Pyruvic acid sensing chip of Prussian blue nano frame / gold nano composite material

Abstract

The invention belongs to the technical field of pyruvic acid biological detection, and relates to a pyruvic acid sensing chip of a Prussian blue nano frame / gold nano composite material. The method comprises the following steps: adding soluble metal salt containing ferricyanide ions into water; the method comprises the following steps: adding soluble ferric salt and a complexing agent into water, mixing, and carrying out hydrothermal synthesis reaction to obtain PB powder; adding PB powder into ethanol, adding ammonia water for reaction, mixing with conductive carbon paste to obtain nano printing paste, and printing the printing paste on a substrate to form a working electrode; the preparation method comprises the following steps: preparing a working electrode, drying the working electrode, placing the working electrode in a chloroauric acid solution for constant potential electrodeposition, mixing a PBS solution of pyruvate oxidase, a polylysine solution and an EDC solution, dropwise adding the mixture on the surface of the working electrode, and drying to obtain the pyruvic acid sensing chip. The obtained pyruvic acid sensor can reach the sensitivity of 50.42 [mu] A.mM <-1 >. Cm <-2 >, and is simple in preparation process and relatively low in cost.

Description

Technical Field

[0001] The invention belongs to the technical field of pyruvic acid biological detection and relates to a pyruvic acid sensor chip of a Prussian blue nano-framework / gold nano-composite material. Background Art

[0002] Pyruvate is a key intermediate in both anaerobic and aerobic metabolism in most organisms and serves as an intermediate between glycolysis and the tricarboxylic acid cycle. During glycolysis, one glucose molecule breaks down into two pyruvate molecules, which are further oxidized through aerobic respiration to release energy for cellular use. In medicine, measuring pyruvate levels can be used to monitor disease progression. For example, blood pyruvate concentrations can be used as a diagnostic indicator for diabetes. New platforms for pyruvate detection have been reported, for example, for assessing the quality of food and alcohol products, particularly the flavor of different onion and garlic varieties; for evaluating fermentation processes and key process parameters during microbial fermentation; and for real-time monitoring of pyruvate intermediate metabolites in winemaking to redirect metabolic activity toward ethanol production, thereby increasing ethanol yield. Furthermore, the industrial production of pyruvate has gradually shifted from chemical synthesis to microbial fermentation. Therefore, pyruvate detection is crucial in clinical diagnostics, the food industry, and microbial fermentation. Currently, a variety of methods are available for pyruvate determination, including spectrophotometry, colorimetry, enzyme fluorescence, and high-performance liquid chromatography. However, these methods usually require strict sample preparation steps, expensive testing instruments, and complex determination methods. Summary of the Invention

[0003] Aiming at the problems existing in traditional pyruvic acid detection, the present invention proposes a novel pyruvic acid sensor chip of Prussian blue nanoframe / gold nanocomposite material.

[0004] In order to achieve the above object, the present invention is implemented by adopting the following technical solutions: A method for preparing a pyruvic acid sensor chip of a Prussian blue nanoframe / gold nanocomposite material, comprising the following steps: (1) A soluble metal salt containing ferrocyanide ions is added to water to obtain solution A; a soluble iron salt and a complexing agent are added to water to obtain a mixed solution B; solution A and mixed solution B are mixed and subjected to a hydrothermal synthesis reaction. After the reaction is completed, the mixture is filtered, and the obtained solid is centrifuged, washed, and dried to obtain PB powder.

[0005] (2) PB powder is added to ethanol and mixed evenly to obtain a dispersion, and ammonia water is added to the dispersion to obtain a reaction solution. After the reaction, the mixture is centrifuged, washed, and dried to obtain open-framework Prussian blue particles.

[0006] (3) Open-frame Prussian blue particles are mixed with conductive carbon paste to obtain nanoprinting paste.

[0007] (4) Screen printing the conductive carbon paste onto the substrate to form a counter electrode and a connector, screen printing the silver / silver chloride paste onto the substrate to form a reference electrode, and screen printing the nano-printing paste onto the substrate to form a working electrode; drying and curing the mixture to obtain an electrode chip.

