understand the effect of their own blood sugar control in real time, and thus lack the internal motivation to maintain lifestyle changes. The development of a continuous, non-invasive and convenient blood sugar monitoring system has become an urgent need for diabetic patients.

[0003] Human sweat contains a variety of chemicals such as glucose, lactate and uric acid, which reflect nutritional and metabolic status and provide a comprehensive insight into the health of the body. Human sweat is easy to obtain, and there is a strong correlation between sweat glucose concentration and blood glucose level, making it the most suitable medium for non-invasive self-monitoring of blood glucose in real time. Compared with the millimolar level of glucose in blood, the glucose content in sweat is at the micromolar level, which means that the sensitivity requirement for detecting glucose content in sweat as a sample is higher, that is, the linear range is lower. Electrochemical sensing technology is a measurement principle based on biosensor technology. It can capture the electrochemical signal changes generated on the electrode surface through high-sensitivity sensing technology, and can also convert the captured electrochemical signal into a detectable electrical signal, so as to accurately and quantitatively detect low-concentration analytes. Electrochemical sensing technology has the advantages of portability, fast response, high sensitivity and low cost, so it is most commonly used in the determination of trace substances. Screen printing is a technology widely used to manufacture miniaturized electrochemical sensors. Because of its easy assembly, good repeatability and low preparation cost, it provides a good practical basis for wearable detection of glucose in sweat.

[0004] At present, conventional enzymatic detection of glucose requires large-scale biochemical equipment and venipuncture to collect serum for detection. Even if this method has high sensitivity and specificity in catalyzing glucose, it cannot fully meet the needs of low sample volume and low concentration glucose detection. Precious metal materials (such as Au, Pd, Ag and Pt) have certain capabilities in non-enzymatic electrochemical catalysis of low concentration glucose, among which gold nanoparticles (Au NPs) are the most widely used. Au NPs produce gluconic acid while catalyzing glucose to produce H2O2, and gluconic acid will attach to gold nanoparticles and reduce their activity until they lose their activity in catalyzing glucose. Therefore, how to utilize the high sensitivity and specificity of enzymes while achieving enzyme electrodes with low sample volume and low concentration detection limits has become the key to realizing wearable electrochemical methods for detecting glucose in sweat. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the present invention provides a working electrode, a preparation method thereof, an electrochemical biosensor, a detection kit and a micro electrochemical workstation to detect the glucose content in sweat with high sensitivity and specificity.

[0006] To achieve the above objectives, the scheme of this application is as follows:

[0007] In a first aspect, the present invention provides a working electrode, comprising a substrate electrode and a metal nanozyme and glucose oxidase located on the substrate electrode, wherein the metal nanozyme has peroxidase-like activity.

[0008] Optionally, the substrate electrode is selected from screen-printed electrodes.

[0009] Optionally, the metal nanozyme is selected from palladium / boron / phosphorus metal wire network nanocomposite materials.

[0010] Optionally, the method for preparing the metal nanozyme comprises the following steps:

[0011] Ammonia water, ammonium fluoride, a water-soluble salt of palladium, boric acid and hypophosphite are added to an aqueous solution of a surfactant, incubated, and then a water-soluble salt of boron is added, reacted, centrifuged, and washed to obtain the metal nanozyme.

[0012] Optionally, the surfactant is selected from polyoxyethylene polyoxypropylene ether block copolymers.

[0013] Optionally, the water-soluble salt of palladium is selected from at least one of palladium chloride, palladium nitrate and palladium sulfate.

[0014] Optionally, the hypophosphite is selected from at least one of sodium hypophosphite, potassium hypophosphite and ammonium hypophosphite.

[0015] Optionally, the water-soluble salt of boron is selected from dimethylamine borane.

[0016] Optionally, the molar ratio of the surfactant, ammonia water, ammonium fluoride, water-soluble salt of palladium, boric acid, hypophosphite and water-soluble salt of boron is 1:1-1.2:0.3-0.04:0.005-0.01:0.08-0.15:0.03-0.04:0.05-0.15.

[0017] Optionally, the incubation temperature is 80-85° C., and the incubation time is 8-12 min.

