A metal organic framework hybrid nanosheet and a method for real-time detection of glucose and uric acid content in metabolites

Abstract

The application belongs to the technical field of electrochemical detection and analysis, and relates to a kind of metal organic framework hybrid nanosheet and a method for real-time non-invasive detection of glucose and uric acid content in metabolites.The metal organic framework hybrid nanosheet is a Cu-TCPP(Fe) / Mxene composite material.The application replaces the traditional electrolytic cell with a paper-based electrode, further reducing experimental costs and improving the portability and operability of the experiment;By first applying Cu-TCPP(Fe) / Mxene composite material to paper-based electrode, the speed of electron transfer and the specific surface area of reaction are improved.Therefore, the method has the advantages of rapidity, portability, high sensitivity, wide detection range and good specificity, and has important practical significance for the ultra-sensitive non-invasive detection of glucose and uric acid, and good guiding significance for realizing on-site rapid and ultra-sensitive detection technology.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical detection and analysis technology, and more specifically, relates to a working electrode of a metal-organic framework composite material modified three-electrode dual-channel paper-based electrode, and a detection method for detecting glucose and uric acid using the paper-based electrode. Background Technology

[0002] With continuous social development and improved living standards, an increasing number of people suffer from both diabetes and gout, both chronic diseases requiring long-term treatment. Patients with both diabetes and gout generally need long-term medication and regular blood glucose and uric acid monitoring. Accurate and long-term monitoring of blood glucose / uric acid concentrations is crucial for assessing the effectiveness of treatment for patients with both diabetes and gout. Currently, the commonly used method for detecting glucose and uric acid involves using blood glucose meters and uric acid meters. However, for patients with both diabetes and uric acid, this method requires two blood draws for separate testing and monitoring, doubling the workload in the self-management of chronic diseases.

[0003] Therefore, there is a strong interest and desire to develop highly sensitive and reliable biosensors for the detection of multiple analytes from human bodily fluids. Electrochemical multicomponent analysis has become a mainstream method for the simultaneous detection of multiple analytes, widely used in biochemistry, immunosensors, and molecular diagnostics. Studies have shown that composite electrochemical sensors with planar electrochemical reaction channels offer a promising solution for the rapid development of the big data health market. While biosensors have been reported to detect multiple targeted biomolecules, simultaneous non-invasive detection of blood glucose and uric acid for point-of-care (POC) testing in diabetic and gout patients has not yet been reported. Considering the large number of such patients and the unnecessary pain caused by the need for two punctures in daily blood glucose and uric acid monitoring, it is necessary to develop a non-invasive, highly sensitive, rapid, simple, and low-cost detection method for real-time monitoring of glucose and uric acid. Summary of the Invention

[0004] The purpose of this invention is to provide a metal-organic framework hybrid nanosheet, a working electrode based on a three-electrode dual-channel paper-based electrode modified with a metal-organic framework composite material, a paper-based electrochemical biosensor based on a Cu-TCPP(Fe) / Mxene composite material and an oxidase, and a detection method for detecting glucose and uric acid using this paper-based electrode or sensor. This method can detect the glucose and uric acid content in metabolites with high sensitivity.

[0005] To achieve the above objectives, a first aspect of the present invention provides a metal-organic framework hybrid nanosheet, wherein the metal-organic framework hybrid nanosheet is a Cu-TCPP(Fe) / Mxene composite material.

[0006] According to a preferred embodiment of the present invention, the preparation method of the Cu-TCPP(Fe) / Mxene composite material includes the following steps:

[0007] (1) Copper nitrate trihydrate, polyvinylpyrrolidone and trifluoroacetic acid are mixed to obtain a first mixture. Under stirring, tetrakis(4-carboxyphenyl) porphyrin (III) chloride dissolved in N,N-dimethylformamide / ethanol solution is added dropwise to the first mixture to obtain a second mixture. The second mixture is subjected to ultrasonic treatment and then heated for reaction.

[0008] (2) After the reaction, the system was cooled and centrifuged, washed and dried to obtain Cu-TCPP(Fe) powder;

[0009] (3) Mix the Mxene aqueous solution with the Cu-TCPP(Fe) powder obtained in step (2), stir and centrifuge to remove the uncomposite material, and vacuum dry the resulting solid to obtain the Cu-TCPP(Fe) / Mxene composite material.

[0010] Specifically, in step (1), compared to 1 mg of copper nitrate trihydrate, the amount of polyvinylpyrrolidone added is 4-5 mg, the amount of trifluoroacetic acid added is 16-17 μL, the amount of tetrakis(4-carboxyphenyl)porphyrin iron(III) chloride added is 1.5-2 mg, and the amount of N,N-dimethylformamide / ethanol solution added is 4-6 mL; the volume ratio of N,N-dimethylformamide to ethanol in the N,N-dimethylformamide / ethanol solution is 2.5-3.5:1; the heating reaction temperature is 75-85℃, and the time is 3-5 h. More specifically, in step (1), compared to 1 mg of copper nitrate trihydrate, the amount of polyvinylpyrrolidone added is 4.17 mg, the amount of trifluoroacetic acid added is 16.67 μL, the amount of tetrakis(4-carboxyphenyl)porphyrin iron(III) chloride added is 1.83 mg, and the amount of N,N-dimethylformamide / ethanol solution added is 5 mL; the volume ratio of N,N-dimethylformamide to ethanol in the N,N-dimethylformamide / ethanol solution is 3:1; the heating reaction temperature is 80 °C, and the time is 4 h.

