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CN-121994887-A - Preparation method and application of glycerin biosensor based on hollow cuprous sulfide

CN121994887ACN 121994887 ACN121994887 ACN 121994887ACN-121994887-A

Abstract

The invention belongs to the technical field of glycerol detection devices in fermentation, food analysis, clinical medicine and bioengineering, and relates to a preparation method of a glycerol biosensor. According to the invention, the hollow cuprous sulfide nanocubes with the surfaces provided with the stacked nanosheets are synthesized by etching the cuprous sulfide with the sodium sulfide, so that the conductivity and the active site of the material are increased, and rich binding sites are provided for loading glycerol-3-phosphate oxidase and glycerol kinase. The nano material is fixed on a glassy carbon electrode, and glycerin-3-phosphate oxidase and glycerin kinase are dripped on the electrode, so that the high-performance glycerin biosensor is obtained. The method has higher performance in the detection of a real sample, has simple preparation process and lower cost, can be used for large-scale production, and can realize the real-time monitoring of the glycerol in the fermentation liquor.

Inventors

  • CHU ZHENYU
  • XU CHENLONG
  • ZHANG SHAOQI
  • PANG JUN

Assignees

  • 南京工业大学

Dates

Publication Date
20260508
Application Date
20260302

Claims (6)

  1. 1. The preparation method of the glycerol biosensor based on hollow cuprous sulfide is characterized by comprising the following steps: (1) Adding sodium citrate and copper sulfate pentahydrate into deionized water, uniformly mixing, adding sodium hydroxide solution, uniformly mixing, adding ascorbic acid solution, reacting at normal temperature, filtering after the reaction is finished, centrifuging the obtained solid, washing and drying to obtain Cu 2 O powder; (2) Adding Cu 2 O powder and sodium sulfide into deionized water, uniformly mixing to obtain a mixed solution, reacting at normal temperature, and centrifuging after the reaction is finished to obtain Cu 2 S powder; (3) Adding Cu 2 S powder into deionized water and uniformly dispersing to obtain a dispersion liquid, and dripping the dispersion liquid onto a glassy carbon electrode to obtain a working electrode; (4) And sequentially fixing glycerol-3-phosphate oxidase and glycerol kinase on the surface of a working electrode, and drying to obtain the glycerol biosensor.
  2. 2. The method for preparing the hollow cuprous sulfide-based glycerin biosensor according to claim 1, wherein in the step (1), the molar ratio of sodium citrate to cupric sulfate pentahydrate to sodium hydroxide to ascorbic acid is (5-15): 5-25): 50-100): 40-60, the concentration of the ascorbic acid solution is 0.5-1.5mol/L, and the reaction time is 1-3 hours.
  3. 3. The preparation method of the glycerol biosensor based on hollow cuprous sulfide according to claim 1, wherein the molar ratio of Cu 2 O to sodium sulfide in the mixed solution in the step (2) is (1-3) (8-15), the concentration of Cu 2 O in the mixed solution is 10-30mmol/L, and the reaction time is 1-3h.
  4. 4. The method for preparing a hollow cuprous sulfide-based glycerin biosensor according to claim 1, wherein the concentration of Cu 2 S in the dispersion in step (3) is 0.5-10mg/mL, and the dropping amount is 2-6 μl.
  5. 5. The method for preparing a hollow cuprous sulfide-based glycerol biosensor according to claim 1, wherein the loading amount of glycerol-3-phosphate oxidase on the working electrode in step (4) is 10-100 μl cm -2 , the loading amount of glycerol kinase is 10-100 μl cm -2 , and the drying temperature is 0-10 ℃.
  6. 6. Use of a sensor prepared by the method of any one of claims 1-5 in glycerol detection.

