Preparation method and application of glycerol biosensor based on hollow cuprous sulfide

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. Cuprous oxide is etched through sodium sulfide, a hollow cuprous sulfide nanocube with nanosheets stacked layer by layer on the surface is synthesized, the conductivity and active sites 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 glycerol-3-phosphate oxidase and glycerol kinase are dropwise added on the electrode, so that the glycerol biosensor with high performance is obtained. The method has the advantages of simple preparation process and low cost, has higher performance in real sample detection, can be used for large-scale production, and can realize real-time monitoring of glycerol in fermentation liquor.

Description

Technical Field

[0001] This invention belongs to the field of glycerol detection technology, and relates to a method for preparing and applying a glycerol biosensor based on hollow cuprous sulfide. Background Technology

[0002] Glycerol is a major byproduct of biodiesel production. With increasing global emphasis on sustainable development and resource recycling, 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. Microbial uptake and metabolism of glycerol are key steps in the fermentation reaction. Optimal glycerol concentration provides sufficient carbon to support normal microbial growth and metabolism. However, excessively high glycerol levels can cause osmotic stress, inhibiting microbial growth and slowing the fermentation process, while insufficient glycerol may fail to meet the microbial needs, leading to premature fermentation termination. Therefore, real-time online monitoring of the glycerol concentration in the fermentation broth is crucial for improving fermentation performance, ensuring product yield, guaranteeing process stability, and achieving industrial economic feasibility.

[0003] In industry, traditional fermentation analysis methods, including high-performance liquid chromatography (HPLC), gas chromatography (GC), and spectrophotometry, can detect the concentration of components in the fermentation broth, but they suffer from drawbacks such as being time-consuming, using bulky instruments, providing delayed results, and being complex to operate. In this context, these methods are difficult to apply for rapid and on-site monitoring of glycerol level fluctuations, enabling timely and accurate control of microbial metabolic states during the fermentation reaction. Compared to traditional glycerol detection methods, online detection using electrochemical sensors offers advantages such as simple operation, rapid efficiency, and automation, achieving greater transparency. Summary of the Invention

[0004] This invention addresses the problems existing in traditional glycerol detection by proposing a novel method for preparing and applying a glycerol biosensor based on hollow cuprous sulfide.

[0005] To achieve the above objectives, the present invention is implemented using the following technical solution: A method for preparing a glycerol biosensor based on hollow cuprous sulfide comprises the following steps: (1) Sodium citrate and copper sulfate pentahydrate were added to deionized water and mixed evenly. Sodium hydroxide solution was added and mixed evenly. Ascorbic acid solution was added and the reaction was carried out at room temperature. After the reaction was completed, the mixture was filtered, and the obtained solid was centrifuged, washed, and dried to obtain Cu2O powder.

[0006] (2) Add Cu2O powder and sodium sulfide to deionized water and mix evenly to obtain a mixture. React at room temperature. After the reaction is completed, centrifuge to obtain Cu2S powder.

[0007] (3) Add Cu2S powder to deionized water and disperse it evenly to obtain a dispersion. Add the dispersion to a glassy carbon electrode to obtain a working electrode.

[0008] (4) Glycerol-3-phosphate oxidase and glycerol kinase were sequentially immobilized on the surface of the working electrode and dried to obtain a glycerol biosensor.

[0009] As a preferred embodiment, in step (1), the molar ratio of sodium citrate: copper sulfate pentahydrate: sodium hydroxide: ascorbic acid is (5-15):(5-25):(50-100):(40-60), the concentration of ascorbic acid solution is 0.5-1.5mol / L, and the reaction time is 1-3h.

[0010] Preferably, in step (2), the molar ratio of Cu2O to sodium sulfide in the mixture is (1-3):(8-15), the concentration of Cu2O in the mixture is 10-30 mmol / L, and the reaction time is 1-3 h.

[0011] Preferably, the concentration of Cu2S in the dispersion in step (3) is 0.5-10 mg / mL, and the amount added is 2-6 μL.

[0012] Preferably, the loading of glycerol-3-phosphate oxidase on the working electrode in step (4) is 10-100 μL·cm⁻¹. -2 The glycerol kinase loading capacity is 10-100 μL·cm. -2 The drying temperature is 0-10℃.

[0013] This invention proposes the application of the sensor prepared by the above method in glycerol detection.

