Anti-interference molecular imprinting sensor, preparation method thereof and application of anti-interference molecular imprinting sensor in pentachlorophenol detection

Abstract

The invention provides an anti-interference molecular imprinting sensor, a preparation method thereof and application of the anti-interference molecular imprinting sensor in pentachlorophenol detection. The preparation method comprises the following steps: providing an Au NPs / GCE electrode; dropwise coating hydroxyproline helical peptide PHHP on the surface of the Au NPs / GCE electrode, and incubating under the irradiation of an infrared lamp to obtain a polypeptide modified electrode interface; preparing an electrolyte containing a polymer monomer and a PCP molecular imprinting template, and performing electrochemical polymerization on the polypeptide modified electrode interface; and washing the surface of the electrode, eluting, and finally removing the PCP template molecules to obtain the anti-interference molecularly imprinted sensor. The Au NPs coating is beneficial to enhancing volt-ampere response, and the optimal arrangement of the MIP layer provides higher peak change. The prepared electrochemical sensor is used for detecting PCP in water, and has the advantages of high specificity, strong stability and good anti-interference capability.

Description

Technical Field

[0001] The invention relates to the technical field of electrochemical sensing, in particular to an anti-interference molecular imprinting sensor and a preparation method thereof and application thereof in detecting pentachlorophenol. Background Art

[0002] Pentachlorophenol (PCP) is a widely used organochlorine pesticide and wood preservative. Due to its persistence and bioaccumulation in water and soil, it poses a serious threat to human health and the ecological environment. The monitoring and control of PCP is an important part of environmental protection and ecological risk assessment. However, although traditional methods for detecting PCP in groundwater, such as liquid chromatography, have high sensitivity and accuracy, they usually have problems such as expensive equipment, complex operation, cumbersome pretreatment, and susceptibility to interference from other substances, which limits its wide application in rapid on-site detection and environmental monitoring. Molecular imprinting sensor is a sensor with specific recognition function prepared based on molecular imprinting technology. By preparing imprinting cavities that match the shape, size and functional groups of target molecules (such as pentachlorophenol), highly selective recognition of target molecules is achieved. It has the advantages of high selectivity, high affinity, good stability and simple preparation.

[0003] In recent years, molecular imprinting technology, as an emerging molecular recognition technology, has shown great application potential in chemical sensing, drug analysis, environmental monitoring and other fields. However, the preparation process of pentachlorophenol sensors based on molecular imprinting technology still faces some challenges, especially in complex environmental samples, such as groundwater, where there are a large number of compounds with similar structures to pentachlorophenol. These compounds may interfere with the detection of pentachlorophenol and reduce the sensitivity and accuracy of the sensor.

[0004] Therefore, this study aims to develop a method for preparing an anti-interference molecular imprinting sensor for pentachlorophenol detection. By optimizing the selection of template molecules, the design of functional monomers, the optimization of cross-linking agents, and the stability and anti-interference of the imprinted cavity, a pentachlorophenol molecular imprinting sensor with high selectivity, high affinity, good stability and anti-interference is prepared. This sensor will be able to achieve rapid and accurate detection of pentachlorophenol, providing strong technical support for environmental protection and ecological risk assessment. Summary of the invention

[0005] To solve the above technical problems, the present invention provides an anti-interference molecular imprinting sensor and a preparation method thereof and application thereof in detecting pentachlorophenol. The anti-interference molecular imprinting sensor for pentachlorophenol detection of the present invention has the advantages of high specificity, strong stability and good anti-interference ability.

[0006] The preparation method of the anti-interference molecular imprinting sensor of the present invention comprises four steps: preparation of gold nanoparticles / glassy carbon electrode (Au NPs / GCE), construction of polypeptide anti-interference interface (PHHP / Au NPs / GCE), preparation of o-phenylenediamine molecular imprinting sensitive membrane (MIP / PHHP / Au NPs / GCE) and PCP analysis and detection. The present invention develops an ultra-sensitive electrochemical sensor for detecting PCP in groundwater by modifying Au NPs, PHHP polypeptide and MIP thin coating, wherein the MIP film is prepared by electrochemical polymerization of o-phenylenediamine (O-PD) and PCP on a glassy carbon electrode, wherein O-PD is used as a functional monomer for the formation of MIP and PCP is used as a template molecule. The AuNPs coating helps to enhance the voltammetric response, and the optimized setting of the MIP layer provides a higher peak change. The prepared electrochemical sensor is used to detect PCP in water, and has the advantages of high specificity, strong stability and good anti-interference ability.

