3D-rGO-CS-BSA Composite Modified Electrode for Electrochemical Chiral Recognition, Its Preparation Method and Application

Abstract

The present invention belongs to the field of analytical chemistry, and relates to a 3D-rGO-CS-BSA composite modified electrode for electrochemistry chiral recognition, its preparation method and application. The preparation includes the following steps: preparing a three-dimensional reduced graphene oxide, chitosan and bovine serum albumin (3D-rGO-CS-BSA) composite solution; preparing a 3D-rGO-CS-BSA modified electrode; and identifying the enantiomers of 1-Boc-3-hydroxypyrrolidine by electrochemistry method. The beneficial effects of the present invention are: the 3D-rGO-CS-BSA modified electrode is simple and easy to operate, has high stability, and has good chiral recognition effect.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry, and particularly relates to a 3D-rGO-CS-BSA composite modified electrode for electrochemical chiral recognition, a preparation method thereof, and an application thereof. Background Art

[0002] Chirality exists ubiquitously in nature. Many molecular substances are mirror images of each other but cannot be superimposed, just like a person's left and right hands. This non-superimposable property is called chirality, and these two molecules that are mirror images of each other but cannot be superimposed are called enantiomers. Although the chiral enantiomers of the same substance have the same elemental composition, they sometimes exhibit different pharmacological activities. As the most fundamental biological macromolecules in life activities, such as proteins, polysaccharides, nucleic acids, and enzymes, etc., are almost all chiral. Therefore, the research on the recognition of chiral drugs has important theoretical significance and application prospects in aspects such as drug design, food technology, and biomedicine. Developing simple, rapid, and accurate recognition has become a hot and frontier direction in analytical chemistry in recent years, and has important research significance. Electrochemical methods have attracted wide attention due to their advantages such as simple operation and rapid detection.

[0003] Reduced graphene oxide (3D-rGO) has a large effective specific surface area, which is conducive to the loading of chiral selectors, has good thermal stability, many active sites, and high conductivity. Chitosan (CS), as a natural polysaccharide, has excellent optical activity and a large number of chiral sites, excellent hydrophilicity, easy film-forming property, and good adhesion, and is widely used in the construction of electrochemical chiral sensors. Bovine serum albumin (BSA) has abundant chiral sites, and its adsorption with chitosan can change its spatial configuration, making its chiral sites fully exposed. Therefore, we constructed an electrochemical chiral surface by adsorbing and binding chitosan and bovine serum albumin into the three-dimensional cavity structure of reduced graphene oxide, and has a good recognition effect on the enantiomers of 1-Boc-3-hydroxypyrrolidine. Summary of the Invention

[0004] The object of the present invention is to provide a 3D-rGO-CS-BSA composite modified electrode for the electrochemical chiral recognition of 1-Boc-3-hydroxypyrrolidine enantiomers, a preparation method thereof, and an application thereof. Three-dimensional reduced graphene oxide has a large specific surface area, can significantly improve conductivity, and the framework-open and interconnected porous structure provides an effective substrate for chitosan and bovine serum albumin. The preparation process is simple, has high stability, and significantly improves the recognition ability of enantiomers.

[0005] A 3D-rGO-CS-BSA composite modified electrode for electrochemistry chiral recognition provided by the present invention comprises an electrode and a 3D-rGO-CS-BSA composite modified on the surface of the electrode, which is obtained by dropping a 3D-rGO-CS-BSA suspension on the surface of the electrode and then drying it.

[0006] The preparation method of the above 3D-rGO-CS-BSA composite modified electrode for electrochemistry chiral recognition comprises the following steps:

[0007] (1) Preparation of 3D-rGO powder: Weigh a certain amount of GO and disperse it in deionized water by ultrasonic; add L-cysteine to the above solution, and then quickly add ammonia water dropwise. Place the mixture in an oil bath and heat it (usually for 4 hours). After cooling to room temperature, wash the black product with deionized water and dry it to obtain three-dimensional reduced graphene oxide (3D-rGO).

[0008] (2) Preparation of 3D-rGO-CS-BSA composite: Weigh an appropriate amount of 3D-rGO powder to prepare a dispersion. After activating the carboxylic acid groups in 3D-rGO with EDC·HCl (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), disperse it in a mixed solution of BSA and CS and ultrasonic to obtain a 3D-rGO-CS-BSA suspension;

[0009] (3) Preparation of 3D-rGO-CS-BSA modified electrode: Take the 3D-rGO-CS-BSA suspension and drop it on the surface of the electrode, and dry it under an infrared lamp to prepare a 3D-rGO-CS-BSA composite modified electrode.