[0011] Specifically, in step (2), the centrifugation speed is 8000-12000 rpm, and the centrifugation time is 10-20 min; a red solid is obtained after centrifugation; the washing method includes: adding ethanol, shaking to dissolve the solid, then ultrasonic treatment, and centrifugation separation. More specifically, in step (2), the centrifugation speed is 10000 rpm, and the centrifugation time is 15 min; a red solid is obtained after centrifugation; the washing method includes: adding water, shaking to dissolve the solid, then ultrasonic treatment, and centrifugation separation.

[0012] Specifically, in step (3), the Mxene aqueous solution and Cu-TCPP(Fe) powder are stirred and mixed at room temperature. The concentration of the Mxene aqueous solution is 8-12 mg / L, and the amount of Cu-TCPP(Fe) powder added is 800-12000 mg compared to 1 mg Mxene. The centrifugation conditions include: a rotation speed of 8000-12000 rpm and a time of 10-20 min. More specifically, in step (3), the Mxene aqueous solution and Cu-TCPP(Fe) powder are stirred and mixed at room temperature. The concentration of the Mxene aqueous solution is 10 mg / L, and the amount of Cu-TCPP(Fe) powder added is 1000 mg compared to 1 mg Mxene. The centrifugation conditions include: a rotation speed of 10000 rpm and a time of 15 min.

[0013] According to the present invention, the metal-organic framework composite material comprises 2D-MOF and Mxene, wherein Mxene has good electrocatalytic performance, and the combination of metalloporphyrin-based MOF nanosheets and Mxene can simulate enzyme cascade reactions.

[0014] The principle of this invention is as follows: First, a Cu-TCPP(Fe) / Mxene metal-organic framework composite material is modified onto the working electrode by drop-coating. Then, glucose oxidase and uric acid oxidase are modified onto the working electrode by drop-coating. In the presence of the target analyte, the oxidase oxidizes the target analyte into gluconic acid, allantoic acid, and H2O2. The Cu-TCPP(Fe) / Mxene composite material then catalyzes H2O2, producing O2 and H2O, and simultaneously generating electrons, thereby causing a change in current. The magnitude of the generated current can be detected by connecting to a portable electrochemical workstation. The current value is positively correlated with the concentration of the target analyte, allowing for quantitative detection and the creation of a standard curve. The same method is used to measure the sample to be tested, and the measured current value is substituted into the standard curve to obtain the concentrations of glucose and uric acid.

[0015] Based on the above principles of this invention, a paper-based electrode with a three-electrode dual-channel system can be designed. The ink for the electrode can be supplied separately or printed onto an NC film using screen printing technology.

[0016] The metal-organic framework composite material of the present invention has excellent electrochemical catalytic oxidation performance of H2O2, thereby causing a change in current.

[0017] In this invention, the oxidase must have the ability to oxidize glucose and uric acid to produce H2O2. Based on the characteristics of enzymes having high specificity and high catalytic efficiency for their substrates, the oxidase is preferably glucose oxidase and uric acid oxidase.

[0018] Based on the principles of electrochemical reactions and the above-described principles of this invention, those skilled in the art can design suitable electrode systems, including three-electrode systems and two-electrode systems. According to a preferred embodiment of this invention, the electrode adopts a three-electrode system, including a working electrode, a reference electrode, and a counter electrode.

[0019] Specifically, a second aspect of the present invention provides a paper-based electrode based on a metal-organic framework composite material and an oxidase-modified three-electrode dual-channel system, the electrode comprising the following components:

[0020] (1) Working electrode: modified carbon and graphene electrode;

[0021] (2) Reference electrode: Ag / AgCl;

[0022] (3) Counter electrode: carbon and graphene electrode;

[0023] The modified carbon-plus-graphene electrode is an electrode that has been sequentially modified with the Cu-TCPP(Fe) / Mxene composite material and oxidase described above. The modified carbon-plus-graphene electrode is capable of performing redox reactions on glucose and uric acid.

[0024] The oxidases include glucose oxidase and urate oxidase.

[0025] According to a preferred embodiment of the present invention, the modification method includes:

[0026] (a) Dissolve glucose oxidase and uricase oxidase in PBS solution;

[0027] (b) Fabrication of a three-electrode dual-channel paper-based electrode using screen printing technology;

[0028] (c) The Cu-TCPP(Fe) / Mxene solution was drop-coated onto the working electrode of the paper-based electrode obtained in step (b) and placed in a refrigerator at 4°C to dry.