[0018] In a second aspect, the present application also provides a method for preparing the working electrode as described above, comprising the following steps:

[0019] The metal nanozyme is dripped onto the surface of the substrate electrode and dried, and glucose oxidase is dripped onto the surface of the substrate electrode and dried again to obtain the working electrode.

[0020] In a third aspect, the present application also provides an electrochemical biosensor, which includes the working electrode as described above or the working electrode prepared according to the method as described above.

[0021] In a fourth aspect, the present application also provides a detection kit for detecting the glucose content in sweat, wherein the detection kit comprises the working electrode as described above or the working electrode prepared according to the method as described above or the electrochemical biosensor as described above.

[0022] In a fifth aspect, the present application also provides a micro electrochemical workstation, which includes the working electrode as described above or the working electrode prepared according to the method as described above or the electrochemical biosensor as described above.

[0023] Beneficial effects of the present invention:

[0024] The present application uses palladium (Pd) as the main alloy, and adds boron (B) and phosphorus (P) elements through a hydrothermal method to explore a multi-element three-dimensional palladium / boron / phosphorus metal wire mesh nanocomposite material with simple structure and electron transfer advantages. The introduction of boron (B) and phosphorus (P) reduces the interatomic gap of Pd, plays a role in efficient electron transfer, optimizes the electrocatalytic performance of the material, and obtains a multi-element three-dimensional palladium / boron / phosphorus metal wire mesh nanocomposite material with peroxidase-like catalytic activity. Based on this, the present application utilizes the peroxidase-like catalytic activity, higher catalytic activity and stability of the material, combines electrochemical sensing technology and the introduction of screen-printed electrodes and wearable devices with sweat-promoting properties to achieve high-sensitivity detection of glucose content in sweat, and provides new ideas for clinical real-time, non-invasive and convenient glucose monitoring research. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0026] Figure 1 Schematic diagram of the construction process and detection principle of the electrochemical biosensor of the present application, Mixing means mixing, Washing means washing, GLU means glucose, Without means no, With means yes, the same below;

[0027] Figure 2 To characterize the result diagram;

[0028] Figure 3 The results of enzyme dynamics, electrochemical impedance spectroscopy and cyclic voltammetry characterization of modification steps and feasibility verification are shown in Figure 1. Time represents time, Absorbance represents absorbance, Potential represents voltage, and Current represents current, the same below.

[0029] Figure 4 This is the concentration of glucose oxidase and the optimization result of the measurement buffer pH. Concentration indicates the concentration.

[0030] Figure 5 This is a sweat-specific result graph, Blank indicates blank control group, UA indicates uric acid, Bun indicates urea, βL indicates β-lactose, and Mix indicates mixed;

[0031] Figure 6 It is the DPV differential pulse response curve and linear regression relationship curve for detecting different concentrations of glucose.

[0032] Figure 7 This is the schematic diagram of the micro electrochemical workstation, Voltage regulator represents voltage regulator, Battery represents battery, Boost converter represents boost converter, MCU represents micro control unit, DAC represents digital-to-analog converter, ADC represents analog-to-digital converter, UART represents universal asynchronous receiver-transmitter, User interface represents smartphone interface, CE represents counter electrode, RE represents reference electrode, WE represents working electrode, and IP represents voltage stimulation electrode. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] like Figure 1 As shown, the principle of the present application is: using palladium (Pd) as the main alloy, adding boron (B) and phosphorus (P) elements through a hydrothermal method, and synthesizing a multi-element three-dimensional palladium / boron / phosphorus metal wire mesh nanocomposite material with simple structure and electron transfer advantages, wherein the introduction of boron (B) and phosphorus (P) reduces the interatomic gap of Pd, plays a role in efficient electron transfer, optimizes the electrocatalytic performance of the material, and obtains a multi-element three-dimensional palladium / boron / phosphorus metal wire mesh nanocomposite material with peroxidase-like catalytic activity. Based on this, the present application utilizes the peroxidase-like catalytic activity, higher catalytic activity and stability of the material, combined with electrochemical sensing technology and the introduction of screen-printed electrodes and wearable devices with sweat-promoting properties, to achieve high-sensitivity detection of glucose content in sweat, providing new ideas for clinical real-time, non-invasive and convenient glucose monitoring research.