[0008] (5) The electrode chip is placed in a chloroauric acid solution for constant potential electrodeposition to obtain a modified electrode chip.

[0009] (6) The PBS solution of pyruvate oxidase (composition: 0.5 M potassium chloride, 0.3 M potassium dihydrogen phosphate, 0.26 M potassium dihydrogen phosphate, solvent: water) was mixed evenly with the ƹ-polylysine solution and the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution to obtain an enzyme solution. The enzyme solution was added dropwise to the working electrode surface of the modified electrode chip and dried to obtain a pyruvate sensor chip.

[0010] Preferably, the solute of solution A in step (1) is K4Fe(CN)6 or K3Fe(CN)6, the concentration of solution A is 15-25mM, the soluble iron salt in the mixed solution B is any one of FeSO4, FeCl2, and FeCl3, the complexing agent is citric acid or sodium citrate, the concentration of iron ions in the mixed solution B is 25-35mM, the concentration of the complexing agent is 150-200mM, and the volume ratio of solution A to mixed solution B is 1:1; the hydrothermal synthesis reaction temperature is 45-65°C, and the hydrothermal synthesis reaction time is 20-24h.

[0011] Preferably, in step (2), the mass fraction of ammonia water is 1.4-3.4%, the concentration of the dispersion is 0.2-0.6 mg / mL, the volume ratio of ammonia water to the dispersion is 5:1-5, and the reaction time is 10-25 min.

[0012] Preferably, in step (3), the mass ratio of the open-frame Prussian blue particles to the conductive carbon slurry is 1:1-50.

[0013] Preferably, in step (5), the concentration of the chloroauric acid solution is 6-12 mM, the electrodeposition voltage is -0.2 V, and the electrodeposition time is 200-400 s.

[0014] Preferably, the concentration of pyruvate oxidase in the enzyme solution of step (6) is 200-2000 U·mL-1, the mass fraction of the ƹ-polylysine solution is 0.1-0.28%; the concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is 2-9 mM, and the volume ratio of the PBS solution of pyruvate oxidase: ƹ-polylysine solution: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is 1:1:1; the addition amount is 2-10 μL, the drying temperature is 0-20°C, and the drying time is at least 12 h.

[0015] The present invention proposes the application of the sensor chip prepared by the above method in pyruvic acid detection.

[0016] When detecting pyruvate, pyruvate oxidase (Pox) is used as a biorecognition agent to specifically react with pyruvate to produce H2O2, which is then decomposed into O2 and H + Ions and electrons (e - ) is released and detected in the form of current. The PB used in the present invention is capable of reducing hydrogen peroxide at a low potential. The present invention further utilizes ammonia water to etch to obtain an open framework morphology. The framework nanostructure can provide a very high high-reactivity specific surface area, which is not available in solid solid nanoparticles. The framework nanostructure prepared by the present invention has a concave surface inside it, which is more suitable as a site for oxygen reduction reaction than a convex surface, and the 3D open structure allows molecules or atoms to enter the inner surface, which has higher electrocatalytic activity. The present invention also modifies the surface of PB NF by gold nanoparticles, further improving the conductivity and sensing performance of the material. The prepared sensing electrode has good reusability, accuracy, and anti-interference properties at low potential, and can be used to monitor the concentration of pyruvate in the actual ethanol fermentation process.

[0017] Compared with the prior art, the advantages and positive effects of the present invention are: The present invention uses framework nanoparticles as electrode materials and modifies them with chloroauric acid solution to further control the micro-nanostructure. A composite material with high conductivity and high electrocatalytic activity is designed for the preparation of screen-printed electrode chips. The resulting pyruvic acid sensor can reach 50.42μA·mM -1 cm -2 The sensitivity is high, the preparation process is simple, the cost is low, and it can be used for large-scale production to achieve accurate real-time detection of pyruvate concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG1 is a TEM electron microscope image of the open frame Prussian blue in Example 1.

[0019] FIG2 is a scanning electron micrograph of the open-frame Prussian blue-loaded gold nanocubes in Example 1. DETAILED DESCRIPTION

[0020] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described below in conjunction with specific embodiments. It should be noted that, in the absence of conflict, the embodiments of the present application and the features therein can be combined with each other.