[0029] (d) The oxidase solution from step (a) is applied to the working electrode after drying in step (c) by drop coating to obtain the modified carbon-plus-graphene electrode. The modified carbon-plus-graphene electrode can be stored in a refrigerator at 4°C for drying.

[0030] A third aspect of the present invention provides a paper-based electrochemical biosensor based on Cu-TCPP(Fe) / Mxene composite material and oxidase, comprising:

[0031] i) The Cu-TCPP(Fe) / Mxene composite material mentioned above;

[0032] ii) Oxidases, including glucose oxidase and uricase oxidase;

[0033] iii) The paper-based electrode of the three-electrode dual-channel system is preferably prepared by screen printing technology;

[0034] iv) Portable electrochemical workstation.

[0035] A fourth aspect of the present invention provides a method for real-time detection of glucose and uric acid content in a living fluid, the method being based on the electrode described above or the electrochemical biosensor described above, and comprising the following steps:

[0036] (1) Obtaining metal-organic framework nanosheets Cu-TCPP(Fe);

[0037] (2) React Cu-TCPP(Fe) with Mxene solution to form Cu-TCPP(Fe) / Mxene composite solution;

[0038] (3) Dissolve glucose oxidase and uricase oxidase in PBS solution;

[0039] (4) Paper-based electrodes with a three-electrode dual-channel system were prepared by screen printing technology;

[0040] (5) The Cu-TCPP(Fe) / Mxene composite material solution obtained in step (2) is drop-coated onto the working electrode of the paper-based electrode obtained in step (4) and placed in a refrigerator at 4°C to dry;

[0041] (6) Modify the oxidase solution prepared in step (3) onto the working electrode after drying in step (5) by drop coating to obtain the modified electrode, which can be placed in a refrigerator at 4°C for drying and storage.

[0042] (7) Take glucose and uric acid standard solutions respectively and add them to the working area of ​​the electrode modified in step (6). This area is a hydrophilic area.

[0043] (8) Measure the current signal of the reaction product solution obtained in step (7) and establish a standard curve of glucose and uric acid standard solution concentration and current signal;

[0044] (9) Perform the above test on the test solution containing glucose and uric acid, substitute the obtained current signal value into the standard curve of step (8), and calculate the concentration of glucose and uric acid in the test solution.

[0045] In this invention, the glucose oxidase and urate oxidase can be aliquoted using conventional methods in the art. For example, the glucose oxidase and urate oxidase can be diluted to 1 U / μL with 1×PBS buffer solution, and then aliquoted into centrifuge tubes of 10 μL each under aseptic conditions. After centrifugation, the aliquots are stored at -20°C. This avoids repeated freeze-thaw cycles that could lead to oxidase inactivation.

[0046] According to the present invention, a standard curve is established using a series of glucose and uric acid standard solutions with concentration gradients, the concentration range of which can be 0.001 nM-5 mM or 0.025 nM-5 mM.

[0047] The conditions for measuring the current signal of the reaction product solution obtained in step (8) are related to the concentration of the solution being measured, and cyclic voltammetry can be used. Preferred measurement conditions include: a maximum current of 10A, a minimum current of 1pA, and a measurement time of 100s.

[0048] In the method of the present invention, determining the volume of Cu-TCPP(Fe) / Mxene modifying the working electrode includes the following steps:

[0049] (1) Place the Cu-TCPP(Fe) / Mxene suspension, glucose standard solution and uric acid standard solution at room temperature;

[0050] (2) Take 6 paper-based electrodes and number them ①, ②, ③, ④, ⑤, ⑥. Modify the working electrodes with 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, and 70 μL of Cu-TCPP(Fe) / Mxene suspension and 5 μL of glucose oxidase and 10 μL of urate oxidase, respectively, and store them in a refrigerator at 4℃.

[0051] (3) Turn on the electrochemical workstation with Cu-TCPP(Fe) / Mxene hybrid nanosheets modified with the paper-based electrode of the oxidase connected to the paper-based electrode, set the parameters, and preheat for 20 minutes.

[0052] (4) Measure 100 μL of 1 mM glucose and uric acid standard solutions and add them to the working area of ​​the paper electrode to wet the working electrode, reference electrode and counter electrode. Then connect the green electrode to the working electrode, the white electrode to the reference electrode, the red electrode to the counter electrode and the yellow electrode to the other working electrode. After connecting, click start and compare the current values ​​of different volumes of Cu-TCPP(Fe) / Mxene to determine the optimal reaction volume as 50 μL of glucose and 40 μL of uric acid.

[0053] In the method of the present invention, determining the volume of the oxidase modifying the working electrode includes the following steps:

[0054] (1) Place the Cu-TCPP(Fe) / Mxene suspension, glucose standard solution and uric acid standard solution at room temperature;

[0055] (2) Take 6 paper-based electrodes and number them ①, ②, ③, ④, ⑤, ⑥. Modify 50 μL and 40 μL of Cu-TCPP(Fe) / Mxene suspension and 3 μL, 4 μL, 5 μL, 6 μL, 7 μL and 8 μL of glucose oxidase and 7 μL, 8 μL, 9 μL, 10 μL, 11 μL and 12 μL of glucose oxidase on the working electrodes respectively and store them in a refrigerator at 4℃.

