Preparation method of ciprofloxacin molecularly imprinted electrochemical sensor

Abstract

The invention belongs to the technical field of sensor preparation, and particularly relates to a preparation method of a ciprofloxacin molecularly imprinted electrochemical sensor. A mixed solution containing template molecule ciprofloxacin and a functional monomer L-cysteine is used as an electroplating solution, a gold electrode is electroplated by using cyclic voltammetry, then eluent elution and drying are performed to obtain the ciprofloxacin molecularly imprinted electrochemical sensor, the ciprofloxacin molecularly imprinted electrochemical sensor is used for ciprofloxacin detection, and under the condition that the CPX concentration range is 5-90 mmol / L, the detection sensitivity of the ciprofloxacin molecularly imprinted electrochemical sensor is greatly improved. The peak current and CPX concentration of the molecularly imprinted electrode are in a good linear relationship, and the molecularly imprinted electrode has excellent selectivity and stability, high sensitivity, simple manufacturing method, wide application market and excellent application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of sensor preparation, and more specifically relates to a preparation method of a ciprofloxacin molecularly imprinted electrochemical sensor. Background Art

[0002] Ciprofloxacin (CPX) is a third-generation quinolone antibacterial drug that inhibits bacterial DNA gyrase, thereby hindering bacterial replication. Therefore, it can kill bacteria inside cells, destroy the cell membrane of bacteria, and cause the contents inside the cells to disperse, leading to the death of bacteria. Due to the role of ciprofloxacin in inhibiting bacterial DNA gyrase, ciprofloxacin has the effect of inhibiting bacterial replication; ciprofloxacin has strong antibacterial properties and has strong antibacterial activity against almost all bacteria, and has good antibacterial effects on Pseudomonas aeruginosa, Neisseria gonorrhoeae, Haemophilus influenzae, Enterobacter, Legionella, Streptococcus, Staphylococcus aureus, etc. It is 2-4 times more antibacterial than norfloxacin and enoxacin, and has strong antibacterial effects on both the antibacterial ability and antibacterial power of bacteria. If used improperly or in excess, the residual CPX in livestock and poultry meat will spread to the human body through the food chain, resulting in harm to the human body such as cancer, birth defects, and impaired organ function, seriously threatening human health.

[0003] At present, there are various methods for detecting CPX, including high-performance liquid chromatography, microbiological method, spectrophotometry, capillary electrophoresis, etc. However, these detection methods usually require expensive instrument equipment and professional personnel, and are time-consuming and laborious. Therefore, it is very important to establish a simple and effective method to measure the content of CPX. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method of a ciprofloxacin molecularly imprinted electrochemical sensor. By using ciprofloxacin and L-cysteine as electroplating materials (where ciprofloxacin is the template molecule and L-cysteine is the functional monomer), a ciprofloxacin molecularly imprinted electrochemical sensor is prepared by cyclic voltammetry (CV), and ciprofloxacin drugs are detected by square wave voltammetry (SWV) to solve the problems existing in the above-mentioned prior art and achieve simple and effective measurement of CPX.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention: Provide a preparation method of a ciprofloxacin molecularly imprinted electrochemical sensor, and the steps include:

[0007] Using a mixed solution containing the template molecule ciprofloxacin and the functional monomer L-cysteine as the electroplating solution, electroplating the gold electrode by cyclic voltammetry, and then eluting with an eluent and drying to obtain the ciprofloxacin molecularly imprinted electrochemical sensor.

[0008] Further, the preparation steps of the electroplating solution include: dissolving the L-cysteine in PBS solution, and then adding ciprofloxacin to obtain a mixed solution; adjusting the pH value of the mixed solution to 2-6 with hydrochloric acid solution under stirring to obtain the electroplating solution.

[0009] Optionally, the concentration of ciprofloxacin in the mixed solution is 4.44 g / L.

[0010] Optionally, the concentration of L-cysteine in the mixed solution is 1.2 g / L.