[0010] Further, the mass concentration ratio of EDC·HCl to NHS is 1.2:1.

[0011] Further, the mass ratio of CS to BSA in the 3D-rGO-CS-BSA suspension is 1:1.

[0012] Further, the preparation method of the mixed solution of BSA (bovine serum albumin) and CS (chitosan) comprises: dissolving CS with 2% dilute acetic acid solution to prepare a CS solution; dissolving BSA with deionized water to prepare a BSA solution, and then uniformly mixing the CS solution and the BSA solution.

[0013] More specifically, weigh CS and place it in a 10 ml volumetric flask, add 2% dilute acetic acid solution to dissolve and dilute to the scale; weigh 50 mg of BSA and place it in a 10 ml volumetric flask, add deionized water to dissolve and dilute to the scale; then mix the two to obtain a CS-BSA solution.

[0014] The concentrations of CS and BSA in the composite solution are 5 mg / ml; the ratio of CS to BSA is 1:1.

[0015] The dropwise addition amount of the 3D-rGO-CS-BSA suspension is 8 μL.

[0016] The present invention also provides the application of the 3D-rGO-CS-BSA composite modified electrode for electrochemical chiral recognition in chiral recognition of 1-Boc-3-hydroxypyrrolidine enantiomers, including the following steps: Weigh a certain amount of R- / S-1-Boc-3-hydroxypyrrolidine and place it in a volumetric flask, add a certain amount of the prepared phosphate buffer solution PBS, shake well to obtain the enantiomer solution, immerse the 3D-rGO-CS-BSA composite modified electrode in the enantiomer solution, take it out, and dry it under an infrared lamp to prepare the 1-Boc-3-hydroxypyrrolidine enantiomer electrode;

[0017] Using a three-electrode system, the 1-Boc-3-hydroxypyrrolidine enantiomer electrode is used as the working electrode, the platinum sheet electrode is used as the counter electrode, and the saturated calomel electrode is used as the reference electrode. Immerse this three-electrode system in the prepared 0.1 M K3Fe(CN)6 solution at room temperature for differential pulse testing, and the potential range is -0.2 to 0.6 V.

[0018] Furthermore, the concentrations of the prepared (R)-1-Boc-3-hydroxypyrrolidine and (S)-1-Boc-3-hydroxypyrrolidine solutions are both 50 mM, the pH of the PBS solution is 7, the concentration is 0.1 M, and the standing time is 20 min.

[0019] The beneficial effects of the present invention are: The preparation process of the 3D-rGO-CS-BSA modified electrode is simple and easy to operate, has high stability, and the modified electrode has obvious recognition effect on enantiomers. Description of the Drawings

[0020] Figure 1 It is the scanning electron microscope image of 3D-rGO in Example 1.

[0021] Figure 2 It is the scanning electron microscope image of the 3D-rGO-CS-BSA composite in Example 1.

[0022] Figure 3 It is the differential pulse voltammogram of the 3D-rGO, CS and BSA composite modified electrode for chiral recognition of 1-Boc-3-hydroxypyrrolidine enantiomers in Example 1.

[0023] Figure 4 It is the effect diagram of the stability investigation of the 3D-rGO, CS and BSA composite for chiral recognition of 1-Boc-3-hydroxypyrrolidine enantiomers in Example 2.

[0024] Figure 5 Differential pulse voltammograms of CS and BSA complex modified electrodes with different ratios for chiral recognition of 1-Boc-3-hydroxypyrrolidine enantiomers in Example 3.

[0025] Figure 6 Effect of the content of 3D-rGO in the 3D-rGO-CS-BSA modified electrode of the present invention on the peak current value I R / I S of. Detailed implementation mode

[0026] The present invention will now be further described in conjunction with specific embodiments. The following embodiments are intended to illustrate the present invention rather than further limit the present invention.

[0027] The present invention recognizes 1-Boc-3-hydroxypyrrolidine enantiomers by modifying an electrode with a reduced graphene oxide, chitosan and bovine serum albumin complex according to the following method:

[0028] R R / S = I R / I S

[0029] △E = E R - E S

[0030] In the formula, R R / S represents the oxidation peak current ratio of 1-Boc-3-hydroxypyrrolidine enantiomers, △E represents the oxidation peak potential difference of 1-Boc-3-hydroxypyrrolidine enantiomers, I R and I S respectively represent the oxidation peak currents of R-1-Boc-3-hydroxypyrrolidine and S-1-Boc-3-hydroxypyrrolidine, and E R and E S respectively represent the oxidation peak potentials of R-1-Boc-3-hydroxypyrrolidine and S-1-Boc-3-hydroxypyrrolidine.