[0056] (3) Turn on the electrochemical workstation that is modified with Cu-TCPP(Fe) / Mxene composite material and connected to the paper-based electrode of the oxidase, set the parameters, and preheat for 20 minutes.

[0057] (4) Measure 100 μL of 1 mM glucose and uric acid standard solutions and add them to the working area of ​​the paper electrode to wet the working electrode, reference electrode and counter electrode. Then connect the green electrode to the working electrode, the white electrode to the reference electrode, the red electrode to the counter electrode and the yellow electrode to the other working electrode. After connecting, click start and compare the current values ​​of different volumes of oxidase to determine the optimal volume of glucose oxidase as 5 μL and the optimal volume of uric acid oxidase as 10 μL.

[0058] In the method of the present invention, determining the pH value of the buffer solution includes the following steps:

[0059] (1) Place the Cu-TCPP(Fe) / Mxene suspension at room temperature;

[0060] (2) Take 6 paper-based electrodes and number them ①, ②, ③, ④, ⑤, ⑥. Modify the working electrodes with 50 μL and 40 μL of Cu-TCPP(Fe) / Mxene suspension, 5 μL of glucose oxidase, and 10 μL of uric acid oxidase, respectively, and store them in a refrigerator at 4℃.

[0061] (3) Turn on the electrochemical workstation, set the parameters, and preheat for 20 minutes;

[0062] (4) Take 100 μL of 1×PBS buffer solution with pH=4.40, pH=5.40, pH=6.40, pH=7.40, pH=8.40 and pH=9.40 respectively and add it to the working area of ​​the paper electrode so that it wets the working electrode, reference electrode and counter electrode. Then connect the green electrode to the working electrode, the white electrode to the reference electrode, the red electrode to the counter electrode and the yellow electrode to the other working electrode. After connecting, click start directly and compare the current value of 1×PBS buffer solution with different pH values ​​to determine that pH=7.40 is the best test condition for glucose and pH=5.40 is the best test condition for uric acid.

[0063] This invention replaces the traditional electrolytic cell with a paper-based electrode, further reducing experimental costs and improving portability and operability. By adding glucose and uric acid oxidases and a Cu-TCPP(Fe) / Mxene composite material, the electron transfer rate and the specific surface area of ​​the reaction are increased. Therefore, the method of this invention has advantages such as high sensitivity, wide detection range, and good specificity, and has significant practical implications for the ultrasensitive detection of glucose and uric acid. It also provides excellent guidance for realizing rapid, ultrasensitive on-site detection technology.

[0064] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0065] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0066] Figure 1 Scanning electron microscope image of Cu-TCPP(Fe) two-dimensional metal-organic framework.

[0067] Figure 2 This is a scanning electron microscope image of the Cu-TCPP(Fe) / Mxene composite material.

[0068] Figure 3 This is a transmission electron microscope (TEM) image of the Cu-TCPP(Fe) / Mxene composite material.

[0069] Figure 4a and Figure 4b The standard curves are plotted with the logarithms of the concentrations of glucose and uric acid standards on the x-axis and the corresponding current values ​​on the y-axis.

[0070] Figure 5 The results show the stability test results of the paper-based electrochemical detection method for glucose and uric acid oxidase based on Cu-TCPP(Fe) / Mxene composite material. The measured concentration was 1 mM. Figure a shows the stability of glucose detection, and Figure b shows the stability of uric acid detection. Detailed Implementation

[0071] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0072] In the following examples, copper nitrate and its trihydrate were purchased from Shanghai Maclean Biotechnology Co., Ltd.; polyvinylpyrrolidone (PVP) reagent was purchased from Sigma-Aldrich; N,N-dimethylformamide (DMF) was purchased from Röhn Reagents; meso-tetra(4-carboxyphenyl)porphyrin ferric chloride was purchased from Shanghai Maclean Biotechnology Co., Ltd.; glucose and uric acid standards used in the experiments were purchased from Alfaesa (China) Chemical Co., Ltd.; and glucose, uricase, artificial sweat, urine, and saliva were purchased from Shanghai Yuanye Biotechnology Co., Ltd. The electrochemical workstation was purchased from Shanghai Huachen. Unless otherwise specified, conditions in the examples were performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers were not specified were all commercially available products.

[0073] In the following examples, the volume of glucose in the Cu-TCPP(Fe) / Mxene modified working electrode was 50 μL, and the volume of uric acid was 40 μL. The volume of glucose oxidase in the modified working electrode was 5 μL, and the volume of uric acid oxidase was 10 μL. The pH of the 1×PBS buffer solution used to prepare the glucose solution was 7.40, and the pH of the 1×PBS buffer solution used to prepare the uric acid solution was 5.40.

