Preparation method of an electrochemical sensor based on a dual molecularly imprinted membrane for chloramphenicol

Abstract

The present invention relates to a preparation method of a chloramphenicol double molecular imprinted membrane electrochemical sensor. The present invention uses chloramphenicol as a template molecule and N-(4-pentenoyl) isoleucyl-chitosan oligosaccharide as a functional monomer oligomer. Under the action of a cross-linking agent and an initiator, a chloramphenicol molecular imprinted membrane is first formed by thermal polymerization on the surface of a glassy carbon electrode. Then, electrochemical polymerization is carried out in a pyrrole solution containing chloramphenicol to form a composite double molecular imprinted membrane. When the sensor prepared by the present invention is used for chloramphenicol concentration determination, specificity, sensitivity and stability are all excellent. The electroactive probe during detection can be potassium ferrocyanide, ferrocenylmethanol, etc.; the prepared sensor is a double molecular imprinted performance, contrasting the molecular imprinted membrane of a monolayer, significantly improving the recognition performance of the composite molecular imprinted membrane to chloramphenicol, and the detection limit reaches nM level.

Description

Technical Field

[0001] The invention relates to the field of electrochemical sensors, in particular to a method for preparing a chloramphenicol double molecular imprinted membrane electrochemical sensor. Background Art

[0002] Chloramphenicol was once a potent drug for treating bacterial infections. In clinical practice, it was only used for influenza and streptococcal infections, and had a good therapeutic effect on patients allergic to penicillin. It was also effective in treating various infections caused by typhoid and paratyphoid fever. However, subsequent studies have revealed significant side effects on the human hematopoietic system, such as agranulocytosis, myelopoiesis, and aplastic anemia. These effects are particularly severe in the elderly and newborns.

[0003] Currently, the main method for detecting chloramphenicol is high-performance liquid chromatography (HPLC) combined with ultraviolet (UV) detection. However, because chloramphenicol analogs share the same chromophore, these methods often lack specificity. In some studies, they cannot accurately determine the stability of their metabolites or the stability of the substance itself. Therefore, it is necessary to develop detection methods with high specificity and sensitivity.

[0004] Molecular imprinting technology (MIP) has been successfully used in electrochemical analysis methods for chloramphenicol due to its simple preparation, high specificity, reusability, and low cost. Molecular imprinting technology involves forming a host-guest complex with a template molecule and a specific functional monomer through intermolecular interactions. This complex is then polymerized into a high molecular weight polymer by adding a specific amount of a crosslinker and the functional monomer. After the template molecule is removed, the cavities in the rigid polymer retain the configuration of the template molecule, and the precise arrangement of the functional groups within the cavities complements that of the template molecule, resulting in a high recognition capability for the specific template molecule. However, these MIPs are typically prepared using a one-shot polymerization method using small-molecule functional monomers. Although these methods are simple and convenient, the sensitivity of imprinting is limited by the rigidity of the imprinted polymer chain or the thickness of the membrane. That is, the thickness of the molecularly imprinted polymer membrane prepared by bulk polymerization cannot be controlled. The imprinted membrane formed by direct electrochemical polymerization (such as polypyrrole, etc.) has a low degree of match between the position space of the template molecule during imprinting and the configuration of the template molecule itself due to the rigidity of the polymer chain and the small number of polar groups it carries. Therefore, a larger thickness is required to achieve the imprinting effect.

[0005] In previous research, the applicant proposed a method for preparing an electrochemical molecular imprinting sensor based on a chitosan oligosaccharide derivative as a functional monomer, that is, a primary molecular imprinting membrane is prepared with a chitosan oligosaccharide derivative as the functional monomer, and then electrochemically induced secondary polymerization is deposited in the gaps of the primary molecular imprinting membrane, so that the composite imprinting membrane obtains a very high sensitivity. This method is mainly applicable to some water-insoluble template molecules. Obviously, although the electrochemically induced secondary polymer improves the imprinting effect of the primary imprinting membrane, it has no imprinting effect itself. Therefore, if a polymer with an imprinting effect is used in the secondary polymerization, a composite double molecular imprinting membrane can be formed, which can greatly improve the sensitivity of the electrochemical sensor. Here, in order for the secondary polymer to have a certain imprinting effect, the template molecule must have a certain solubility (i.e., slightly soluble). Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a double molecular imprinting membrane sensor and a preparation method of an electrochemical molecular imprinting sensor with high specificity, sensitivity and stability for chloramphenicol detection.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for preparing a chloramphenicol double molecular imprinted membrane electrochemical sensor comprises the following steps:

[0009] 1) Chloramphenicol and N-(4-pentenoyl)isoleucyl-chitosan oligosaccharide (PICO) are added to a mixed solvent consisting of DMF and H2O in a volume ratio of 1 / 2 to 2 / 1, and dissolved by ultrasonication at room temperature. A crosslinker and an initiator are then added, and the mixture is allowed to stand for 5 to 24 hours, followed by removal of dissolved oxygen with nitrogen to obtain a mixed solution;

[0010] 2) Drop the mixed solution onto the surface of a clean glassy carbon electrode, then cover it with a clean cover glass, and heat it in an oven at 55-75°C for 5-20 hours. After removing the cover glass, a transparent primary polymer film is formed on the surface of the glassy carbon electrode.

[0011] 3) The reaction was carried out in 2 mL of a 0.1-0.5 M electrolyte solution containing 5-30 mM pyrrole and a saturated amount of chloramphenicol, using a three-electrode system with a primary polymer membrane electrode as the working electrode, a platinum wire electrode as the control electrode, and a saturated calomel electrode as the reference electrode. The potential range was 0-1.2 V, and the scan rate was 5-20 mVs -1 , Cyclic voltammetry was performed for 4 to 10 cycles to obtain a glassy carbon electrode modified with a secondary polymerized chloramphenicol molecular imprinted film;

[0012] 4) The glassy carbon electrode modified with the secondary polymerized chloramphenicol molecular imprinted membrane is eluted with a methanol solution containing 10-70% acetic acid by mass to elute the imprinted chloramphenicol molecules in the molecular imprinted membrane, thereby obtaining a composite chloramphenicol double molecular imprinted membrane electrochemical sensor.

[0013] Furthermore, in the step 1), the mass ratio of chloramphenicol, N-(4-pentenoyl)isoleucyl-chitosan oligosaccharide, cross-linking agent and initiator is 3-5:5-10:20-50:1-5.

[0014] Furthermore, the cross-linking agent is ethylene glycol dimethacrylate, and the initiator is azobisisobutyronitrile.

[0015] Furthermore, the step 3) is carried out in an acetate buffer solution or a phosphate buffer solution, and the pH value of the solution is 5.0 to 7.5.

[0016] The working principle of the present invention is to first coat the surface of a clean glassy carbon electrode with a primary molecularly imprinted membrane using chloramphenicol as the template molecule, N-(4-pentenoylisoleucyl)-chitosan oligosaccharide as the functional monomer oligomer, and ethylene glycol dimethacrylate as the crosslinking agent through thermal polymerization. During the thermal polymerization process, because water in the mixed solvent has a low boiling point and a fast volatilization rate, while the functional monomer oligomer has a low solubility in organic solvents and a rigid structure, polymerization occurs while the crosslinking reaction occurs and the oligomer precipitates, forming a surface structure with a very large specific surface area on the electrode surface. The amino-derived ends of the chitosan oligosaccharide are distributed on both sides of the chitosan oligosaccharide with a large spacing, resulting in a membrane structure with good permeability. However, when used in electrochemical measurements, this molecularly imprinted membrane generates a large background current, which weakens the difference in DPV signals caused by membrane elution and adsorption of the template molecule. Therefore, the gaps in the primary imprinted membrane must be sealed to reduce the excessive background current. Since chloramphenicol contains a nitro group, it will be reduced under negative voltage, but it can withstand a certain positive voltage. Therefore, it is considered to use electrochemical polymerization of pyrrole as a blocker under positive voltage. Because chloramphenicol is slightly soluble in water, the pyrrole solution will dissolve away the chloramphenicol in a portion of the primary imprinted membrane, resulting in a reduction in imprinted sites. Therefore, a saturated amount of chloramphenicol must be added to the pyrrole solution to minimize the impact of the chloramphenicol in the primary imprinted membrane. The polypyrrole obtained by secondary polymerization contains a certain amount of chloramphenicol, which can also produce corresponding imprinted sites after elution, thus forming a double imprinting effect. The imprinted sites of the entire composite imprinted membrane are significantly more than the gaps in the simply enclosed primary imprinted membrane.