[0035] In the following description, numerous details are discussed to provide a more thorough explanation of the embodiments of the present invention; however, it is obvious to those skilled in the art that the embodiments of the present invention can be implemented without these specific details.

[0036] Experimental materials: Palladium chloride (PdCl2) was purchased from Adamas-beta (Shanghai, China), namely Adamas Reagent Co., Ltd., boric acid (H3BO3) was purchased from Sangon Biotech (Shanghai, China), namely Sangon Biotech (Shanghai) Co., Ltd., polyoxyethylene polyoxypropylene ether block copolymer F-127, glucose oxidase and ammonia water were purchased from Macklin (Shanghai, China), namely Shanghai Macklin Biochemical Technology Co., Ltd., ammonium fluoride (NH4F) and hydrogen peroxide (H2O2) were purchased from Sigma-Aldrich (StLouis, USA), sodium hypophosphite (NaH2PO2) and 3,3',5,5'-tetramethylbenzidine (TMB) were purchased from Adamas-beta (Shanghai, China), namely Adamas Reagent Co., Ltd., dimethylamine borane (DMAB) was purchased from Acros Organics (Geel, Belgium) were purchased from Beijing Inokai Technology Co., Ltd. Hydrochloric acid (HCl), sodium hydroxide (NaOH), and anhydrous ethanol were purchased from Greagent (Shanghai, China) and Shanghai Titan Technology Co., Ltd. Horseradish peroxidase (HRP) and 96-well plates were purchased from Sangon (Shanghai, China) and Bioengineering (Shanghai) Co., Ltd. Screen-printed electrodes were purchased from Poten (Shandong, China) and Poten Technology (Weihai) Co., Ltd. All chemicals used in the experiments were of analytical grade, and deionized water was prepared in a Millipore Mill-Q purification system with a resistivity of 18 MΩ cm -1 .

[0037] Detection instruments: The electrochemical experiments were measured by a DONGHUA DH7000 electrochemical working system (Jiangsu, China), the absorbance was measured by an infinite E Plex multifunctional microplate reader from Switzerland, and the transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) were performed using a Tencnai G2 F30 from FEI, USA (with an accelerating voltage of 300 kV).

[0038] (I) Synthesis of Pd / B / P metal wire network nanocomposites (PdBP NWs nanozymes)

[0039] 0.0887 g of palladium chloride powder was added to 0.5 mL of 2 M concentrated hydrochloric acid for sufficient reaction, and the volume was made up to 50 mL with deionized water, and mixed evenly to prepare a palladium chloride solution with a concentration of 10 mM;

[0040] Dissolve 0.1248 g of ammonium fluoride powder in 10 mL of deionized water and stir to prepare an ammonium fluoride solution with a concentration of 0.337 M;

[0041] Dissolve 0.0624 g of boric acid powder in 10 mL of deionized water and stir to prepare a boric acid solution with a concentration of 0.101 M;

[0042] Dissolve 0.0360 g of sodium hypophosphite monohydrate crystals in 10 mL of deionized water and stir to prepare a sodium hypophosphite solution with a concentration of 0.034 M;

[0043] Dissolve 0.0589 g of dimethylamine borane crystals in 10 mL of deionized water and stir to prepare a dimethylamine borane solution with a concentration of 0.1 M;

[0044] 45 mL of deionized water was added to a round-bottom flask containing a magnetic stirring bar, and the mixture was placed in a heater, maintained at 85° C. and a rotation speed of 750 rpm. 90 mg of surfactant F-127 (polyoxyethylene polyoxypropylene ether block copolymer F-127), 2.4 mL of palladium chloride solution, 2.4 mL of 2.5 wt% ammonia water, 3 mL of ammonium fluoride solution, 3 mL of boric acid solution and 3 mL of sodium hypophosphite solution were added to the round-bottom flask in sequence. After 10 minutes, 3 mL of dimethylamine borane solution was added, and the reaction solution quickly changed from colorless to black. After continuous heating and stirring for 5 minutes, heating and stirring were stopped, and the precipitate was collected by centrifugation. The precipitate was washed with 50% ethanol, and then washed twice with deionized water, and then placed in 2.4 mL of deionized water and refrigerated for storage (i.e., palladium / boron / phosphorus metal wire mesh nanocomposite material).