[0074] Example 1

[0075] This embodiment provides a method for real-time detection of glucose and uric acid content in a living fluid based on Cu-TCPP(Fe) / Mxene composite material and a paper-based electrochemical biosensor for glucose and uric acid oxidase, comprising the following steps:

[0076] (1) Synthesis method of Cu-TCPP(Fe)2D MOF:

[0077] ① Weigh out 7.2 mg of copper nitrate trihydrate, 30 mg of polyvinylpyrrolidone (PVP), and 120 μL of trifluoroacetic acid (0.1 M), and place them in a 250 mL round-bottom flask.

[0078] ② While stirring, add dropwise 13.2 mg of tetra(4-carboxyphenyl)porphyrin(III) chloride dissolved in 36 mL of N,N-dimethylformamide (DMF):ethanol (3:1) solution.

[0079] ③ Sonicate the mixture for 10 minutes.

[0080] ④ Place the round-bottom flask in a water bath, heat to 80°C, and then maintain the reaction for 4 hours.

[0081] ⑤ Remove and place at room temperature, then centrifuge for 15 minutes at a speed of 10,000 rpm to obtain a red solid.

[0082] ⑥ Wash the obtained solid three times with 20 mL of ethanol, add water, shake to dissolve, sonicate for 5-10 min to completely dissolve, and centrifuge for 15 min at 10000 rpm.

[0083] ⑦ The solid obtained by centrifugation was dried in a vacuum drying oven to obtain Cu-TCPP(Fe)2D MOF, which was then stored in a sealed container at 4℃. The scanning electron microscope image of the obtained material is shown below. Figure 1 As shown.

[0084] (2) Synthesis method of Cu-TCPP(Fe) / Mxene composite material:

[0085] ① Measure 10 mL of Mxene aqueous solution (10 mg / L) and add it to a test tube, then add 100 mg of Cu-TCPP(Fe) powder and stir at room temperature for 6 h.

[0086] ②Place the above mixed solution into a centrifuge and centrifuge for 15 minutes at a speed of 10,000 rpm to remove the uncomposite material.

[0087] ③ Place the above mixed solution into a centrifuge and centrifuge for 15 minutes at a speed of 10,000 rpm. Remove the supernatant to obtain a solid.

[0088] ④ The obtained solid was placed in a vacuum drying oven and dried to obtain the Cu-TCPP(Fe) / Mxene composite material. It was then stored in a sealed container at 4℃. The scanning electron microscope (SEM) and transmission electron microscope (TEM) images of the Cu-TCPP(Fe) / Mxene composite material are shown below. Figure 2 , 3 As shown.

[0089] (3) Preparation of paper-based electrodes:

[0090] ① Print the wax onto plain paper using a printer and treat it at 90°C for 5 minutes to allow the wax to penetrate the paper evenly, creating a hydrophobic pattern designed specifically for this range.

[0091] ②Then, three electrodes are printed using a screen printer. The working electrode and the counter electrode are printed using ink mixed with carbon and graphene in a certain proportion, while the reference electrode is printed using Ag / AgCl ink. The resulting working electrode has a width of 2 mm.

[0092] (4) Preparation of modified paper-based electrodes:

[0093] ① Remove the Cu-TCPP(Fe) / Mxene suspension from the 4℃ refrigerator and let it stand at room temperature.

[0094] ② Measure 40 μL and 50 μL of the suspension and modify the working electrode by dripping. Take 25 μL each time and modify it in three times. After each modification, place the electrode in a refrigerator at 4°C to dry for 2 hours.

[0095] ③ Take glucose and uric acid oxidase out of the -20℃ freezer, thaw them, and then measure 5μL of glucose oxidase and 10μL of uric acid oxidase to modify the working electrode by dripping. After modification, place the electrode in a 4℃ freezer to dry and store it.

[0096] (5) Take the glucose and uric acid standard solutions out of the 4°C freezer and prepare solutions of 0.000013μM, 0.00004μM, 0.00012μM, 0.0004μM, 0.001μM, 0.003μM and 0.01μM respectively with 1×PBS buffer.

[0097] (6) Turn on the electrochemical workstation, set the parameters as follows: maximum current 10A, minimum current 1pA, time 100s, and preheat for 20min.

[0098] (7) Take 100 μL of glucose and uric acid solutions of different concentrations prepared in step (5) and add them dropwise to the working area of ​​the paper electrode so that the solution wets the working electrode, reference electrode and counter electrode.

[0099] (8) Connect the green electrode to the working electrode, the white electrode to the reference electrode, the red electrode to the counter electrode, and the yellow electrode to the other working electrode. After connecting, click Start.

[0100] (9) Detect different concentrations of glucose (0.001nM, 0.005nM, 3.125nM, 78.125nM, 29295nM, 732375nM, 5000000nM) and different concentrations of uric acid (0.025nM, 0.125nM, 15.625nM, 78.125nM, 146475nM, 1000000nM, 5000000nM) and obtain the current value of each concentration.