[0017] The polypyrrole formed by the secondary polymerization further matches the primary imprinted membrane to the template molecule and its spatial location. After removing the template molecule, the specificity and sensitivity of the molecular imprinted membrane for chloramphenicol molecules are greatly improved. Differential pulse voltammetry is a highly sensitive detection method in electrochemical analysis, and thus a highly sensitive molecular imprinted sensor for chloramphenicol can be obtained.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. For the first time, a functional monomer oligomer, N-(4-pentenoyl)isoleucyl-chitosan oligosaccharide, capable of forming multi-site interactions with chloramphenicol, was used in a chloramphenicol molecularly imprinted sensor, significantly improving the specificity of the molecularly imprinted membrane for chloramphenicol.

[0020] 2. The secondary polymerization solution uses a saturated chloramphenicol pyrrole solution, which can effectively reduce the impact of the secondary polymerization solution on the chloramphenicol in the primary blot membrane;

[0021] 3. The secondary polymerization liquid is electrochemically polymerized under a positive voltage. The advantage of electrochemical polymerization is that the thickness of polypyrrole can be well controlled, thereby controlling the background current of the membrane electrode.

[0022] 4. The prepared sensor achieved the double molecular imprinting effect in the molecular imprinting electrochemical sensor for the first time. Compared with the single-layer molecular imprinting membrane, the recognition performance of the double molecular imprinting membrane for chloramphenicol was significantly improved, and the detection limit reached the nM level. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The flowchart of the molecular imprinting sensor preparation process of the present invention is shown in FIG.

[0024] Figure 2 This is a computer simulation diagram of the molecular structure coordination of the functional monomer PILEGA and CAP.

[0025] Figure 3 UV test spectra of CAP and PICO solutions with different ratios.

[0026] Figure 4 This is a scanning electron micrograph of the chloramphenicol double molecular imprinted membrane electrochemical sensor.

[0027] Figure 5 Cyclic voltammograms of different modified electrodes.

[0028] Figure 6 This is the DPV response curve of the chloramphenicol double molecular imprinted membrane electrochemical sensor to different CAP concentrations.

[0029] Figure 7 The calibration curve of the current response of the chloramphenicol double molecularly imprinted membrane electrochemical sensor versus chloramphenicol concentration is shown.

[0030] Figure 8 This is the differential pulse voltammetry peak current (difference) response diagram of the molecularly imprinted polymer film modified glassy carbon electrode and the non-molecularly imprinted membrane modified electrode to 2.0 μM chloramphenicol and its analogues. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0033] Example 1:

[0034] 1) 3 mg of chloramphenicol (CAP) and 6 mg of N-(4-pentenoyl)isoleucyl-chitosan oligosaccharide (PICO) were added to 5 ml of a mixed solvent consisting of equal volumes of DMF and HO and dissolved by sonication at room temperature. 50 mg of ethylene glycol dimethacrylate (EGDMA) and 1.5 mg of azobisisobutyronitrile (AIBN) were then added. After standing for 12 h, dissolved oxygen was removed with nitrogen to obtain a mixed solution.

[0035] 2) Drop 5 μl of the mixed solution onto the surface of a clean glassy carbon electrode, then cover it with a clean cover glass and heat it in an oven at 55-75°C for 12 hours. After removing the cover glass, a transparent primary polymer film is formed on the surface of the glassy carbon electrode.

[0036] Example 2:

[0037] The reaction was carried out in 2 mL of a 0.1-0.5 M electrolyte solution containing 5-30 mM pyrrole (PR) and a saturated amount of chloramphenicol. A three-electrode system was used, with a primary polymer membrane electrode as the working electrode, a platinum wire electrode as the control electrode, and a saturated calomel electrode as the reference electrode. The potential range was 0-1.2 V, and the scan rate was 5-20 mVs. -1 , cyclic voltammetry was scanned for 5 cycles to obtain a glassy carbon electrode modified with a secondary polymerized chloramphenicol molecular imprinted membrane; then a methanol solution containing 30% acetic acid was used as an eluent to elute the imprinted molecule chloramphenicol in the molecular imprinted membrane to obtain a composite chloramphenicol double molecular imprinted membrane electrochemical sensor.