[0007] The present invention is achieved through the following technical solutions:

[0008] The first object of the present invention is to provide a method for preparing an anti-interference molecular imprinting sensor, comprising the following steps:

[0009] Provide an Au NPs / GCE electrode;

[0010] The hydroxyproline helical peptide PHHP was drop-coated on the surface of the Au NPs / GCE electrode and incubated under infrared light to obtain a polypeptide-modified electrode interface;

[0011] An electrolyte containing polymer monomers and PCP molecular imprinting templates is prepared, and electrochemical polymerization is performed on the polypeptide-modified electrode interface; the electrode surface is rinsed and eluted, and finally the PCP template molecules are removed to obtain the anti-interference molecular imprinting sensor.

[0012] In some embodiments of the present invention, the sequence of the hydroxyproline helical peptide PHHP is CPPP(HYP) 8 .

[0013] In some embodiments of the present invention, the concentration of the hydroxyproline helical peptide PHHP is 0.5-5 mM.

[0014] In some embodiments of the present invention, the incubation time in the constant temperature box is 20 to 100 minutes; and the temperature of the constant temperature box is 25°C.

[0015] In some embodiments of the present invention, the molar ratio of the polymer monomer to the PCP molecular imprinting template in the electrolyte is (1:1) to (10:1).

[0016] In some embodiments of the present invention, the polymer monomer is selected from o-phenylenediamine (o-PD).

[0017] In some embodiments of the present invention, the electrochemical polymerization is performed using cyclic voltammetry (CV).

[0018] In some embodiments of the present invention, the cyclic voltammetry conditions are: voltage of 0 to +1.0 V, CV polymerization cycles of 20 to 40, and polymerization scan rate of 25 to 300 mV / s.

[0019] The second object of the present invention is to provide an interference-resistant molecular imprinting sensor prepared by the preparation method.

[0020] The third object of the present invention is to provide the use of the anti-interference molecular imprinting sensor in detecting pentachlorophenol.

[0021] The above technical solution of the present invention has the following advantages compared with the prior art:

[0022] 1. The present invention uses GCE (glassy carbon electrode) as a carrier, and through a carefully designed anti-interference sensing interface and molecular imprinting recognition technology, successfully developed an ultra-sensitive electrochemical voltammetric sensor for pentachlorophenol (PCP) in groundwater. This sensor uses Au NPs (gold nanoparticles) to modify the surface of the GCE electrode, which significantly enhances the electrochemical signal. At the same time, PHHP polypeptide is used to construct the sensing interface, which effectively prevents interference from other organic matter and ions in groundwater. In addition, the introduction of MIP (molecular imprinting polymer) film provides highly specific recognition capabilities for PCP, and further improves the detection sensitivity through statistical optimization. The performance of this sensor in detecting PCP in groundwater is comparable to that of high-performance liquid chromatography tandem mass spectrometry, showing its excellent analytical capabilities. The entire sensor preparation process includes four key steps: preparation of gold nanoparticles / glassy carbon electrode (Au NPs / GCE) S1, construction of anti-interference sensing interface (PHHP / Au NPs / GCE) S2, assembly of anti-interference molecular imprinting sensor MIP / PHHP / Au NPs / GCE S3, and analysis and detection of pentachlorophenol (PCP) S4. These steps make the sensor have significant advantages such as simple operation, rapid response, high sensitivity, strong specificity, good stability and excellent anti-interference when detecting PCP in groundwater.