[0011] Further, the scanning range of the cyclic voltammetry is -0.1 V to 1.2 V, the scanning speed is 50 mV / s, and the number of scanning cycles is 20.

[0012] Further, the oxidation potential of the gold electrode is not higher than 100 mV.

[0013] Further, the eluent is obtained by mixing ethanol and sodium hydroxide solution in a volume ratio of 3:1.

[0014] Optionally, the concentration of the sodium hydroxide solution is 1 mol / L.

[0015] Further, the elution time is 0.5-3 h.

[0016] The second technical solution of the present invention: provides a ciprofloxacin molecularly imprinted electrochemical sensor prepared by the above preparation method.

[0017] The third technical solution of the present invention: provides an application of the above ciprofloxacin molecularly imprinted electrochemical sensor in the detection of ciprofloxacin.

[0018] The present invention discloses the following technical effects:

[0019] In the present invention, L-cysteine is used as a functional monomer and CPX molecule as a template molecule, and molecularly imprinted polymer is formed by electro-polymerization through cyclic voltammetry (CV), and then CPX molecularly imprinted electrochemical sensor is obtained after elution with eluent. In the concentration range of 5-90 mmol / L of CPX, the peak current of the molecularly imprinted electrode has a good linear relationship with the CPX concentration, and its linear equation is: y = -0.1306x + 26.7471, and the linear coefficient R 2 = 0.9745. This sensor is simple to fabricate, sensitive in response, has excellent selectivity and stability, provides a method for the detection of CPX in actual samples, and has a wide application market and application prospect. Description of the Drawings

[0020] The accompanying drawings, which form a part of this application, are used to provide a further understanding of this application. The schematic embodiments and descriptions thereof of this application are used to explain this application and do not constitute an improper limitation of this application. In the drawings:

[0021] Figure 1 is the cyclic voltammogram in step S2 of Example 1;

[0022] Figure 2 are the cyclic voltammetry curves of the gold electrode (bare electrode) used in step S2 of Example 1 and the electrode electroplated with the CPX molecularly imprinted polymer film prepared in step S2 (electroplated electrode) in PBS solution;

[0023] Figure 3 are the SWV curves of the bare electrode, the imprinted electrode, and the non-imprinted electrode;

[0024] Figure 4 is the SWV curve diagram of the electrode electroplated with the CPX molecularly imprinted polymer film and its adsorption and elution;

[0025] Figure 5 is the influence of different elution times on the response current of the ciprofloxacin molecularly imprinted electrochemical sensor;

[0026] Figure 6 is the influence of different pH values of the electroplating solution on the response current of the ciprofloxacin molecularly imprinted electrochemical sensor;

[0027] Figure 7 In it, A is the SWV curve after adsorption in CPX solutions with different concentrations, and B is the relationship diagram between the peak current and the concentration of the CPX solution. Detailed Embodiments

[0028] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0030] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to those documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0031] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0032] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0033] The raw materials and reagents used in the specific embodiments of this invention are all commercially available products.

[0034] Electrode treatment: Polish the gold electrode with metallographic paper, then polish the surface of the gold electrode with Al3O4 powder. Subsequently, wash the Al3O4 powder on the surface with distilled water, and then wash it with an ultrasonic cleaner for 5 minutes in the order of distilled water - absolute ethanol - distilled water. Then connect it to an electrochemical workstation for electrochemical cleaning. Immerse the gold electrode in a 0.5 mol / L sulfate solution, and clean the electrode with high potential, low potential, and cyclic voltammetry in sequence. Finally, characterize it in a potassium ferricyanide solution, and it is okay if the potential width of the oxidation-reduction peak is not higher than 100 mV.

[0035] Preparation of potassium ferrocyanide characterization solution:

[0036] Weigh 0.2111 g of potassium ferrocyanide solid and 4.3565 g of potassium sulfate solid, add a small amount of distilled water and stir for 2 min to dissolve, and then make up the volume to 250 mL to obtain a K3Fe(CN)6 solution (concentration: 5.0 mmol / L).