[0038] The preparation process of Examples 1 and 2 can be as follows Figure 1As shown in the figure, a represents a glassy carbon electrode (sensor) modified with a chloramphenicol molecular imprinting membrane after secondary polymerization, and b represents the chloramphenicol double molecular imprinting membrane electrochemical sensor finally prepared after removing the template molecule.

[0039] Using computer simulations of the molecular structure of CAP and the PICO monomer (i.e., N-(4-pentenoylisoleucyl)-glucosamine, abbreviated as PILEGA) under vacuum conditions, the terminal hydroxyl oxygen of CAP formed a hydrogen bond with the hydroxyl group on the third carbon of the pyranose ring of PILEGA. The hydrogen and oxygen on the amide group of CAP also formed hydrogen bonds with the oxygen and hydrogen on the two amide groups of PILEGA, respectively.

[0040] Example 3: Performance Test

[0041] 1. UV characterization

[0042] like Figure 3 As shown, chloramphenicol (CAP) exhibits a strong UV absorption peak at 281 nm. When the ratio of PICO to chloramphenicol (based on monomeric PILEGA) reaches 1:0.45, the 281 nm absorption peak increases compared to CAP. As the amount of PICO increases to a ratio of 1:0.79, the absorption peak decreases. This indicates that within the 1:0.45 to 1:0.79 range, CAP and PICO can form a strong complex, confirming the existence of a strong molecular interaction between CAP and PICO monomers.

[0043] 2. Scanning electron microscopy characterization:

[0044] The scanning electron microscopy characterization of chloramphenicol double molecular imprinted membrane electrochemical sensor is shown in Figure 4 ,from Figure 4 It can be seen that the electrode surface of the modified imprinted membrane (after eluting the template molecules) has many relatively small surface depressions, which significantly increases the surface area of the membrane and is beneficial to the adsorption of the template molecule chloramphenicol by the molecularly imprinted membrane.

[0045] 3. Cyclic voltammetry test of different modified molecularly imprinted electrodes:

[0046] The bare glassy carbon electrode (a), the glassy carbon electrode modified with the primary polymer film prepared in Example 1 (b), the glassy carbon electrode modified with the secondary polymerized chloramphenicol molecular imprinting membrane prepared in Example 2 (c), the double molecular imprinting membrane electrochemical sensor after elution (d), and the molecular imprinting electrode after re-adsorption of chloramphenicol (e) were used as working electrodes, a saturated calomel electrode was used as a reference electrode, and a platinum electrode was used as an auxiliary electrode; the electrolyte was 10 ml of 0.25 M acetate buffer (pH = 6.5) containing 1.0 mmol / L ferrocene methanol; and the scanning potential range was -0.1 to 0.5 V.

[0047] The molecularly imprinted electrode (e) after re-adsorbing chloramphenicol is obtained by immersing the molecularly imprinted electrode after eluting chloramphenicol in a 0.2M chloramphenicol ethanol solution to adsorb chloramphenicol until saturation.

[0048] The test results are as follows Figure 5 As shown in the figure, it can be seen that curve a, that is, the bare glassy carbon electrode, has the largest peak current; the peak current of curve b has decreased significantly, but is still large, which indicates that there are more gaps in the film on the electrode surface for conductivity; after electrochemical polymerization of pyrrole, it can be seen that the peak current of curve (c) becomes very small, and the peak shape is almost invisible. When the molecular imprinting membrane modified electrode removes the template molecule (chloramphenicol), the peak current of the voltammetric curve increases significantly (curve d). When the sensor adsorbs the template molecule (chloramphenicol) again, the peak current of the voltammetric curve decreases again (curve e).

[0049] 4. Calibration curve of chloramphenicol double molecular imprinted membrane electrochemical sensor

[0050] The chloramphenicol double molecular imprinted membrane electrochemical sensor was used as the working electrode, the reference electrode was a saturated calomel electrode, and the auxiliary electrode was a platinum electrode; the electrolyte was 1.0 mmol / L ferrocene methanol in 0.25 M acetate buffer (pH = 6.5); the scanning potential range was -0.5 to 0.4 V; the sensor was placed in the electrolyte and differential pulse voltammetry (DPV) scanning was performed. The results are shown in Figure 2. Figure 6 .Depend on Figure 6 It can be seen that the sensor has different DPV responses to chloramphenicol at different concentrations (a→i corresponds to 0, 0.04, 0.08, 0.12, 0.16, 1.0, 2.0, 3.0, and 4.0 μM, respectively).