[0023] 2. Electrochemical impedance spectroscopy (EIS) tests and cyclic voltammetry (CV) were used to further explore the electrochemical properties of GCE, Au NPs / GCE, and PHHP / Au NPs / GCE. Differential pulse voltammetry (DPV) was used to further analyze the electrochemical responses of Au NPs / GCE, PHHP / Au NPs / GCE in removing PCP and MIP / PHHP / Au NPs / GCE in identifying PCP. In addition, DPV also helped to explore the trend of electrical signal changes in the PCP concentration range of 1nM to 10μM, and the corresponding calibration curve was drawn. The entire preparation process has extremely low requirements for experimental equipment and conditions, is relatively low in cost, and is safe and easy to operate. The developed electrochemical sensor exhibits excellent current response, strong stability, high repeatability, and operability. By optimizing the electropolymerization process of MIP, we successfully reduced the potential passivation risk of the electrode surface and reduced the possibility of analyte saturation. These advantages make the sensor of great significance for on-site detection of PCP in a variety of sample matrices such as surface water, seawater, wastewater and sludge. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0025] Figure 1 The electrochemical impedance spectroscopy and cyclic voltammograms of PHHP / Au NPs / GCE, Au NPs / GCE and GCE obtained in Example 1 of the present invention are shown.

[0026] Figure 2 This is the cyclic voltammogram of MIP / PHHP / Au NPs / GCE polymerization obtained in Example 1 of the present invention.

[0027] Figure 3 The differential pulse voltammograms of AuNPs / GCE, PHHP / AuNPs / GCE, MIP / PHHP / AuNPs / GCE (PCP removed) and MIP / PHHP / AuNPs / GCE (after PCP identification) obtained in Example 1 of the present invention.

[0028] Figure 4 The differential pulse voltammogram and calibration curve of the MIP / PHHP / AuNPs / GCE sensor obtained in Example 2 of the present invention in the PCP concentration range of 1 nM to 10 μM.

[0029] Figure 5 The differential pulse voltammogram and calibration curve of the MIP / AuNPs / GCE sensor obtained in the comparative example of the present invention in the PCP concentration range of 1 nM to 1 μM are shown.

[0030] Figure 6 Comparison of the spiked recoveries of the MIP / PHHP / AuNPs / GCE and MIP / AuNPs / GCE sensors in real water samples in the comparative example of the present invention.

[0031] Figure 7 The figure is a comparison of the anti-interference performance of the MIP / PHHP / AuNPs / GCE and MIP / AuNPs / GCE sensors in humic acid and salt ion solutions in the comparative example of the present invention.

[0032] Figure 8 This is the specificity evaluation of the MIP / PHHP / AuNPs / GCE sensor in the comparative example of the present invention. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0034] Embodiment 1:

[0035] The invention provides a method for preparing an anti-interference molecular imprinting sensor for pentachlorophenol detection, comprising the steps of preparing a gold nanoparticle / glassy carbon electrode (Au NPs / GCE) S1, constructing a polypeptide (PHHP) anti-interference interface (PHHP / Au NPs / GCE) S2, and forming an o-phenylenediamine (o-PD) molecular imprinting sensitive membrane S3.

[0036] Preparation of (MIP / PHHP / Au NPs / GCE) S3 and analytical detection of pentachlorophenol S4.

[0037] 1. Preparation of gold nanoparticles / glassy carbon electrode (Au NPs / GCE) S1 The operation steps are as follows:

[0038] (1) The GCE electrode was polished to a mirror surface using 0.05 μm alumina slurry as the polishing material, and then ultrasonically treated in 75% anhydrous ethanol and ultrapure water for 10 min to remove impurities on the surface of the gold electrode;

[0039] (2) The GCE electrode was immersed in 5 mL of 0.5 MH 2 SO 4 In the solution, the electrode was scanned and cleaned for 400 s using an electrochemical workstation, rinsed with deionized water, and placed upright to dry at room temperature;

[0040] (3) 0.5 mL of chloroauric acid stock solution was added to 8.5 mL of deionized water. The Au core was formed by chronoamperometry (CA) at an initial potential of 0.8 V for 15 s. Then, cyclic voltammetry (CV) was used to scan 50 cycles in the range of -0.4 to +0.3 V at a speed of 50 mV / s to obtain a gold nanoparticle / glassy carbon electrode (Au NPs / GCE).