[0021] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0022] Example 1 Prepare 100 mL of 20 mM potassium ferrocyanide solution A; then prepare 100 mL of solution B containing 30 mM ferrous sulfate and 170 mM sodium citrate; mix solution A and solution B evenly and add them to a hydrothermal synthesis reactor, seal it and react at 60°C. After reacting for 24 hours, centrifuge the reaction solution at a centrifugal speed of 5000 r / min -1 The centrifugation time was 6 minutes. After centrifugation, the supernatant was removed with a pipette and discarded. Approximately 5 mL of deionized water was then added dropwise with a pipette to wash the solids adsorbed on the centrifuge tube wall. After washing, the deionized water was removed with a pipette and discarded. Approximately 5 mL of ethanol was then added dropwise and washed using the same method, alternating between deionized water and ethanol three times. The solids adsorbed on the centrifuge tube wall were scraped off and dried in a 60°C oven for 6 hours to obtain an iron-based Prussian blue-like material (PB powder).

[0023] 2) Take 28% ammonia water and dilute it to 2.8% with deionized water. Then add 20 mg of PB powder to 40 mL of ethanol and mix well to obtain a dispersion. Add 100 mL of 2.8% ammonia water to the dispersion and stir the reaction system at room temperature for 15 minutes at a stirring rate of 200 rpm. After the reaction, centrifuge at a centrifugal speed of 3000 r / min. -1 The centrifugation time was 5 minutes, and the supernatant was aspirated with a dropper and discarded; then about 5 mL of deionized water was added with a dropper to wash the solid adsorbed on the wall of the centrifuge tube. After washing, the solid was scraped off and placed in an oven at 60°C for 6 hours to obtain the product open framework Prussian blue, which was recorded as PB NF.

[0024] 3) 0.5 g of PB NF material and 5 g of conductive carbon slurry (Shenzhen Jieyongcheng Technology Co., Ltd.) were mixed and stirred to obtain the working electrode slurry, which was then dried at 60°C for 30 min.

[0025] 4) Using screen printing technology, a working electrode paste, silver / silver chloride paste (Shanghai Julong Electronic Technology Co., Ltd.), and carbon paste (Shenzhen Jieyongcheng Technology Co., Ltd.) were respectively printed on different locations of a PET substrate (Jiangsu Xianfeng Nanomaterial Technology Co., Ltd.). The conductive carbon paste was screen-printed onto the PET substrate to form the counter electrode and connector, the silver / silver chloride paste was screen-printed onto the PET substrate to form the reference electrode, and the working electrode paste was screen-printed onto the PET substrate to form the working electrode, thus fabricating a three-electrode chip.

[0026] 5) The electrode chip obtained in step (4) was placed in a 10 mM chloroauric acid solution for constant potential electrodeposition at a deposition voltage of -0.2 V and a deposition time of 300 s to obtain a PB NF / Au chip.

[0027] 6) Dissolve 40 U of pyruvate oxidase (purchased from Sigma Biotechnology Co., Ltd.) in 20 μL of PBS solution (containing 0.5 M potassium chloride, 0.3 M potassium dihydrogen phosphate, 0.26 M potassium dihydrogen phosphate, and water). Take 10 μL and add it to a 0.2 mL centrifuge tube. Then, add 10 μL of a 0.18% solution of ƹ-polylysine (CAS No. 28211-04-3) and 10 μL of a 3 mM solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, CAS No. 25952-53-8). After mixing thoroughly, take 3 μL and place it on the working electrode. After drying at 5°C for 24 h, the pyruvate biosensor chip was obtained.

[0028] Example 2 This embodiment is consistent with the embodiment 1 unless otherwise specified.

[0029] 1) Prepare 100 mL of 15 mM potassium ferrocyanide solution A; then prepare 100 mL of solution B containing 25 mM ferrous sulfate and 150 mM sodium citrate; mix solution A and solution B evenly, react at 45°C for 20 hours, and then centrifuge the reaction solution at 5000 rpm. -1 The centrifugation time was 6 min. After the centrifugation, the supernatant was removed, and the obtained precipitate was washed alternately with deionized water and ethanol three times, and then dried at 60°C for 6 h to obtain an iron-based Prussian blue material.