[0014] In glycerol detection, glycerol undergoes oxidation under the action of enzymes to generate H2O2, which in turn produces an electrical signal that is detected in the form of current. The semiconductor transition metal sulfide Cu2S used in this invention is a p-type semiconductor with variable stoichiometry, accessible valence states, and diverse nanostructures, possessing strong enzyme immobilization sites. These characteristics enable Cu2S to serve as an electrocatalytic platform for the stable immobilization of glycerol-3-phosphate oxidase and glycerol kinase, thus constructing an electrochemical glycerol biosensor. However, the morphological defects of Cu2S itself affect the sensor's detection performance. This invention utilizes sodium sulfide to etch its hollow morphology, resulting in synthesized hollow Cu2S with a larger internal pore volume and specific surface area. This improves the surface active sites and electrocatalytic activity, significantly enhancing the final sensor performance and yielding a highly sensitive glycerol biosensor.

[0015] Compared with the prior art, the advantages and positive effects of the present invention are as follows: 1. This invention designs hollow cuprous sulfide nanoparticles with high conductivity and high electrocatalytic activity as electrode materials. The material has a rough nanosheet surface, which provides abundant enzyme binding sites, which is conducive to enzyme immobilization and provides a short path for electron transfer, thus meeting the requirements for glycerol concentration detection in fermentation systems.

[0016] 2. The high-performance glycerol sensor obtained by this invention has high detection sensitivity, simple preparation process, and low cost, and can be used for large-scale production to achieve accurate real-time detection of glycerol concentration. Attached Figure Description

[0017] Figure 1 is a TEM image of hollow cuprous sulfide in Example 1.

[0018] Figure 2 is an SEM image of the hollow cuprous sulfide in Example 1. Detailed Implementation

[0019] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described below with reference to specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0020] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification. Example 1

[0021] 1) Dissolve 15 mmol copper sulfate pentahydrate and 5 mmol sodium citrate in 80 mL of deionized water to obtain solution A. Dissolve 100 mmol sodium hydroxide in 20 mL of deionized water to obtain solution B. Dissolve 50 mmol ascorbic acid in 50 mL of deionized water to obtain solution C. Add solutions B and C sequentially to solution A and react at room temperature. After reacting for 1 hour, centrifuge the reaction mixture and wash it three times alternately with 10 mL of ethanol and deionized water each time. Centrifuge at 8000 r·min. -1 The centrifugation time was 6 min. After centrifugation and washing, the product was dried at 60℃ for 6 h to obtain Cu2O powder.

[0022] 2) Place 2.5 mmol of Cu₂O powder in a beaker, add 100 mL of deionized water, and stir manually with a glass rod for 1 min. Place 10 mmol of sodium sulfide in a beaker, add 40 mL of deionized water, stir, and stir manually with a glass rod for 1 min to dissolve. Mix the two solutions and react at room temperature. After reacting for 1 hour, centrifuge the reaction solution, wash three times with 15 mL of deionized water each time, and centrifuge at 10000 r·min. -1 The centrifugation time was 6 min. After centrifugation and washing, the product was dried at 60℃ for 6 h to obtain Cu2S powder.

[0023] 3) Take 5 mg of Cu2S powder and add it to 1 mL of deionized water. Sonicate for 5 min to disperse the powder evenly to obtain a Cu2S dispersion. Take 4 μL of the Cu2S dispersion and drop it onto the surface of a glassy carbon electrode (Shanghai Ciyue Industrial Co., Ltd.). Dry the electrode at room temperature for 24 h to obtain the working electrode.

[0024] 4) Add 0.6 g of chitosan to 100 mL of 2% acetic acid solution and stir with a glass rod for 2 min to obtain a chitosan dispersion. Take 15 mg of 300 U of glycerol-3-phosphate oxidase (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) and add it to 30 μL of PBS buffer (containing 0.5 M potassium chloride, 0.3 M potassium dihydrogen phosphate, and 0.26 M dipotassium hydrogen phosphate, with water as the solvent, the same below). Take 15 μL and add it to a 0.2 mL centrifuge tube. Then add 15 μL of 5 mg / mL chitosan solution to dilute the glycerol-3-phosphate oxidase solution. After mixing well, take 5 μL and drop it onto the working electrode (electrode area 0.07065 cm²). 2 The glycerol biosensor was dried at 0℃ for 12 h. 15 mg and 300 U of glycerol kinase (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) were taken out and dispersed in 30 μL of PBS buffer. 15 μL of the glycerol kinase solution was added to a 0.2 mL centrifuge tube, and then 15 μL of 5 mg / mL chitosan solution was added to dilute the glycerol kinase solution. After mixing evenly, 5 μL of the solution was added dropwise to the working electrode. After drying at 0℃ for 12 h, the glycerol biosensor was obtained.