[0101] The standard curve calculated using software is F = 0.6005lgC + 5.6720(R). 2 =0.9987), F=3.2817lgC+8.2856(R 2 =0.9994), the linear detection ranges are 0.001 nM-5 mM and 0.025 nM-5 mM, and the detection limits are 1.88 aM / L and 5.80 pM / L (S / N=3). A standard curve was plotted with the current value on the ordinate and the concentrations of glucose and uric acid standards on the abscissa, as shown below. Figure 4a and Figure 4b As shown.

[0102] Example 2

[0103] A spiked recovery experiment for the detection of glucose and uric acid in metabolites based on Cu-TCPP(Fe) / Mxene composite material and a paper-based electrochemical biosensor for glucose and uric acid oxidase includes the following steps:

[0104] (1) Glucose and uric acid were added to artificial sweat at four different concentrations: 0, 5 μmol / L, 50 μmol / L and 500 μmol / L. The artificial sweat, glucose and uric acid were mixed in test tubes to obtain four different concentrations of test samples, which were then stored in a 4°C refrigerator for later use.

[0105] (2) Take the Cu-TCPP(Fe) / Mxene suspension out of the 4℃ refrigerator and let it stand at room temperature.

[0106] (3) Measure 40 μL and 50 μL of suspension and modify the working electrode by dripping. Take 25 μL each time and modify it three times. After each modification, place the electrode in a 4℃ refrigerator to dry for 2 hours.

[0107] (4) Take out glucose and uric acid oxidase from the -20℃ refrigerator, thaw them, and then measure 5μL of glucose oxidase and 10μL of uric acid oxidase to modify the working electrode by dripping. After modification, place the electrode in a 4℃ refrigerator to dry and store it.

[0108] (5) Turn on the electrochemical workstation, set the parameters to the maximum current of 10A, the minimum current of 1pA, the time of 100s, and preheat for 20min.

[0109] (6) Add 100 μL of glucose and uric acid solutions with different spiking concentrations prepared in step (1) to the working area of ​​the electrode modified in step (4) so ​​that the solution wets the working electrode, reference electrode and counter electrode.

[0110] (7) Connect the green electrode to the working electrode, the white electrode to the reference electrode, the red electrode to the counter electrode, and the yellow electrode to the other working electrode. After connecting, click Start.

[0111] (8) By comparing the detected concentration with the actual added concentration using the standard curve, the recovery rate of glucose ranged from 95% to 103%, and the recovery rate of uric acid ranged from 100% to 103%, with RSDs of 5.42% to 6.94% and 7.54% to 9.30%, respectively. The results indicate that this detection method can be applied to actual sweat detection, and the pretreatment is simple.

[0112] Example 3

[0113] A spiked recovery experiment for the detection of glucose and uric acid in metabolites based on Cu-TCPP(Fe) / Mxene composite material and a paper-based electrochemical biosensor for glucose and uric acid oxidase includes the following steps:

[0114] (1) Glucose and uric acid were added to artificial urine at four different concentrations: 0, 5 μmol / L, 50 μmol / L and 500 μmol / L. The artificial urine, glucose and uric acid were mixed in test tubes to obtain four different concentrations of test samples, which were then stored in a 4°C refrigerator for later use.

[0115] (2) Take the Cu-TCPP(Fe) / Mxene suspension out of the 4℃ refrigerator and let it stand at room temperature.

[0116] (3) Measure 40 μL and 50 μL of suspension and modify the working electrode by dripping. Take 25 μL each time and modify it three times. After each modification, place the electrode in a 4℃ refrigerator to dry for 2 hours.

[0117] (4) Take out glucose and uric acid oxidase from the -20℃ refrigerator, thaw them, and then measure 5μL of glucose oxidase and 10μL of uric acid oxidase to modify the working electrode by dripping. After modification, place the electrode in a 4℃ refrigerator to dry and store it.

[0118] (5) Turn on the electrochemical workstation, set the parameters to the maximum current of 10A, the minimum current of 1pA, the time of 100s, and preheat for 20min.

[0119] (6) Add 100 μL of glucose and uric acid solutions with different spiking concentrations prepared in step (1) to the working area of ​​the electrode modified in step (4) so ​​that the solution wets the working electrode, reference electrode and counter electrode.

[0120] (7) Connect the green electrode to the working electrode, the white electrode to the reference electrode, the red electrode to the counter electrode, and the yellow electrode to the other working electrode. After connecting, click Start.

[0121] (8) By comparing the detected concentration with the actual added concentration using the standard curve, the recovery rate of glucose ranged from 95% to 103%, and the recovery rate of uric acid ranged from 100% to 103%, with RSDs of 5.42% to 6.94% and 7.54% to 9.30%, respectively. The results indicate that the detection method can be applied to actual urine detection, and the pretreatment is simple.

[0122] Example 4

[0123] Stability experiments were conducted on the real-time detection method for glucose and uric acid metabolites based on Cu-TCPP(Fe) / Mxene composite materials and a paper-based electrochemical biosensor for glucose and uric acid oxidase. The method included the following steps:

[0124] (1) Take out the glucose and uric acid standard solutions from the 4°C refrigerator and prepare 1mM standard solutions with 1×PBS buffer.