[0037] Preparation of eluent:

[0038] Mix absolute ethanol and 1 mol / L NaOH solution according to a volume ratio of 3:1 to obtain it.

[0039] Preparation of detection solution:

[0040] It is obtained by mixing the above-mentioned K3Fe(CN)6 solution with a concentration of 5.0 mmol / L and KCl solution with a concentration of 0.1 mol / L in a volume ratio of 1:1.

[0041] Preparation of PBS solution with pH = 7:

[0042] Weigh 0.1146 g of solid Na2HPO4, 0.0321 g of solid NaH2PO4, and 0.4383 g of solid NaCl. After mixing, dissolve them with a little distilled water, and then make up the volume to 50 mL to obtain it.

[0043] Example 1

[0044] The preparation steps of the ciprofloxacin molecularly imprinted electrochemical sensor are as follows:

[0045] S1. Weigh 0.0060 g of L-cysteine and dissolve it with 5 mL of the above-mentioned PBS solution with pH = 7. Then, add 0.0222 g of ciprofloxacin under stirring conditions, and adjust the pH value to 3 with hydrochloric acid solution to obtain the electroplating solution.

[0046] S2. Connect the above-mentioned cleaned gold electrode (bare electrode) to the electrochemical workstation, immerse it in the electroplating solution, and electroplate the gold electrode using cyclic voltammetry to obtain an electrode electroplated with a CPX molecularly imprinted polymer film. Among them, the scanning range of cyclic voltammetry is -0.1 V to 1.2 V, the scanning speed is 50 mV / s, and the number of scanning cycles is 20.

[0047] S3. Wash the electrode electroplated with the CPX molecularly imprinted polymer film with distilled water, then soak it in the above-prepared eluent for 2.5 h. After the elution is completed, wash it with distilled water and dry it to obtain the ciprofloxacin molecularly imprinted electrochemical sensor.

[0048] Comparative Example 1

[0049] The preparation steps of the electrochemical sensor are as follows:

[0050] S1. Weigh 0.0060 g of L-cysteine and dissolve it with 5 mL of the above-mentioned PBS solution with pH = 7, and adjust the pH value to 3 with hydrochloric acid solution to obtain the electroplating solution.

[0051] S2. Connect the above-mentioned cleaned gold electrode (bare electrode) to the electrochemical workstation, immerse it in the electroplating solution, and electroplate the gold electrode using cyclic voltammetry to obtain an electroplated electrode. Among them, the scanning range of cyclic voltammetry is -0.1 V to 1.2 V, the scanning speed is 50 mV / s, and the number of scanning cycles is 20.

[0052] S3. Clean the electroplating electrode with distilled water, then soak it in the above-prepared eluent and elute for 2.5 h. After the elution is completed, wash it with distilled water and air-dry it to obtain an electrochemical sensor, denoted as a non-imprinted electrode.

[0053] Test Example

[0054] In Step S2 of Example 1, when the gold electrode was electroplated by cyclic voltammetry to prepare an electrode electroplated with a CPX molecularly imprinted polymer film, the cyclic voltammogram is as Figure 1 shown.

[0055] Figure 1 is the cyclic voltammogram in Step S2 of Example 1. It can be seen from the figure that a pair of redox peaks are clearly observed on the cyclic voltammogram. And as the scanning time becomes longer, the peak potential of the electrode will gradually decrease, and the current in the next cycle shows a decreasing trend compared with the current in the previous cycle, indicating that during the electropolymerization process, non-conductive substances are generated on the electrode surface, further proving that a non-conductive molecularly imprinted film is formed by CPX and L-cysteine on the electrode surface, indicating the success of the electroplating of CPX and L-cysteine.