[0030] 2) After diluting 28% ammonia water to 1.4% by mass, 200 mL was added to 40 mL of ethanol containing 8 mg of PB powder. The reaction system was stirred at room temperature for 10 minutes, then washed with deionized water and centrifuged. After washing, it was dried to obtain the product open-framework Prussian blue, which was recorded as PB NF.

[0031] 3) Mix 1 g of PB NF nanomaterial and 5 g of conductive carbon slurry to obtain a working electrode slurry, and dry the slurry at 60°C for 30 min.

[0032] 4) The working electrode paste, silver / silver chloride paste, and carbon paste were printed on different positions of the PET substrate using screen printing technology to produce a three-electrode system chip.

[0033] 5) The electrode chip was placed in a 6 mM chloroauric acid solution for constant potential electrodeposition at a deposition voltage of -0.2 V for 200 s to obtain a PB NF / Au chip.

[0034] 6) Dissolve 40 U of pyruvate oxidase in 20 μL of PBS solution (containing 0.5 M potassium chloride, 0.3 M potassium dihydrogen phosphate, 0.26 M potassium dihydrogen phosphate, and water). Add 10 μL to a 0.2 mL centrifuge tube. Then, add 10 μL of 0.1% polylysine and 10 μL of 2 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Mix well. Place 7 μL on the working electrode and dry at 20°C for 12 h to obtain a pyruvate biosensor chip.

[0035] Example 3 This embodiment is consistent with the embodiment 1 unless otherwise specified.

[0036] 1) Prepare 100 mL of 20 mM potassium ferrocyanide solution A; then prepare 100 mL of solution B containing 30 mM ferrous sulfate and 175 mM sodium citrate; after mixing solution A and solution B evenly, react at 50°C for 22 hours, and then centrifuge the reaction solution at a speed of 5000 rpm. -1 The centrifugation time was 6 min. After the centrifugation, the supernatant was removed, and the obtained precipitate was washed alternately with deionized water and ethanol three times, and then dried at 60°C for 6 h to obtain an iron-based Prussian blue material.

[0037] 2) Dilute 28% ammonia water to 2.4% by mass, then add 50 mL to 40 mL of ethanol containing 8 mg of PB powder. Stir the reaction system at room temperature for 20 minutes, then rinse with deionized water, centrifuge, and dry to obtain the open-framework Prussian blue product, designated PB NF.

[0038] 3) Mix 1 g of PB NF nanomaterial and 5 g of conductive carbon slurry to obtain a working electrode slurry, and dry the slurry at 60°C for 30 min.

[0039] 4) The working electrode paste, silver / silver chloride paste, and carbon paste were printed on different positions of the PET substrate using screen printing technology to produce a three-electrode system chip.

[0040] 5) The electrode chip was placed in an 8 mM chloroauric acid solution for constant potential electrodeposition at a deposition voltage of -0.2 V for 350 s to obtain a PB NF / Au chip.

[0041] 6) Dissolve 40 U of pyruvate oxidase in 20 μL of PBS solution (containing 0.5 M potassium chloride, 0.3 M potassium dihydrogen phosphate, 0.26 M potassium dihydrogen phosphate, and water). Take 10 μL and add it to a 0.2 mL centrifuge tube. Then, add 10 μL of 0.2% polylysine and 10 μL of 6 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). After mixing, take 7 μL and place it on the working electrode. After drying at 5°C for 24 h, the pyruvate biosensor chip was obtained.

[0042] Example 4 This embodiment is consistent with the embodiment 1 unless otherwise specified.

[0043] 1) Prepare 100 mL of 25 mM potassium ferrocyanide solution A; then prepare 100 mL of solution B containing 35 mM ferrous sulfate and 200 mM sodium citrate; mix solution A and solution B evenly and react at 65°C for 24 hours. Centrifuge the reaction solution at 5000 rpm. -1 The centrifugation time was 6 min. After the centrifugation, the supernatant was removed and the precipitate was washed alternately with deionized water and ethanol three times, and then dried at 60 ° C for 6 h to obtain PB powder.