[0125] (2) Take the Cu-TCPP(Fe) / Mxene suspension out of the 4℃ refrigerator and let it stand at room temperature.

[0126] (3) Measure 40 μL and 50 μL of suspension and modify the working electrode by dripping. Take 25 μL each time and modify it three times. After each modification, place the electrode in a 4℃ refrigerator to dry for 2 hours.

[0127] (4) Take out glucose and uric acid oxidase from the -20℃ refrigerator, thaw them, and then measure 5μL of glucose oxidase and 10μL of uric acid oxidase to modify the working electrode by dripping. After modification, place the electrode in a 4℃ refrigerator to dry and store it.

[0128] (5) Turn on the electrochemical workstation, set the parameters to the maximum current of 10A, the minimum current of 1pA, the time of 100s, and preheat for 20min.

[0129] (6) Add 100 μL of the glucose and uric acid solution prepared in step (1) to the working area of ​​the electrode modified in step (4) so ​​that the solution wets the working electrode, reference electrode and counter electrode.

[0130] (7) Connect the green electrode to the working electrode, the white electrode to the reference electrode, the red electrode to the counter electrode, and the yellow electrode to the other working electrode. After connecting, click Start.

[0131] (8) The voltage of each solution was measured using an electrochemical workstation. Then, the voltage values ​​of the glucose and uric acid solutions prepared according to step (1) of the above method were selected at 30, 40, 50, 60, 80, and 100 days. The results were then processed using software to obtain a bar chart, with the horizontal axis representing the number of days and the vertical axis representing the voltage value. Figure 5 As shown.

[0132] To verify the stability of the detection method of this invention, tests were conducted at 30, 40, 50, 60, 80, and 100 days. Figure 5 It can be seen that the detection method of the present invention has good stability, and the measurement results on day 1 and day 30 are almost the same. By day 100, the voltage value has decreased, but it is within an acceptable range. Overall, the current value does not change much within 100 days.

[0133] Example 5

[0134] The reproducibility of a real-time detection method for glucose and uric acid in metabolites based on Cu-TCPP(Fe) / Mxene composite material and a paper-based electrochemical biosensor for glucose and uric acid oxidase includes the following steps:

[0135] (1) Take the Cu-TCPP(Fe) / Mxene suspension and 1mM glucose and uric acid solution out of the 4℃ refrigerator and let them stand at room temperature.

[0136] (2) Measure 40 μL and 50 μL of suspension and modify the working electrode by dripping. Take 25 μL each time and modify it three times. After each modification, place the electrode in a 4℃ refrigerator to dry for 2 hours.

[0137] (3) Take out glucose oxidase and urate oxidase from the -20℃ refrigerator, thaw them, and then measure 5μL of glucose oxidase and 10μL of urate oxidase to modify the working electrode by dripping. After modification, place the electrode in a 4℃ refrigerator to dry and store it.

[0138] (4) Turn on the electrochemical workstation, set the parameters to the maximum current of 10A, the minimum current of 1pA, the time of 100s, and preheat for 20min.

[0139] (5) Add 100 μL of 1 mM glucose and uric acid solution to the modified electrode working area in step (4) so ​​that the solution wets the working electrode, reference electrode and counter electrode.

[0140] (6) Connect the green electrode to the working electrode, the white electrode to the reference electrode, the red electrode to the counter electrode, and the yellow electrode to the other working electrode. After connecting, click Start.

[0141] (7) Repeat the measurement 5 times, record the current value each time, and calculate the relative standard deviation. The results are shown in Table 1 and Table 2.

[0142] Table 1. Repeatability and Reproducibility of Uric Acid

[0143]

[0144] Table 2. Repeatability and Reproducibility of Glucose

[0145]

[0146] Example 6

[0147] The reproducibility of a real-time detection method for glucose and uric acid in metabolites based on Cu-TCPP(Fe) / Mxene composite material and a paper-based electrochemical biosensor for glucose and uric acid oxidase includes the following steps:

[0148] (1) Take the Cu-TCPP(Fe) / Mxene suspension and 1mM glucose and uric acid solution out of the 4℃ refrigerator and let them stand at room temperature.

[0149] (2) 40 μL and 50 μL of suspension were measured and modified by dripping to three different working electrodes. 25 μL was measured each time and modified in three batches. After each modification, the electrodes were placed in a 4°C refrigerator to dry for 2 hours.

[0150] (3) Take out glucose oxidase and urate oxidase from the -20℃ refrigerator, thaw them, and then measure 5 μL of glucose oxidase and 10 μL of urate oxidase to modify the three different working electrodes by dripping. After modification, place the electrodes in a 4℃ refrigerator to dry and store them.

[0151] (4) Turn on the electrochemical workstation, set the parameters to the maximum current of 10A, the minimum current of 1pA, the time of 100s, and preheat for 20min.