[0056] Figure 2 are the cyclic voltammograms of the gold electrode (bare electrode) used in Step S2 of Example 1 and the electrode electroplated with a CPX molecularly imprinted polymer film (electroplated electrode) prepared in Step S2 in PBS solution. It can be seen from the figure that the redox peaks of the two are different, and the redox peaks of the two change at 0.5 V, which can prove that CPX and L-cysteine are successfully electroplated onto the gold electrode.

[0057] Square wave voltammetry (SWV) was used for determination, with potassium ferricyanide as an electron probe, to study the electrochemical properties of the bare electrode, the electrode electroplated with a CPX molecularly imprinted polymer film (imprinted electrode), and the electroplated electrode (non-imprinted electrode), as Figure 3 shown.

[0058] Figure 3 are the SWV curves of the bare electrode, the imprinted electrode, and the non-imprinted electrode. It can be seen from the figure that the results show that the peak current of the bare electrode is the largest, and the peak current of the imprinted electrode is the lowest. This is because a molecularly imprinted film is formed on the electrode surface, and the large amount of CPX molecules contained therein causes the current to decrease significantly compared with the bare electrode. For the non-imprinted electrode, since a small amount of L-cysteine adheres to the electrode surface and does not encapsulate the CPX target molecule, the peak current decreases slightly compared with the bare electrode. The change in the peak current indicates the successful preparation of the imprinted electrode in Example 1.

[0059] The performance of the molecularly imprinted electrode prepared in the experiment was tested by square wave voltammetry (SWV), and the change in current on the electrode was detected using potassium ferricyanide as an electron probe. The results are as Figure 4 shown.

[0060] Figure 4 It is the SWV curve graphs of the electrode electroplated with CPX molecularly imprinted polymer film before and after adsorption and elution. It can be seen from the figure that the peak current magnitude of the CPX molecularly imprinted polymer film electrode follows the order of after elution > after adsorption > after electroplating. The reason for the relatively small peak current after electroplating (11 μA) is that the formation of the molecularly imprinted film on the gold electrode surface and the encapsulation of the target molecule CPX will hinder the redox reaction of potassium ferricyanide on the electrode surface. After elution, a large amount of CPX on its surface is washed off, enhancing the conductivity of the electrode surface, enabling potassium ferricyanide to have space for redox reaction on the electrode surface, that is, the peak current rises to 28 μA. After adsorption, CPX will be re-adsorbed on the gold electrode surface, causing its peak current to drop to 16 μA. Thus, it can be seen that the prepared molecularly imprinted electrode has a very sensitive electrochemical response to CPX and can be used for the detection of CPX.

[0061] The results of the influence of elution time on the sensing performance of CPX detection during preparation are as Figure 5 shown.

[0062] Figure 5 It is the influence of different elution times on the response current of the ciprofloxacin molecularly imprinted electrochemical sensor. It can be seen from the figure that as the elution time prolongs, the value of the response current ΔI gradually increases. When the elution time is 2.5 h, the response current value is the largest, indicating that when the CPX molecularly imprinted polymer is eluted for 2.5 h, the most molecularly imprinted sites are formed. When the elution time is prolonged again, the response current ΔI will decrease slightly. This is because the molecularly imprinted sites are damaged due to too long elution time, resulting in the loss of the ability to recognize molecular imprinting.

[0063] Figure 6 It is the influence of different pH values of the electroplating solution on the response current of the ciprofloxacin molecularly imprinted electrochemical sensor. It can be seen from the figure that as the pH of the electroplating solution increases, the response current ΔI shows a trend of first rising and then falling. When pH = 3, the response current ΔI is the largest at this time. When the pH is too low, the base solution will provide too many H + ; as the pH increases, the polymerization base solution provides more and more OH−, which will hinder the formation of hydrogen bonds between the more electronegative O, N, F in CPX and the H atoms in the L-cysteine monomer, thus affecting the number of molecularly imprinted sites.