[0025] Step 2) The Cu2S nanocube TEM characterization image obtained is as follows Figure 1 As shown in the figure, Cu₂S exhibits a hollow morphology. The SEM characterization image is shown below. Figure 2 As shown in the figure, the surface of Cu2S nanocube has layers of stacked nanosheets. Example 2

[0026] Unless otherwise specified, this embodiment is consistent with Embodiment 1.

[0027] 1) Dissolve 25 mmol copper sulfate pentahydrate and 8 mmol sodium citrate in 80 mL of deionized water to obtain solution A. Dissolve 100 mmol sodium hydroxide in 20 mL of deionized water to obtain solution B. Dissolve 40 mmol ascorbic acid in 50 mL of deionized water to obtain solution C. Add solutions B and C sequentially to solution A and react at room temperature. After reacting for 1 hour, centrifuge the reaction mixture and wash it three times alternately with 10 mL of ethanol and deionized water each time. Centrifuge at 8000 r·min. -1 The centrifugation time was 6 min. After centrifugation and washing, the product was dried at 60℃ for 6 h to obtain Cu2O powder.

[0028] 2) Place 3 mmol of Cu₂O powder in a beaker, add 100 mL of deionized water, and stir manually with a glass rod for 1 min; place 9 mmol of sodium sulfide in a beaker, add 40 mL of deionized water, and stir to dissolve; mix the two solutions and react at room temperature. After reacting for 1 hour, centrifuge the reaction solution, wash three times with 15 mL of deionized water each time, and centrifuge at 10000 r·min. -1 The centrifugation time was 6 min. After centrifugation and washing, the product was dried at 60℃ for 6 h to obtain Cu2S powder.

[0029] 3) Take 10 mg of Cu2S powder and add it to 1 mL of deionized water. Sonicate for 5 min to disperse the powder evenly to obtain a Cu2S dispersion. Take 2 μL of the Cu2S dispersion and add it dropwise onto the surface of a glassy carbon electrode. Dry the electrode at room temperature for 24 h to obtain the working electrode.

[0030] 4) Add 0.6 g of chitosan to 100 mL of 2% acetic acid solution and stir with a glass rod for 2 min to obtain a chitosan dispersion. Take 15 mg and 300 U of glycerol-3-phosphate oxidase and add it to 30 μL of PBS buffer. Take 15 μL of the dispersion and add it to a 0.2 mL centrifuge tube. Then add 15 μL of chitosan solution with a concentration of 5 mg / mL to dilute the glycerol-3-phosphate oxidase solution. After mixing well, take 7 μL and add it to the working electrode. Dry at 0 °C for 12 h. Take 15 mg and 300 U of glycerol kinase and disperse it in 30 μL of PBS buffer. Take 15 μL of the dispersion and add it to a 0.2 mL centrifuge tube. Then add 15 μL of chitosan solution with a concentration of 5 mg / mL to dilute the glycerol kinase solution. After mixing well, take 7 μL and add it to the working electrode. Dry at 0 °C for 12 h to obtain a glycerol biosensor. Example 3

[0031] Unless otherwise specified, this embodiment is consistent with Embodiment 1.

[0032] 1) Dissolve 10 mmol copper sulfate pentahydrate and 10 mmol sodium citrate in 80 mL of deionized water to obtain solution A. Dissolve 80 mmol sodium hydroxide in 20 mL of deionized water to obtain solution B. Dissolve 40 mmol ascorbic acid in 50 mL of deionized water to obtain solution C. Add solutions B and C sequentially to solution A and react at room temperature. After reacting for 1 hour, centrifuge the reaction mixture and wash it three times alternately with 10 mL of ethanol and deionized water each time. Centrifuge at 8000 r·min. -1 The centrifugation time was 6 min. After centrifugation and washing, the product was dried at 60℃ for 6 h to obtain Cu2O powder.