[0044] 2) After diluting 28% ammonia water to 3.4% by mass, 40 mL was added to 40 mL of ethanol containing 24 mg of PB powder. The reaction system was stirred at room temperature for 25 minutes, then washed with deionized water and centrifuged. After washing, it was dried to obtain the product open-framework Prussian blue, which was recorded as PB NF.

[0045] 3) Mix 1 g of PB NF nanomaterial and 5 g of conductive carbon slurry to obtain a working electrode slurry, and dry the slurry at 60°C for 30 min.

[0046] 4) The working electrode paste, silver / silver chloride paste, and carbon paste were printed on different positions of the PET substrate using screen printing technology to produce a three-electrode system chip.

[0047] 5) The electrode chip was placed in a 12 mM chloroauric acid solution for constant potential electrodeposition at a deposition voltage of -0.2 V for 400 s to obtain a PB NF / Au chip.

[0048] 6) Dissolve 60 U of pyruvate oxidase in 20 μL of PBS solution (containing 0.5 M potassium chloride, 0.3 M potassium dihydrogen phosphate, 0.26 M potassium dihydrogen phosphate, and water). Take 10 μL and add it to a 0.2 mL centrifuge tube. Then, add 10 μL of 0.28% polylysine and 10 μL of 9 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). After mixing, take 7 μL and place it on the working electrode. After drying at 10°C for 20 h, the pyruvate biosensor chip was obtained.

[0049] First, the relationship between the current response detected by the sensor chip and the pyruvic acid concentration in the detection cell was determined, and a calibration curve was prepared.

[0050] PBS electrolyte (composition: 0.5M potassium chloride, 0.3M potassium dihydrogen phosphate, 0.26M potassium hydrogen phosphate, solvent: water, the same below) was added to the electrolytic cell, and then pyruvic acid was added to prepare pyruvic acid standard solutions with concentrations of 0 M (pure PBS electrolyte), 0.05mM, 0.1mM, 0.15mM, 0.2mM, and 0.25mM, respectively. The current changes (∆I) of the above standard solutions were tested in sequence using the constant current IT method of the Shanghai Chenhua CHI660E electrochemical workstation. For each test, 40μL of sample was added to 40mL of electrolyte for test, and then calculated. The results are shown in Table 1 below.

[0051] Table 1 Current response values ​​at various concentrations According to the results in Table 1, a calibration curve was formed by fitting, and the linear relationship between the current response value and the concentration was obtained as follows: ∆I=3.76C-0.008, R 2 =0.99. In the formula, C represents the concentration of pyruvate, in M, and ∆I is the difference between the current response value at different concentrations and the current response value of pure electrolyte, in μA.

[0052] Based on the standard curve, the performance test of the chip prepared in each example was performed as follows.

[0053] 1. Linear range and sensitivity test The pyruvate biosensor chip prepared in Examples 1-4 was used to measure pyruvate using the constant current (IT) method on a Shanghai Chenhua CHI660E electrochemical workstation. PBS was also used in the electrolytic cell, and the voltage was -0.2V. The conductive substrate (working electrode), platinum wire (counter electrode), and Ag / AgCl (reference electrode) were connected to the corresponding electrode wires on the electrochemical workstation. 40 μL of PBS solutions of varying pyruvate concentrations were added to 40 mL of the electrolyte for detection and analysis. The IT test revealed a sensitivity of 50.42 μA·mM for pyruvate. -1 •cm -2 The linear detection range was 5-895 μM, with a lower detection limit of 0.2 μM. After 30 days of storage at 4°C, the chip's steady-state response current retained over 50% of its initial sensitivity, demonstrating the excellent stability of the prepared pyruvate enzyme biosensor chip.

[0054] The specific test results of the sensors prepared in each embodiment are as follows.

[0055] Table 2 Linear detection range of the detection sensor obtained in each embodiment As can be seen from Table 2, the detection performance of the sensors prepared in Examples 2-4 is not much different from that of Example 1.

[0056] 2. Real sample testing and accuracy verification The pyruvate biosensor chip prepared in Example 1-4 was applied to the fermentation broth to detect the pyruvate concentration.