[0064] Prepare CPX solutions with concentrations of 5 mmol / L, 15 mmol / L, 30 mmol / L, 45 mmol / L, 55 mmol / L, 75 mmol / L, and 90 mmol / L. Immerse the ciprofloxacin molecularly imprinted electrochemical sensor prepared in Example 1 into each of the above seven different concentrations of CPX solutions for 10 min for adsorption. After adsorption, wash the sensor with distilled water. Then, connect the electrode to an electrochemical workstation and use square wave voltammetry (SWV) to detect it, and record the magnitude of its peak current. Analyze the relationship between the magnitude of the peak current and the change in CPX concentration.

[0065] Figure 7 In Figure, A is the SWV curve after adsorption in CPX solutions with different concentrations, and B is the relationship diagram between the peak current and the concentration of the CPX solution. Figure 7 It shows that the higher the concentration of CPX in the adsorption solution, the greater the decrease in the peak current on the imprinted electrode. This is because the imprinted electrode specifically recognizes CPX and can adsorb CPX. As the concentration of CPX increases, the amount of CPX adsorbed by it is also more, and the molecularly imprinted membrane becomes denser, resulting in a stronger blocking effect on the potassium ferricyanide probe, and thus the smaller the peak current on the molecularly imprinted electrode. Figure 7 B in Figure shows that in the concentration range of 5 mmol / L to 90 mmol / L, there is a linear relationship between the peak current on the molecularly imprinted electrode and the concentration of the CPX solution. The relevant linear equation is obtained as y = -0.1306x + 26.7471, and the linear coefficient R 2 = 0.9745.

[0066] Prepare solutions of commercially available ciprofloxacin drugs with concentrations of 20 mmol / L, 40 mmol / L, and 80 mmol / L, and use the ciprofloxacin molecularly imprinted electrochemical sensor prepared in Example 1 for detection. Calculate the content of CPX in the detection solution using the linear regression equation, and measure each concentration in parallel 3 times. The results are shown in Table 1.

[0067] Table 1

[0068]

[0069] As can be seen from the content of Table 1, the standard deviations are 1.29%, 1.04%, and 1.35% respectively, indicating that this method can be used for the determination of actual samples.

[0070] In this specification, each embodiment is described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same and similar parts among the embodiments, reference can be made to each other.

[0071] The foregoing description of the disclosed embodiments enables those skilled in the art to practice or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A preparation method of a ciprofloxacin molecularly imprinted electrochemical sensor, characterized in that the steps Including: Using a mixed solution containing the template molecule ciprofloxacin and the functional monomer L-cysteine as the electroplating solution, electroplating the gold electrode by cyclic voltammetry, and then eluting with an eluent and drying to obtain the ciprofloxacin molecularly imprinted electrochemical sensor.

2. The preparation method according to claim 1, wherein The preparation steps of the electroplating solution include: dissolving the L-cysteine in a PBS solution, adding ciprofloxacin to obtain a mixed solution; adjusting the pH value of the mixed solution to 2-6 with a hydrochloric acid solution under stirring to obtain the electroplating solution.

3. The preparation method according to claim 2, characterized in that, The concentration of ciprofloxacin in the mixed solution is 4.44 g / L; and / or The concentration of L-cysteine in the mixed solution is 1.2 g / L.

4. The preparation method according to claim 1, wherein The scanning range of the cyclic voltammetry is -0.1 V to 1.2 V, the scanning speed is 50 mV / s, and the number of scanning cycles is 20.

5. The preparation method according to claim 1, characterized in that, The oxidation potential of the gold electrode is not higher than 100 mV.

6. The preparation method according to claim 1, characterized in that, The eluent is obtained by mixing ethanol and a sodium hydroxide solution in a volume ratio of 3:

1.

7. The preparation method according to claim 6, characterized in that, The concentration of the sodium hydroxide solution is 1 mol / L.

8. The preparation method according to claim 1, characterized in that, The elution time is 0.5-3 h.

9. A ciprofloxacin molecularly imprinted electrochemical sensor prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the ciprofloxacin molecularly imprinted electrochemical sensor according to claim 9 in the detection of ciprofloxacin.

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