[0031] Example 1

[0032] (1) Preparation of 3D-rGO powder: Weigh 100 mg of GO and disperse it in 50 mL of deionized water and sonicate; add 200 mg of L-cysteine to the above solution, then quickly add 3 mL of 25% ammonia water, heat the mixture in an oil bath for 4 hours, and after cooling to room temperature, wash the black product with deionized water and dry it.

[0033] (2) Preparation of CS-BSA suspension: Weigh 50 mg of CS and place it in a 10-ml volumetric flask. Add 2% dilute acetic acid solution to dissolve and dilute to the mark. Weigh 50 mg of BSA and place it in a 10-ml volumetric flask. Add deionized water to dissolve and dilute to the mark. Mix CS and BSA according to the mass concentration ratio of 1:1 to obtain CS-BSA suspension.

[0034] (3) Preparation of 3D-rGO-CS-BSA composite suspension: Weigh 5 mg of the 3D-rGO powder prepared in step (1) and disperse it in a small amount of deionized water. After adding 6 mg of EDC·HCl and 5 mg of NHS to activate the carboxyl groups, add it to 5 mL of the CS-BSA solution prepared in step (2), and ultrasonicate to obtain 3D-rGO-CS-BSA suspension.

[0035] (4) Preparation of 3D-rGO-CS-BSA modified electrode: Take 8 μL of the suspension prepared in step (3) and drop-coat it on the electrode surface, and dry it under an infrared lamp to obtain 3D-rGO-CS-BSA modified electrode.

[0036] (5) Preparation of 1-Boc-3-hydroxypyrrolidine enantiomer electrode: Weigh 234 mg of R- / S-1-Boc-3-hydroxypyrrolidine and place it in a 50-ml volumetric flask. Add a certain amount of prepared phosphate buffer solution (PBS, pH = 7), shake well. Immerse the 3D-rGO-CS-BSA modified electrode prepared in step (4) in the enantiomer solution for 20 min, take it out, and dry it under an infrared lamp. Then the 1-Boc-3-hydroxypyrrolidine enantiomer electrode can be obtained.

[0037] (6) Electrochemical recognition of 1-Boc-3-hydroxypyrrolidine enantiomers: Use a three-electrode system. The 1-Boc-3-hydroxypyrrolidine enantiomer electrode is the working electrode, the platinum sheet electrode is the counter electrode, and the saturated calomel electrode is the reference electrode. Immerse this three-electrode system in the prepared 0.1 M K3Fe(CN)6 solution at room temperature and perform differential pulse testing. The potential range is -0.2 to 0.6 V.

[0038] Example 2

[0039] (1) Preparation of 3D-rGO powder: Weigh 100 mg of GO and disperse it in 50 mL of deionized water by ultrasonication. Add 200 mg of L-cysteine to the above solution, and then quickly add 3 ml of 25% ammonia water. Place the mixture in an oil bath and heat for 4 hours. After cooling to room temperature, wash the black product with deionized water and dry it.

[0040] (2) Preparation of CS-BSA suspension: Weigh 50 mg of CS and place it in a 10-ml volumetric flask. Add 2% dilute acetic acid solution to dissolve and dilute to the mark. Weigh 50 mg of BSA and place it in a 10-ml volumetric flask. Add deionized water to dissolve and dilute to the mark. Mix CS and BSA in a mass concentration ratio of 1:1 to obtain the CS-BSA suspension.

[0041] (3) Preparation of 3D-rGO-CS-BSA composite suspension: Weigh 5 mg of the 3D-rGO powder prepared in step (1) and disperse it in a small amount of deionized water. After adding 6 mg of EDC·HCl and 5 mg of NHS to activate the carboxyl groups, add it to 5 mL of the CS-BSA solution prepared in step (2), and ultrasonicate to obtain the 3D-rGO-CS-BSA suspension.

[0042] (4) Preparation of 3D-rGO-CS-BSA modified electrode: Simultaneously prepare 5 groups of modified electrodes. Take 8 μL of the suspension prepared in step (3) and drop-coat it on the electrode surface, and dry it under an infrared lamp to obtain the 3D-rGO-CS-BSA modified electrode. Store it in the refrigerator for 3, 6, 9, 12, and 15 days for later use.