[0041] 2. Construction of polypeptide (PHHP) anti-interference interface (PHHP / Au NPs / GCE) S2 operation steps are as follows:

[0042] Hydroxyproline helical peptide (PHHP) with a concentration of 0.5, 1, 2, 3, 4, and 5 mM was drop-coated on the surface of gold nanoparticles / glassy carbon electrode (Au NPs / GCE) and incubated in a constant temperature oven at 25°C for 20, 40, 60, 80, and 100 min to obtain a peptide-modified electrode interface (PHHP / Au NPs / GCE).

[0043] 3. Preparation of molecular imprinted sensitive membrane (MIP / PHHP / Au NPs / GCE) S3 operation steps are as follows:

[0044] (1) preparing an electrolyte containing o-PD (polymer monomer) and PCP (molecular imprinting template), and performing electrochemical polymerization on the surface of the PHHP / Au NPs / GCE electrode between 0 and +1.0 V using cyclic voltammetry (CV);

[0045] (2) Optimization of electrochemical polymerization parameters, including the molar ratio of polymer monomer to molecular imprinting template (o-PD:PCP = 10:1, 5:1, 3:1, 2:1, 1:1), CV polymerization cycles (20, 25, 30, 35, 40), and polymerization scan rate (25, 50, 100, 200, 300 mV / s);

[0046] (3) After the polymerization is completed, the surface of MIP / PHHP / Au NPs / GCE is rinsed with deionized water and eluted in an eluent of methanol: water = 4:1 for 6 minutes to remove the PCP template molecules and obtain the MIP / PHHP / Au NPs / GCE.

[0047] 4. Structural characterization: In 5mM [Fe(CN) 6 ] 3- / 4- The electrochemical behaviors of PHHP / Au NPs / GCE, Au NPs / GCE and GCE were characterized by electrochemical impedance spectroscopy and cyclic voltammograms in 0.1 M KCl solution. Figure 1As shown in the EIS graph, as Au NPs are deposited on the electrode surface, the electron transfer ability of Au NPs / GCE is enhanced and its charge transfer resistance (R ct ) is significantly smaller than GCE, and the CV current increases. With the modification of PHHP peptide on the electrode surface, the electrochemical impedance value increases significantly, and the current value in the CV graph decreases significantly. The polymerization of molecular imprinted membrane is characterized by cyclic voltammogram. The results are shown in Figure 2 As shown, as the number of scanning circles increases, the conductivity of the electrode decreases, indicating that a polymer layer has been formed on the electrode surface.

[0048] 5. PCP analysis and detection S4 operation steps are as follows:

[0049] Differential pulse voltammetry (DPV) was used to measure the prepared MIP / PHHP / Au NPs / GCE sensor in 5 mM [Fe(CN) 6 ] 3- / 4- The measurement was carried out in a mixed solution with 0.1M KCl, and the signal change of the potassium ferrocyanide probe before and after PCP incubation was used to quantitatively analyze the PCP concentration in the water sample. The electrochemical measurement process includes pre-washing, incubation and detection. During pre-washing, MIP / PHHP / Au NPs / GCE was gently rinsed with deionized water to clean the electrode surface; during incubation, it was incubated in 0.5mL of a certain concentration of PCP solution (pH=6.0) for 90 minutes; during detection, it was detected in DPV potassium ferrocyanide solution.

[0050] Differential pulse voltammetry (DPV) was used to characterize the electrochemical responses of Au NPs / GCE, PHHP / Au NPs / GCE in removing PCP and MIP / PHHP / Au NPs / GCE in recognizing PCP. Figure 3 As shown, it can be seen that around 0.220 V, an obvious current response appears, and the current value decreases significantly because the PCP recognition hinders the electron transfer in the MIP cavity.

[0051] Embodiment 2:

[0052] As an improvement on the previous embodiment, the preparation S3 of the MIP / PHHP / Au NPs / GCE did not follow the traditional laboratory incubation method in PCP solution, but chose a method that is closer to the actual application environment, that is, directly immersing it in the standard sample for up to 90 minutes, and then performing DPV tests on these samples immersed in the standard sample according to the established steps and procedures.

[0053] The DPV test has a potential range of -0.2 to 0.6 V (vs Ag / AgCl), a potential amplitude of 4 mV, a potential increment of 50 mV, a sampling width of 50 ms, and a pulse period of 500 ms.