[0045] (II) Nanozyme characterization

[0046] Transmission electron microscopy (TEM) was used to characterize the PdBP NWs nanozyme prepared in (a) to determine the spatial structure and morphology and verify whether the PdBP NWs nanozyme was successfully synthesized. Figure 2 As shown in A to 2D;

[0047] STEM-EDS elemental mapping was used to analyze the chemical composition of nanozyme PdBP NWs. Figure 2 E to 2H.

[0048] Depend on Figure 2 It can be seen that the prepared PdBP NWs nanozyme is in the form of a wire-net composite, and line segments can be seen at 100nm ( Figure 2 A), further magnification, the reticular morphology can be seen at 50nm ( Figure 2 B and 2C), the lattice measurement shows that the spacing is 0.23nm ( Figure 2 D). The chemical composition of PdBP NWs nanozyme was analyzed by STEM-EDS elemental mapping, and the three elements Pd, B and P were clearly observed, indicating that the three-element PdBP NWs nanozyme was successfully synthesized ( Figure 2 E to 2H).

[0049] (III) Verification of catalase activity of PdBP NWs

[0050] Take 2.5×10 -8 5 μL of each of 100 μL 0.2 mM TMB and 100 μL 14 mM H2O2 were added to the microwells of the ELISA plate, and then 100 μL 0.2 mM TMB and 100 μL 14 mM H2O2 were added and mixed. The ELISA plate was placed in a Varioskan LUX multifunctional ELISA reader to measure the absorbance change. The 650 nm absorption peak measurement results are shown in Figure 2. Figure 3 As shown in A.

[0051] like Figure 3 As shown in A, compared with blank ( Figure 3 For curve d in A, HRP( Figure 3 Curve a) in A catalyzes H2O2 to make TMB color development most obvious, followed by PdBP nanozyme ( Figure 3 Curve b in A), followed by Pd nanoparticles ( Figure 3 Curve c in A). This result shows that the PdBP NWs nanozyme has catalase-like activity, and the introduction of B and P elements increases the activity of the Pd nanoparticles of the nanozyme.

[0052] (IV) Preparation and characterization of glucose oxidase@PdBP NWs nanozyme electrodes and feasibility verification of glucose measurement

[0053] 4.1 Take 20 μL of the PdBP NWs nanozyme synthesized in (a) and cast it on the screen-printed electrode, dry it at 37°C until dry, then cast 20 μL of 2.5 mg / mL glucose oxidase (GOX) solution on the screen-printed electrode and dry it at 37°C until dry to prepare a working electrode.

[0054] 4.2 Take 20 μL of the synthesized PdBP NWs nanozyme in (a) and cast it on the screen-printed electrode, and dry it at 37°C to obtain a nanozyme electrode.

[0055] The working electrode, the blank screen-printed electrode identical to that in 4.1 (i.e., the screen-printed electrode without any treatment), and the nanozyme electrode prepared by adding only PdBP NWs nanozyme and drying (i.e., the nanozyme electrode prepared in 4.2) were subjected to electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) characterization. The results are shown in FIG. Figure 3 B and 3C.

[0056] Depend on Figure 3 As shown in B and 3C, the screen-printed electrode is connected to the PdBP NWs nanozyme ( Figure 3 After the modification of curve b) in B, the redox peak current decreased slightly and the electrochemical impedance increased. Subsequently, glucose oxidase was added to further modify ( Figure 3 After curve c) in B, the redox peak current decreased significantly, and the electrochemical impedance increased significantly. This result shows that the two-step modification of PdBP NWs nanozyme and glucose oxidase (GOX) electrode was successfully completed.

[0057] The glucose test solution was diluted with PBS buffer with a pH of 5.5 and a concentration of 0.1M to a glucose solution with a concentration of 2mM. Two working electrodes were prepared according to 4.1, one of which was dripped with glucose solution (experimental group), and the other was dripped with PBS buffer with a pH of 5.5 and a concentration of 0.1M (blank control group). The electrochemical signal was measured using differential pulse voltammetry (DPV). The results are as follows: Figure 3 As shown in D.

[0058] Depend on Figure 3 D shows that, compared with the blank control group, the experimental group has an obvious peak. This result shows that the working electrode prepared in this application can realize glucose detection.