[0043] (5) Preparation of 1-Boc-3-hydroxypyrrolidine enantiomer electrode: Weigh 234 mg of R- / S-1-Boc-3-hydroxypyrrolidine on the 3rd, 6th, 9th, 12th, and 15th days respectively and place it in a 50-ml volumetric flask. Add a certain amount of prepared phosphate buffer solution (PBS, pH = 7), shake well. Immerse the 3D-rGO-CS-BSA modified electrode prepared in step (4) in the enantiomer solution for 20 min, take it out, and dry it under an infrared lamp. The 1-Boc-3-hydroxypyrrolidine enantiomer electrode can be obtained.

[0044] (6) Electrochemical recognition of 1-Boc-3-hydroxypyrrolidine enantiomers: On the 3rd, 6th, 9th, 12th, and 15th days respectively, use a three-electrode system. The 1-Boc-3-hydroxypyrrolidine enantiomer electrode is used as the working electrode, the platinum sheet electrode is used as the counter electrode, and the saturated calomel electrode is used as the reference electrode. Immerse this three-electrode system in the prepared 0.1 M K3Fe(CN)6 solution at room temperature for differential pulse testing. The potential range is -0.2 to 0.6 V. The stability can be evaluated by the change in peak current. Compare the ratio of the peak current values I R / I S measured on the 3rd, 6th, 9th, 12th, and 15th days with the initial value and make an error bar histogram as Figure 4 shown.

[0045] Example 3

[0046] (1) Preparation of 3D-rGO powder: Weigh 100 mg of GO and disperse it in 50 mL of deionized water by ultrasonic treatment; add 200 mg of L-cysteine to the above solution, and then quickly add 3 mL of 25% ammonia water. Heat the mixture in an oil bath for 4 hours. After cooling to room temperature, wash the black product with deionized water and dry it.

[0047] (2) Preparation of CS-BSA suspension: Weigh 100 mg of CS and place it in a 20 mL volumetric flask. Add 2% dilute acetic acid solution to dissolve and dilute to the mark; weigh 100 mg of BSA and place it in a 20 mL volumetric flask. Add deionized water to dissolve and dilute to the mark. Mix according to the mass concentration ratio of CS:BSA of 3:1, 2:1, 1:1, 1:2, 1:3 to prepare suspensions with respective concentration ratios.

[0048] (3) Preparation of 3D-rGO-CS-BSA composite suspension: Weigh 5 mg of the 3D-rGO powder prepared in step (1) and disperse it in a small amount of deionized water. After adding 6 mg of EDC·HCl and 5 mg of NHS to activate the carboxyl groups, add it to 5 mL of the CS-BSA solution prepared in step (2), and perform ultrasonic treatment to obtain the 3D-rGO-CS-BSA suspension.

[0049] (4) Preparation of 3D-rGO-CS-BSA modified electrode: Take 8 μL of the suspension prepared in step (3) and drop-coat it on the electrode surface, and dry it under an infrared lamp to obtain the 3D-rGO-CS-BSA modified electrode.

[0050] (5) Preparation of 1-Boc-3-hydroxypyrrolidine enantiomeric electrode: Weigh 234 mg of R- / S-1-Boc-3-hydroxypyrrolidine and place it in a 50 mL volumetric flask. Add a certain amount of prepared phosphate buffer solution (PBS, pH = 7), shake well. Immerse the 3D-rGO-CS-BSA modified electrode prepared in step (3) in the enantiomeric solution for 20 minutes, take it out, and dry it under an infrared lamp. Thus, the 1-Boc-3-hydroxypyrrolidine enantiomeric electrode is obtained.

[0051] (6) Electrochemical characterization of the loading of chiral surface materials: Adopt a three-electrode system, with the 1-Boc-3-hydroxypyrrolidine enantiomeric electrode as the working electrode, a platinum sheet electrode as the counter electrode, and a saturated calomel electrode as the reference electrode. Immerse this three-electrode system in the prepared 0.1 M K3Fe(CN)6 solution at room temperature and perform differential pulse testing. The potential range is -0.2 to 0.6 V. Plot a graph with the ratio of peak current values I R / I S as shown in Figure 5 as follows.

[0052] To obtain the optimal ratio of the chiral surface, the mass concentration ratio of the chiral surface material 3D-rGO was optimized. Mass concentrations of 3D-rGO: 0.5 mg / ml, 1.0 mg / ml, 1.5 mg / ml, and 2.0 mg / ml were prepared respectively. The solvent was dissolved and diluted using the optimal mass concentration ratio of BSA:CS of 1:1. After loading it onto the electrode surface and reacting with the enantiomers respectively, DPV tests were carried out, and a graph was plotted with the peak current value I R / I S ratio. The optimal mass concentration of 3D-rGO was found to be 1.0 mg / ml, as shown in Figure 6 .