[0051] Peak current I of blank electrolyte scanning DPV p0 Then, the molecular imprinted sensor was placed in a certain concentration of chloramphenicol solution for incubation, and then the peak current I was obtained by scanning. p , then the response current of the sensor is ΔI p =I p0 -I p , within the measurement range ΔI p The value is linearly related to the concentration response of chloramphenicol. Figure 7 There are two linear intervals on the calibration curve, corresponding to the fast adsorption area and the slow adsorption area. The linear range of chloramphenicol determination is 4.0×10 -8 ~4.0×10 -6 mol / L. The sensor was placed in a 4°C environment and retained more than 90% of its response current value after two weeks.

[0052] 5. Performance comparison of chloramphenicol double molecular imprinted membrane electrochemical sensor and non-molecular imprinted membrane modified electrode as electrochemical sensor

[0053] Depend on Figure 8 It can be seen that the DPV peak current decrease value of chloramphenicol by the chloramphenicol electrochemical molecular imprinting sensor (MIM in the figure) for chloramphenicol is much higher than the peak current decrease values of other analogues (curcumin, ribavirin, bisphenol A, Sudan red). Therefore, the chloramphenicol molecular imprinting sensor prepared by this method has a high selectivity for chloramphenicol.

[0054] The differential pulse voltammetry peak current response of the non-molecularly imprinted modified membrane electrode (NIM in the figure) to chloramphenicol and its analogues is much smaller than that of the molecularly imprinted membrane modified electrode, but it is obvious that the non-molecularly imprinted membrane has a larger response to chloramphenicol, indicating that the functional monomer oligomer has a higher binding ability to chloramphenicol molecules.

[0055] In summary, the double molecular imprinting membrane electrochemical sensor constructed in this case has a specific adsorption capacity for chloramphenicol, which far exceeds the nonspecific adsorption of chloramphenicol by non-molecular imprinting. At the same time, the molecular imprinting sensor has obvious selectivity for interfering substances.

[0056] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for preparing a chloramphenicol double molecular imprinted membrane electrochemical sensor, characterized in that: The steps include: 1) Chloramphenicol and N-(4-pentenoyl)isoleucyl-chitosan oligosaccharide were added to a mixed solvent consisting of DMF and H2O in a volume ratio of 1 / 2 to 2 / 1, and dissolved by ultrasonication at room temperature. Then, a crosslinker and initiator were added. After standing for 5 to 24 hours, the dissolved oxygen was removed with nitrogen to obtain a mixed solution. 2) Drop the mixed solution onto the surface of a clean glassy carbon electrode, then cover it with a clean cover glass and heat it in a 55-75°C oven for 5-20 hours. After removing the cover glass, a transparent primary polymer film is formed on the surface of the glassy carbon electrode. 3) The secondary polymerization was carried out in 2 mL of a 0.1–0.5 M electrolyte solution containing 5–30 mM pyrrole and a saturated amount of chloramphenicol. A three-electrode system was used, with the primary polymer film electrode as the working electrode, a platinum wire electrode as the control electrode, and a saturated calomel electrode as the reference electrode. The potential range was 0–1.2 V, and the scan rate was 5–20 mVs. -1 , Cyclic voltammetry was scanned for 4 to 10 cycles to obtain a glassy carbon electrode modified with secondary polymerized chloramphenicol molecular imprinted film; 4) Using a methanol solution containing 10-70% acetic acid as an eluent to elute the imprinted chloramphenicol molecules from the glassy carbon electrode modified with the secondary polymerized chloramphenicol molecular imprinted membrane, thereby obtaining a composite chloramphenicol double molecular imprinted membrane electrochemical sensor; The crosslinking agent is ethylene glycol dimethacrylate, and the initiator is azobisisobutyronitrile.

2. The method for preparing the chloramphenicol double molecularly imprinted membrane electrochemical sensor according to claim 1, wherein: In the step 1), the mass ratio of chloramphenicol, N-(4-pentenoyl)isoleucyl-chitosan oligosaccharide, crosslinking agent and initiator is 3-5:5-10:20-50:1-5.

3. The method for preparing the chloramphenicol double molecularly imprinted membrane electrochemical sensor according to claim 1, wherein: The step 3) is carried out in an acetate buffer or a phosphate buffer, and the pH value of the solution is 5.0-7.5.

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