[0033] 2) Place 1.5 mmol of Cu₂O powder in a beaker, add 100 mL of deionized water, and stir manually with a glass rod for 1 min; place 11 mmol of sodium sulfide in a beaker, add 40 mL of deionized water, and stir to dissolve; mix the two solutions and react at room temperature. After reacting for 1 hour, centrifuge the reaction solution, wash three times with 15 mL of deionized water each time, and centrifuge at 10000 r·min. -1 The centrifugation time was 6 min. After centrifugation and washing, the product was dried at 60℃ for 6 h to obtain Cu2S powder.

[0034] 3) Take 7 mg of Cu2S powder and add it to 1 mL of deionized water. Sonicate for 5 min to disperse the powder evenly to obtain a Cu2S dispersion. Take 5 μL of the Cu2S dispersion and drop it onto the surface of a glassy carbon electrode. Dry the electrode at room temperature for 24 h to obtain the working electrode.

[0035] 4) Add 0.6 g of chitosan to 100 mL of 2% acetic acid solution and stir with a glass rod for 2 min to obtain a chitosan dispersion. Take 15 mg and 300 U of glycerol-3-phosphate oxidase and add it to 30 μL of PBS buffer. Take 15 μL of the dispersion and add it to a 0.2 mL centrifuge tube. Then add 15 μL of 5 mg / mL chitosan solution to dilute the glycerol-3-phosphate oxidase solution. After mixing well, take 7 μL and add it to the working electrode. Dry at 0 °C for 12 h. Take 15 mg and 300 U of glycerol kinase and disperse it in 30 μL of PBS buffer. Take 15 μL of the dispersion and add it to a 0.2 mL centrifuge tube. Then add 15 μL of 5 mg / mL chitosan solution to dilute the glycerol kinase solution. After mixing well, take 7 μL and add it to the working electrode. Dry at 0 °C for 12 h to obtain a glycerol biosensor. Example 4

[0036] Unless otherwise specified, this embodiment is consistent with Embodiment 1.

[0037] 1) Dissolve 5 mmol copper sulfate pentahydrate and 15 mmol sodium citrate in 80 mL of deionized water to obtain solution A. Dissolve 70 mmol sodium hydroxide in 20 mL of deionized water to obtain solution B. Dissolve 60 mmol ascorbic acid in 50 mL of deionized water to obtain solution C. Add solutions B and C sequentially to solution A and react at room temperature. After reacting for 1 hour, centrifuge the reaction mixture and wash it three times alternately with 10 mL of ethanol and deionized water each time. Centrifuge at 8000 r·min. -1 The centrifugation time was 6 min. After centrifugation and washing, the product was dried at 60℃ for 6 h to obtain Cu2O powder.

[0038] 2) Place 2 mmol of Cu₂O powder in a beaker, add 100 mL of deionized water, and stir manually with a glass rod for 1 min; place 12 mmol of sodium sulfide in a beaker, add 40 mL of deionized water, and stir to dissolve; mix the two solutions and react at room temperature. After reacting for 1 hour, centrifuge the reaction solution, wash three times with 15 mL of deionized water each time, and centrifuge at 10000 r·min. -1 The centrifugation time was 6 min. After centrifugation and washing, the product was dried at 60℃ for 6 h to obtain Cu2S powder.

[0039] 3) Take 3 mg of Cu2S powder and add it to 1 mL of deionized water. Sonicate for 5 min to disperse the powder evenly to obtain a Cu2S dispersion. Take 6 μL of the Cu2S dispersion and add it dropwise onto the surface of a glassy carbon electrode. Dry the electrode at room temperature for 24 h to obtain the working electrode.