Claims

1. A 3D-rGO-CS-BSA composite modified electrode for chiral recognition of 1-Boc-3-hydroxypyrrolidine enantiomers, characterized in that: It includes an electrode and a 3D-rGO-CS-BSA composite modified on the electrode surface, which is obtained by drop-coating a 3D-rGO-CS-BSA suspension on the electrode surface and then drying it. The preparation method of the 3D-rGO-CS-BSA suspension includes the following steps: (1) Preparation of 3D-rGO powder: Weigh an appropriate amount of GO and disperse it in deionized water, then ultrasonicate it; then add L-cysteine, and then quickly add ammonia water dropwise. Place the mixture in an oil bath and heat it. After cooling to room temperature, wash the black product with deionized water and dry it to obtain 3D-rGO powder. (2) Preparation of 3D-rGO-CS-BSA composite: Weigh an appropriate amount of 3D-rGO powder to prepare a dispersion. After activating the carboxylic acid groups in 3D-rGO with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, disperse it in a mixed solution of BSA and CS, and ultrasonicate it to obtain a 3D-rGO-CS-BSA suspension. In the 3D-rGO-CS-BSA suspension described in step (2), the mass ratio of CS to BSA is 1:

1. In the 3D-rGO-CS-BSA suspension described in step (2), the content of 3D-rGO powder in the CS-BSA solution is 1 mg / ml.

2. The preparation method of the 3D-rGO-CS-BSA composite modified electrode according to claim 1, characterized in that: It includes the following steps: (1) Preparation of 3D-rGO powder: Weigh an appropriate amount of GO and disperse it in deionized water, then ultrasonicate it; then add L-cysteine, and then quickly add ammonia water dropwise. Place the mixture in an oil bath and heat it. After cooling to room temperature, wash the black product with deionized water and dry it to obtain 3D-rGO powder. (2) Preparation of 3D-rGO-CS-BSA composite: Weigh an appropriate amount of 3D-rGO powder to prepare a dispersion. After activating the carboxylic acid groups in 3D-rGO with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, disperse it in a mixed solution of BSA and CS, and ultrasonicate it to obtain a 3D-rGO-CS-BSA suspension. (3) Preparation of 3D-rGO-CS-BSA modified electrode: Take the 3D-rGO-CS-BSA suspension and drop-coat it on the electrode surface, and dry it under an infrared lamp to obtain a 3D-rGO-CS-BSA composite modified electrode.

3. The preparation method of the 3D-rGO-CS-BSA composite modified electrode according to claim 2, characterized in that: In the 3D-rGO, L-cysteine and ammonia water described in step (1), the mass ratio is 1:2:7.

5.

4. The preparation method of the 3D-rGO-CS-BSA composite modified electrode according to claim 2, characterized in that: The preparation method of the mixed solution of BSA and CS includes: Dissolve CS with a 2% dilute acetic acid solution to prepare a CS solution; dissolve BSA with deionized water to prepare a BSA solution, and then uniformly mix the CS solution and the BSA solution.

5. The preparation method of the 3D-rGO-CS-BSA composite modified electrode according to claim 2, wherein: The drop-coating amount of 3D-rGO-CS-BSA on the electrode surface is 0.0849 mg / cm 2 .

6. Application of the 3D-rGO-CS-BSA composite modified electrode according to claim 1, characterized in that: For chiral recognition of 1-Boc-3-hydroxypyrrolidine enantiomers, it includes the following steps: Weigh a certain amount of R- / S-1-Boc-3-hydroxypyrrolidine and place it in a volumetric flask, add a certain amount of prepared phosphate buffer PBS, shake well to obtain an enantiomer solution. Immerse the 3D-rGO-CS-BSA composite modified electrode in the enantiomer solution, take it out, and dry it under an infrared lamp to obtain a 1-Boc-3-hydroxypyrrolidine enantiomer electrode. A three-electrode system was adopted, with the 1-Boc-3-hydroxypyrrolidine enantiomer electrode as the working electrode, a platinum sheet electrode as the counter electrode, and a saturated calomel electrode as the reference electrode. At room temperature, the three-electrode system was immersed in a prepared 0.1 M K3Fe(CN)6 solution for differential pulse testing, with the potential range being -0.2 to 0.6 V.

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