[0057] Fermentation broth preparation method: Under anaerobic conditions, in the alcohol fermentation process using sugar as the carbon source, sugar is glycolyzed and the final products are alcohol and carbon dioxide.

[0058] Yeast pre-activation: Add 3g of Angel heat-resistant active dry yeast to 50mL of 20g / L sucrose solution and pre-activate in a 34℃ constant temperature water bath for 30 minutes.

[0059] Prepare fermentation medium: the mass concentrations of each substance in the fermentation medium are: glucose 160 g / L, potassium dihydrogen phosphate 1.5 g / L, potassium chloride 1.2 g / L, calcium chloride 0.10 g / L, yeast extract 4.4 g / L, ammonium chloride 1.33 g / L, magnesium sulfate 0.65 g / L, and the solvent is water.

[0060] Take 4 L of fermentation medium, add sucrose to an initial sucrose concentration of 160 g / L, raise the temperature to 30°C and maintain it there. Then, pour the preactivated yeast solution into the fermentation broth at 100 rpm for inoculation. Samples are taken after 5 minutes, followed by 1 hour intervals. After 10 samples have been taken, samples are taken every 12 hours thereafter. Maintain 30°C throughout the fermentation process, then raise the temperature to 32°C after 24 hours and maintain it there. After fermentation begins, aerate the jar with sterile air at a flow rate of 0.2 L / min for 8 hours, then stop aeration and keep the fermenter sealed.

[0061] Sampling method: Every 24 hours, 10 mL of fermentation liquid was taken from the fermentation tank sampling port for testing. After sampling, 10 mL of culture medium was added. The composition of the feed medium was the same as that of the fresh fermentation medium.

[0062] The sensors prepared in each example were used to detect pyruvate content (test values) in the fermentation products described above using the standard addition method. During ethanol fermentation using sugars as substrates, pyruvate is produced as an intermediate metabolite during glycolysis. To verify the accuracy of the prepared sensors in real ethanol fermentation broth, ethanol fermentation broth was selected as a representative system and pyruvate detection was performed using the standard addition method. First, an 8.806 g / L pyruvate standard solution was injected into PBS buffer to generate a standard signal current. After the current signal stabilized, fermentation broth containing various pyruvate concentrations (1.761, 8.806, 17.62, 26.42, and 61.64 g / L) (actual values) was added to obtain the corresponding response currents (test values). Five concentration groups were tested to verify the accuracy of the biosensors prepared in Examples 1-4. The results are shown in Table 2 below. As can be seen from Table 2, the biosensor can accurately detect the content of pyruvate in the fermentation broth. The RSD of these samples is between 1.12% and 1.7% (relative standard deviation), which is capable of detection in complex actual fermentation environments.

[0063] Table 3 Detection results of pyruvic acid in real samples 3. Repeatability Verification Ten sensor chips were prepared using the same process as in Example 1. PBS electrolyte and pyruvate were then added to the detection cell to a 1 mM pyruvate concentration. Each of the ten sensor chips was then used for sequential detection. The results showed that the pyruvate biosensor chips produced essentially identical results, with a pyruvate detection value of 1 ± 0.15 mM. This demonstrates that the pyruvate biosensor chip produced by the method of the present invention exhibits stable performance, high detection accuracy, and is suitable for mass production.

[0064] Comparative Example 1 The difference between this comparative example and Example 1 is that, in step 1), the concentrations of sodium citrate in solution B were changed to 0 mM, 10 mM, and 500 mM, respectively. The other conditions were the same as in Example 1. The detection was carried out using the chronoamperometric method of a Shanghai Chenhua CHI660E electrochemical workstation. The results showed that the detection limits were 9.2 μM, 10.7 μM, and 21.9 μM, respectively.

[0065] Comparative Example 2 This comparative example differs from Example 1 in that, instead of etching with ammonia to form an open frame structure and electroplating gold nanoparticles, solid cubic PB was synthesized directly in solution. Steps (2) and (5) in Example 1 were omitted, and the remaining preparation process remained the same as in Example 1. The prepared PB was dried to obtain a screen-printed sensing electrode, and the remaining conditions remained unchanged. No detection signal was observed.