[0040] 4) Add 0.6 g of chitosan to 100 mL of 2% acetic acid solution and stir with a glass rod for 2 min to obtain a chitosan dispersion. Take 15 mg of 300 U glycerol-3-phosphate oxidase and add it to 30 μL of PBS buffer. Take 15 μL of this dispersion and add it to a 0.2 mL centrifuge tube. Then, add 15 μL of 5 mg / mL chitosan solution to dilute the glycerol-3-phosphate oxidase solution. After mixing well, add 4 μL of this solution to the working electrode and dry at 0 °C for 12 h. Take 15 mg of 300 U glycerol kinase and disperse it in 30 μL of PBS buffer. Take 15 μL of this dispersion and add it to a 0.2 mL centrifuge tube. Then, add 15 μL of 5 mg / mL chitosan solution to dilute the glycerol kinase solution. After mixing well, add 4 μL of this solution to the working electrode and dry at 0 °C for 12 h to obtain the glycerol biosensor. 1. Linear range and sensitivity test Using the working electrode prepared in Example 1, with a Pt electrode as the counter electrode (Shanghai Ciyue Industrial Co., Ltd.) and Ag / AgCl as the reference electrode (Shanghai Ciyue Industrial Co., Ltd.), the changes in glycerol concentration during the fermentation process were tested using a constant current (IT) method on a Shanghai Chenhua CHI660E electrochemical workstation. PBS buffer was also used in the electrolytic cell. After each sampling, 40 μL of sample was added to 40 mL of electrolyte for detection. After detection, the sample concentration was calculated using a standard curve, and the linear formula was Δi = 1.76 + 1.22C. Glycerol R 2 =0.996. Testing showed that the sensor chip prepared in Example 1 achieved a detection sensitivity of 15.63 μA·mM in the detection cell. -1 ·cm -2 The detection linear range is 0.005-0.295 mM, meaning the detection limit reaches 0.005 mM. After storing the chip in a refrigerator at 4°C for 30 days, its steady-state response current still maintains more than 81% of the initial sensitivity, indicating that the prepared glycerase biosensor has good long-term stability.

[0041] 2. Accuracy Verification The sensors prepared in each example were randomly used to detect glycerol content at different stages of the fermentation process (test values). The same samples were tested using a glycerol detection kit (Shanghai Beyotime Biotechnology Co., Ltd.) (actual values) for comparison. The accuracy of the biosensors prepared in Examples 1-4 was verified, and the results are shown in Table 1 below. Table 1 shows that the biosensor can accurately detect the glycerol content in the fermentation broth, and its detection results are not significantly different from those obtained using the glycerol detection kit.

[0042] Table 1. Detection results of pyruvate in real samples Actual value of glycerol (g / L) Glycerol test value (g / L) Relative standard deviation Example 1 30.47 30.81 1.12% Example 2 25.68 26.36 2.65% Example 3 20.18 20.46 1.39% Example 4 9.50 9.28 2.32% The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments for application in other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a glycerol biosensor based on hollow cuprous sulfide, characterized in that, The steps are as follows: (1) Sodium citrate and copper sulfate pentahydrate were added to deionized water and mixed evenly. Sodium hydroxide solution was added and mixed evenly. Ascorbic acid solution was added and the reaction was carried out at room temperature. After the reaction was completed, the mixture was filtered, and the solid obtained was centrifuged, washed, and dried to obtain Cu2O powder. (2) Add Cu2O powder and sodium sulfide to deionized water and mix evenly to obtain a mixture. React at room temperature. After the reaction is completed, centrifuge to obtain Cu2S powder. (3) Add Cu2S powder to deionized water and disperse it evenly to obtain a dispersion. Drop the dispersion onto a glassy carbon electrode to obtain a working electrode. (4) Glycerol-3-phosphate oxidase and glycerol kinase were sequentially immobilized on the surface of the working electrode and dried to obtain a glycerol biosensor.

2. The method for preparing a glycerol biosensor based on hollow cuprous sulfide according to claim 1, characterized in that, In step (1), the molar ratio of sodium citrate: copper sulfate pentahydrate: sodium hydroxide: ascorbic acid is (5-15):(5-25):(50-100):(40-60), the concentration of ascorbic acid solution is 0.5-1.5mol / L, and the reaction time is 1-3h.

3. The method for preparing a glycerol biosensor based on hollow cuprous sulfide according to claim 1, characterized in that, In step (2), the molar ratio of Cu2O to sodium sulfide in the mixture is (1-3):(8-15), the concentration of Cu2O in the mixture is 10-30 mmol / L, and the reaction time is 1-3 h.

4. The method for preparing a glycerol biosensor based on hollow cuprous sulfide according to claim 1, characterized in that, In step (3), the concentration of Cu2S in the dispersion is 0.5-10 mg / mL, and the amount added is 2-6 μL.

5. The method for preparing a glycerol biosensor based on hollow cuprous sulfide according to claim 1, characterized in that, Step (4): The loading capacity of glycerol-3-phosphate oxidase on the working electrode is 10-100 μL·cm. -2 The glycerol kinase loading capacity is 10-100 μL·cm. -2 The drying temperature is 0-10℃.

6. The application of the sensor prepared by any one of claims 1-5 in glycerol detection.

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