Description

Preparation method and application of glycerin biosensor based on hollow cuprous sulfide Technical Field The invention belongs to the technical field of glycerin detection, and relates to a preparation method and application of a glycerin biosensor based on hollow cuprous sulfide. Background Glycerol is a major byproduct of biodiesel production. With increasing emphasis on sustainable development and resource recovery worldwide, the use of glycerol as a low cost carbon source in microbial fermentation can produce a variety of high value chemicals including 1, 3-propanediol, mannitol, extracellular lipases, and single cell proteins, among others. The absorption and metabolism of glycerol by microorganisms is a key step in the fermentation reaction. The optimal glycerol concentration may provide a sufficient carbon source to support normal microbial growth and metabolism. However, too high glycerol levels can create osmotic stress, inhibit microbial growth and slow the fermentation process, while insufficient glycerol may not meet microbial requirements, resulting in premature termination of fermentation. Therefore, in order to improve fermentation performance, real-time on-line monitoring of glycerol concentration in fermentation broth is critical to product yield, ensuring process stability, and achieving industrial economic feasibility. In industry, conventional fermentation analysis methods, including High Performance Liquid Chromatography (HPLC), gas Chromatography (GC) and spectrophotometry, are capable of detecting the concentration of components in a fermentation broth, but suffer from the disadvantages of time consuming, cumbersome equipment, delayed results, and complex operations. In this case, these methods are difficult to apply to rapid and on-site monitoring of glycerol level fluctuations in order to control the microbial metabolic state accurately in time during the fermentation reaction. Compared with the traditional glycerol detection method, the online detection of the electrochemical sensor method has the characteristics of simplicity in operation, rapidness, high efficiency, automation and the like, and can realize transparency. Disclosure of Invention The invention provides a preparation method and application of a novel glycerin biosensor based on hollow cuprous sulfide, aiming at the problems existing in the traditional glycerin detection. In order to achieve the above purpose, the invention is realized by adopting the following technical scheme: a preparation method of a glycerin biosensor based on hollow cuprous sulfide comprises the following steps: (1) Adding sodium citrate and copper sulfate pentahydrate into deionized water, uniformly mixing, adding sodium hydroxide solution, uniformly mixing, adding ascorbic acid solution, reacting at normal temperature, filtering after the reaction is finished, centrifuging the obtained solid, washing and drying to obtain Cu 2 O powder. (2) Adding Cu 2 O powder and sodium sulfide into deionized water, uniformly mixing to obtain a mixed solution, reacting at normal temperature, and centrifuging after the reaction is finished to obtain Cu 2 S powder. (3) Adding Cu 2 S powder into deionized water, dispersing uniformly to obtain a dispersion liquid, and dripping the dispersion liquid onto the glassy carbon electrode to obtain the working electrode. (4) And sequentially fixing glycerol-3-phosphate oxidase and glycerol kinase on the surface of a working electrode, and drying to obtain the glycerol biosensor. Preferably, in the step (1), the molar ratio of sodium citrate to cupric sulfate pentahydrate to sodium hydroxide to ascorbic acid is (5-15), the molar ratio of sodium sulfate pentahydrate to sodium hydroxide to ascorbic acid is (5-25), the molar ratio of sodium sulfate pentahydrate to ascorbic acid is (50-100), the molar ratio of sodium sulfate pentahydrate to ascorbic acid is (40-60), the concentration of the ascorbic acid solution is 0.5-1.5mol/L, and the reaction time is 1-3h. Preferably, the molar ratio of Cu 2 O to sodium sulfide in the mixed solution in the step (2) is (1-3): 8-15, the concentration of Cu 2 O in the mixed solution is 10-30mmol/L, and the reaction time is 1-3h. Preferably, the Cu 2 S concentration in the dispersion in the step (3) is 0.5-10mg/mL, and the drop amount is 2-6 mu L. Preferably, the loading of the glycerol-3-phosphate oxidase on the working electrode of the step (4) is 10-100 mu L cm -2, the loading of the glycerol kinase is 10-100 mu L cm -2, and the drying temperature is 0-10 ℃. The invention provides application of the sensor prepared by the method in glycerin detection. In the case of glycerol detection, the oxidation reaction of glycerol under the action of an enzyme generates H 2O2, and an electrical signal is generated and detected in the form of an electric current. The semiconductor transition metal sulfide Cu 2 S adopted by the invention is a p-type semiconductor, has variable stoichiometric ratio, acc