[0059] (V) Parameter Optimization

[0060] In order to obtain the best analytical performance, the two experimental parameters of glucose oxidase concentration and buffer pH were optimized in turn. The specific steps are as follows:

[0061] According to 4.1, several working electrodes were prepared, and glucose oxidase with concentrations of 0.5, 1.0, 1.5, 2.0, and 2.5 mg / mL were added respectively. After drying, a 2 mM glucose solution diluted with PBS buffer with a pH of 5.5 and a concentration of 0.1 M was added, and the detection was performed respectively. The results are as follows: Figure 4 As shown in A.

[0062] Depend on Figure 4 A shows that the optimal concentration of glucose oxidase is 1.5 mg / mL.

[0063] Prepare several working electrodes according to 4.1, add 1.5 mg / mL glucose oxidase to each electrode, and after drying, add 2 mM glucose solution diluted with 0.1 M PBS buffer with pH values ​​of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 to each electrode, and perform detection. The results are as follows: Figure 4 As shown in B.

[0064] Depend on Figure 4 B shows that the optimal pH value of the buffer solution is 5.5.

[0065] (VI) Specificity and stability test

[0066] To evaluate specificity, this application selected potential interfering substances (uric acid, urea, β-lactose) in human sweat to conduct cross-reaction experiments of electrochemical biosensors. The specific steps are as follows:

[0067] Prepare the working electrode according to 4.1, add 1.5 mg / mL glucose oxidase, use 1 mM glucose solution as reference, then detect 10 mM uric acid, urea and β-lactose respectively, and finally detect the mixed solution containing 1 mM glucose, 10 mM uric acid, 10 mM urea and 10 mM β-lactose, and use PBS buffer with pH value of 5.5 and concentration of 0.1 M as blank control (using this PBS buffer instead of the sample to be tested). The results are as follows: Figure 5 As shown in A;

[0068] To evaluate the stability, the present application conducted a stability experiment of the electrochemical biosensor, and the specific steps were as follows:

[0069] Prepare the working electrode according to 4.1, add 1.5 mg / mL glucose oxidase, and test 50, 100, 250, 500, and 1000 μM glucose solutions (diluted with PBS buffer with a pH of 5.5 and a concentration of 0.1 M). The results are as follows: Figure 5 As shown in B.

[0070] like Figure 5 A shows that the current value significantly increases when glucose is present. Compared with the blank group, the presence of other interfering substances does not significantly change the signal. It can be seen that even in the presence of a higher concentration of interfering substances, the response signal of the electrochemical biosensor of the present application to glucose is substantially unaffected. This result shows that the electrochemical biosensor of the present application has excellent specificity.

[0071] like Figure 5 B shows that there is no significant difference in the detection results between repeated experiments under different concentration conditions. This result shows that the electrochemical biosensor of the present application has excellent stability.

[0072] (VII) Sensitivity test

[0073] In order to explore the linear range of the electrochemical biosensor of the present application, the present application conducted a sensitivity test, the specific steps are as follows:

[0074] Prepare the working electrode according to 4.1, add 1.5 mg / mL glucose oxidase, and after drying, add 2 mM glucose solution diluted with PBS buffer at pH 5.5, and detect different concentrations of glucose. The results are as follows: Figure 6 shown.

[0075] like Figure 6 As shown, the electrochemical biosensor made of the working electrode of the present application detects glucose with a good linear relationship, and the standard curve is: Y = 76.23109lgC + 39.21895, R 2 =0.9887, linear range: 50-2000 μM, detection limit: 4.77 μM (S / N=3). The results show that the electrochemical biosensor made of the working electrode of the present application has a lower detection limit and higher sensitivity, and can be used to detect trace glucose in sweat.

[0076] (VIII) Feasibility test

[0077] In order to explore the feasibility of using the electrochemical biosensor made of the working electrode of the present application in sweat detection, specifically:

[0078] Simulated sweat (specifically: 0.5 g NaCl, 0.22 g NaH2PO4 and 0.05 g L-histidine per 100 mL water) was prepared according to ISO105-E04:2013 and mixed into PBS buffer and 50 / 100 / 1000 μM glucose PBS buffer at a ratio of 10% respectively as the sample to be tested;

[0079] The working electrode was prepared according to 4.1, and the concentration of glucose oxidase was 1.5 mg / mL. After drying, the recovery rate of each sample to be tested was tested. The results are shown in Table 1.