[0066] The above description is merely a preferred embodiment of the present invention and does not constitute any other form of limitation to the present invention. Any person skilled in the art may utilize the technical contents disclosed above to change or modify them into equivalent embodiments with equivalent changes for application in other fields. However, any simple modification, equivalent change, and modification of the above embodiments made in accordance with the technical essence of the present invention without departing from the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a pyruvic acid sensor chip of a Prussian blue nanoframe / gold nanocomposite material, characterized in that: The preparation steps are as follows: (1) A soluble metal salt containing ferrocyanide ions is added to water to obtain solution A; a soluble iron salt and a complexing agent are added to water to obtain a mixed solution B; solution A and mixed solution B are mixed and subjected to a hydrothermal synthesis reaction. After the reaction is completed, the mixture is filtered, and the obtained solid is centrifuged, washed, and dried to obtain PB powder; (2) adding PB powder to ethanol and mixing uniformly to obtain a dispersion, adding ammonia water to the dispersion to obtain a reaction solution, centrifuging and washing after the reaction, and drying to obtain open-framework Prussian blue particles; (3) mixing open-frame Prussian blue particles with conductive carbon paste to obtain nanoprinting paste; (4) screen printing a conductive carbon paste onto a substrate to form a counter electrode and a connector, screen printing a silver / silver chloride paste onto a substrate to form a reference electrode, and screen printing the nano-printing paste onto a substrate to form a working electrode; Drying and curing to obtain an electrode chip; (5) Placing the electrode chip in a chloroauric acid solution for constant potential electrodeposition to obtain a modified electrode chip; (6) The PBS solution of pyruvate oxidase, the ƹ-polylysine solution, and the ‌1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution were mixed evenly to obtain an enzyme solution, and the enzyme solution was dropped onto the working electrode surface of the modified electrode chip and dried to obtain a pyruvate sensor chip.

2. The method for preparing the pyruvic acid sensor chip of the Prussian blue nanoframework / gold nanocomposite material according to claim 1, characterized in that: Step (1) The solute of solution A is K4Fe(CN)6 or K3Fe(CN)6, the concentration of solution A is 15-25mM, the soluble iron salt in the mixed solution B is any one of FeSO4, FeCl2, and FeCl3, the complexing agent is citric acid or sodium citrate, the concentration of iron ions in the mixed solution B is 25-35mM, the concentration of the complexing agent is 150-200mM, and the volume ratio of solution A to mixed solution B is 1:1; the hydrothermal synthesis reaction temperature is 45-65°C, and the hydrothermal synthesis reaction time is 20-24h.

3. The method for preparing the pyruvic acid sensor chip of the Prussian blue nanoframework / gold nanocomposite material according to claim 1, characterized in that: In step (2), the mass fraction of ammonia water is 1.4-3.4%, the concentration of the dispersion is 0.2-0.6 mg / mL, the volume ratio of ammonia water to the dispersion is 5:1-5, and the reaction time is 10-25 min.

4. The method for preparing the pyruvic acid sensor chip of the Prussian blue nanoframework / gold nanocomposite material according to claim 1, characterized in that: In step (3), the mass ratio of the open frame Prussian blue particles to the conductive carbon paste is 1:1-50.

5. The method for preparing the pyruvic acid sensor chip of the Prussian blue nanoframework / gold nanocomposite material according to claim 1, characterized in that: In step (5), the concentration of the chloroauric acid solution is 6-12 mM, the electrodeposition voltage is -0.2 V, and the electrodeposition time is 200-400 s.

6. The method for preparing the pyruvic acid sensor chip of the Prussian blue nanoframework / gold nanocomposite material according to claim 1, characterized in that: The concentration of pyruvate oxidase in the enzyme solution in step (6) is 200-2000 U·mL -1 The mass fraction of the ƹ-polylysine solution is 0.1-0.28%; the concentration of the ‌1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is 2-9 mM; the volume ratio of the pyruvate oxidase PBS solution: ƹ-polylysine solution: ‌1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is 1:1:1; the addition amount is 2-10 μL, the drying temperature is 0-20°C, and the drying time is at least 12 hours.

7. Use of the sensor chip prepared by the method according to any one of claims 1 to 6 in the detection of pyruvic acid.

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