[0152] (5) Add 100 μL of 1 mM glucose and uric acid solution to the three different electrode working areas modified in step (3) so that the solution can wet the working electrode, reference electrode and counter electrode respectively.

[0153] (6) Connect the green electrode to the working electrode, the white electrode to the reference electrode, the red electrode to the counter electrode, and the yellow electrode to the other working electrode. After connecting, click Start.

[0154] (7) The current value of each paper electrode can be obtained by measuring with an electrochemical workstation, and its relative standard deviation can be calculated. The results are shown in Table 1 and Table 2.

[0155] To verify the repeatability and reproducibility of the detection method of this invention, 1 mM glucose and uric acid solutions were used for measurement. Tables 1 and 2 show that the relative standard deviations (RSD%) for repeatability and reproducibility of glucose were 0.016 and 0.042, respectively, and the RSD% for repeatability and reproducibility of uric acid were 0.005 and 0.118, respectively. The results indicate that the prepared Cu-TCPP(Fe) / Mxene composite material and paper-based electrochemical biosensor for glucose and uric acid oxidases exhibit excellent repeatability and reproducibility.

[0156] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A paper-based electrode based on a metal-organic framework composite material and an oxidase-modified three-electrode dual-channel system, characterized in that, The electrode comprises the following components: (1) Working electrode: modified carbon and graphene electrode; (2) Reference electrode: Ag / AgCl; (3) Counter electrode: carbon and graphene electrode; The modified carbon-plus-graphene electrode is an electrode that is sequentially modified with Cu-TCPP(Fe) / Mxene composite material and oxidase. The modified carbon-plus-graphene electrode is capable of redox reactions of glucose and uric acid. The oxidase includes glucose oxidase and uric acid oxidase.

2. The electrode according to claim 1, characterized in that, The preparation method of the Cu-TCPP(Fe) / Mxene composite material includes the following steps: (1) Copper nitrate trihydrate, polyvinylpyrrolidone and trifluoroacetic acid are mixed to obtain a first mixture. Under stirring, tetrakis(4-carboxyphenyl)porphyrin iron(III) dissolved in N,N-dimethylformamide / ethanol solution is added dropwise to the first mixture to obtain a second mixture. The second mixture is subjected to ultrasonic treatment and then heated for reaction. (2) After the reaction, the system was cooled and centrifuged, washed and dried to obtain Cu-TCPP(Fe) powder; (3) Mix the Mxene aqueous solution with the Cu-TCPP(Fe) powder obtained in step (2), stir and centrifuge to remove the uncomposite material, and vacuum dry the resulting solid to obtain the Cu-TCPP(Fe) / Mxene composite material.

3. The electrode according to claim 2, characterized in that, In step (1), compared to 1 mg of copper nitrate trihydrate, the amount of polyvinylpyrrolidone added is 4-5 mg, the amount of trifluoroacetic acid added is 16-17 μL, the amount of tetrakis(4-carboxyphenyl)porphyrin iron(III) chloride added is 1.5-2 mg, and the amount of N,N-dimethylformamide / ethanol solution added is 4-6 mL; the volume ratio of N,N-dimethylformamide to ethanol in the N,N-dimethylformamide / ethanol solution is 2.5-3.5:1; the heating reaction temperature is 75-85℃, and the time is 3-5 h; In step (2), the centrifugation speed is 8000-12000 rpm and the centrifugation time is 10-20 min; A red solid was obtained after centrifugation; the washing method includes: adding ethanol, shaking to dissolve the solid, followed by ultrasonic treatment and centrifugation. In step (3), the Mxene aqueous solution and Cu-TCPP(Fe) powder are stirred and mixed at room temperature. The concentration of the Mxene aqueous solution is 8-12 mg / L, and the amount of Cu-TCPP(Fe) powder added is 800-1200 mg compared to 1 mg Mxene. The centrifugation conditions include: rotation speed of 8000-12000 rpm and time of 10-20 min.

4. The electrode according to claim 1, characterized in that, The modification method includes: (a) Dissolve glucose oxidase and uricase oxidase in PBS solution; (b) Fabrication of a three-electrode dual-channel paper-based electrode using screen printing technology; (c) The solution of Cu-TCPP(Fe) / Mxene composite material was drop-coated onto the working electrode of the paper-based electrode obtained in step (b) and placed in a refrigerator at 4°C to dry. (d) The oxidase solution from step (a) is applied to the working electrode after drying in step (c) by drop coating to obtain the modified carbon-plus-graphene electrode.

5. A paper-based electrochemical biosensor based on Cu-TCPP(Fe) / Mxene composite material and oxidase, characterized in that, include: i) The Cu-TCPP(Fe) / Mxene composite material as described in any one of claims 1-4; ii) Oxidases, including glucose oxidase and uricase oxidase; iii) Paper-based electrodes in a three-electrode dual-channel system; iv) Portable electrochemical workstation.

6. The paper-based electrochemical biosensor according to claim 5, characterized in that, The paper-based electrodes of the three-electrode dual-channel system are prepared using screen printing technology.

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