[0080] Table 1 Recovery test results

[0081]

[0082] As shown in Table 1, the glucose test results of 50μM / 100μM / 1000μM after adding 10% simulated sweat, and the recovery rates calculated from the corresponding concentration glucose test results in (VII) are 91.44%, 107.54%, and 93.76%, respectively. This result shows that the electrochemical biosensor made of the working electrode of the present application can detect trace amounts of glucose in a sweat environment.

[0083] Without limitation thereto, the present application can be extended to match a micro electrochemical workstation capable of stimulating perspiration and measuring differential pulse voltammetry (DPV) electrochemical signals, such as Figure 7As shown in the figure, the micro electrochemical workstation outputs a rated voltage through the two stimulation electrodes of DAC2 to stimulate the skin to sweat. After turning off the stimulation voltage, the measurement mode is turned on. The working potential set on the mobile phone is sent to the MCU through the Bluetooth module. The MCU adjusts the current between the working electrode and the counter electrode through DAC1 to keep the working electrode potential constant. The current flowing through the working electrode is converted into voltage by the transimpedance amplifier, and the voltage peak signal is read through the analog-to-digital converter. The voltage signal collected by the analog-to-digital converter is uploaded to the smartphone through the Bluetooth module and converted into glucose concentration by the smartphone APP. In addition, the rated concentration glucose voltage signal can be measured using a standard product and compared with the built-in standard linear curve of the smartphone APP. The curve can be calibrated by the slope and intercept to ensure the measurement accuracy of different batches of working electrodes.

[0084] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A working electrode, characterized in that The working electrode comprises a substrate electrode and a metal nanozyme and glucose oxidase located on the substrate electrode, wherein the metal nanozyme has peroxidase-like activity.

2. The working electrode according to claim 1, characterized in that The substrate electrode is selected from screen-printed electrodes.

3. The working electrode according to claim 1, characterized in that The metal nanozyme is selected from palladium / boron / phosphorus metal wire mesh nanocomposite materials.

4. The working electrode according to claim 3, characterized in that The preparation method of the metal nanozyme comprises the following steps: Ammonia water, ammonium fluoride, a water-soluble salt of palladium, boric acid and hypophosphite are added to an aqueous solution of a surfactant, incubated, and then a water-soluble salt of boron is added, reacted, centrifuged, and washed to obtain the metal nanozyme.

5. The working electrode according to claim 4, characterized in that The surfactant is selected from polyoxyethylene polyoxypropylene ether block copolymer; And / or, the water-soluble salt of palladium is selected from at least one of palladium chloride, palladium nitrate and palladium sulfate; And / or, the hypophosphite is selected from at least one of sodium hypophosphite, potassium hypophosphite and ammonium hypophosphite; And / or, the water-soluble salt of boron is selected from dimethylamine borane.

6. The working electrode according to claim 4, characterized in that The molar ratio of the surfactant, ammonia water, ammonium fluoride, water-soluble salt of palladium, boric acid, hypophosphite and water-soluble salt of boron is 1:1-1.2:0.3-0.04:0.005-0.01:0.08-0.15:0.03-0.04:0.05-0.15; And / or, the incubation temperature is 80-85° C., and the incubation time is 8-12 min.

7. The method for preparing a working electrode according to any one of claims 1 to 6, characterized in that: The following steps are involved: The metal nanozyme is dripped onto the surface of the substrate electrode and dried, and the glucose oxidase is dripped onto the surface of the substrate electrode and dried again to obtain the working electrode.

8. An electrochemical biosensor, characterized in that: The electrochemical biosensor comprises the working electrode according to any one of claims 1 to 6 or the working electrode prepared according to the method according to claim 7.

9. A test kit for detecting glucose content in sweat, characterized in that: The detection kit comprises the working electrode according to any one of claims 1 to 6 or the working electrode prepared according to the method according to claim 7 or the electrochemical biosensor according to claim 9.

10. A micro electrochemical workstation, characterized in that: The micro electrochemical workstation comprises the working electrode according to any one of claims 1 to 6 or the working electrode prepared according to the method according to claim 7 or the electrochemical biosensor according to claim 9.

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