[0054] By using the above technical solution, the differential pulse voltammogram and calibration curve in the PCP concentration range of 1nM to 10μM are shown in the following table. Figure 4 As shown in the figure, it can be seen that the peak position is approximately at 0.220 V and gradually decreases with the increase of PCP concentration. The logarithmic calibration curve shows that the developed sensor has a high sensitivity to PCP in the range of 1nM to 10μM, and the correlation coefficient R 2 It is 0.9949.

[0055] Comparative Example:

[0056] As a comparison with Example 2, MIP / Au NPs / GCE without peptide modification was prepared. Except for the lack of step S2 of peptide modification, the other preparation steps were the same as those of MIP / PHHP / Au NPs / GCE. It was immersed in the standard sample for 90 minutes, and the DPV test was performed on these samples immersed in the standard sample according to the established steps and procedures.

[0057] The parameters of the DPV test are in the potential range of -0.2 to 0.6 V (vs Ag / AgCl), the potential amplitude is 4 mV, the potential increment is 50 mV, the sampling width is 50 ms, the pulse period is 500 ms, the differential pulse voltammogram and the calibration curve in the PCP concentration range of 1 nM to 1 nM are shown in the following table. Figure 5 As shown in the figure, it can be seen that the peak position is approximately at 0.220 V and gradually decreases with the increase of PCP concentration. The logarithmic calibration curve shows that the developed sensor has a high sensitivity to PCP in the range of 1nM to 1μM, and the correlation coefficient R 2 Compared with Example 2, the sensor modified with the polypeptide has a wider detection range for PCP.

[0058] Afterwards, the standard addition method was used to determine 10, 20, 50, and 100 nM PCP in complex water samples of groundwater (1#, 2#, and 3#) and surface water (4#). PCP was detected using MIP / PHHP / Au NPs / GCE and MIP / Au NPs / GCE sensors, respectively. The results are shown in Figure 6As shown. The average recovery of samples detected using MIP / PHHP / Au NPs / GCE ranged from 96.4% to 104.4%, with relative standard deviations (RSD) ranging from 1.33% to 4.13%; the average recovery of samples using MIP / Au NPs / GCE ranged from 103.15% to 168.90%, with RSDs ranging from 2.31% to 5.12%. This indicates that when detecting pentachlorophenol in complex water samples, the introduction of peptides can prevent the sensor from being interfered with, thereby facilitating the high-precision detection of pentachlorophenol.

[0059] Anti-interference and specificity

[0060] On the basis of the comparative example, in order to further illustrate the anti-interference performance and specificity of the prepared MIP / PHHP / Au NPs / GCE for PCP recognition, anti-interference experiments and specificity experiments were carried out.

[0061] Different concentrations of humic acid solution (1, 10, 25, 50, 100 ppm) and salt ions (Na 2 SO 4 , CaCl 2 , KCl) solutions (10, 50, 100 mM) were used to test the antifouling properties of MIP / Au NPs / GCE and MIP / PHHP / Au NPs / GCE electrodes. The current retention rate was used to evaluate the anti-interference ability of the electrode, and the calculation formula was percentage (I / I 0 ×100%), where I 0 represents the DPV peak current measured before immersion in different solutions, and I represents the DPV peak current of the same value after immersion in the relevant solution for 90 minutes. The DPV test parameters have a potential range of -0.2 to 0.6 V (vs Ag / AgCl), a potential amplitude of 4 mV, a potential increment of 50 mV, a sampling width of 50 ms, and a pulse period of 500 ms. Figure 7 It can be seen that the current retention rate of MIP / PHHP / Au NPs / GCE is always above 90%, while the signal of MIP / Au NPs / GCE decreases significantly.

[0062] By comparing the MIP / PHHP / AuNPs / GCE sensor in salt ions (Na 2 SO 4 , CaCl 2 、FeCl 3 , KCl, K 2 SO 4The specificity of the assay was tested by comparing the results of the assay in the presence of 10 μM PCP (same concentration as PCP / 10-fold excess / 100-fold excess). The specificity was evaluated using the recovery ratio, which was calculated as the ratio (c / c 0 ×100%), where c 0 represents the actual PCP concentration in the solution, c represents the PCP concentration detected by MIP / PHHP / Au NPs / GCE, and the test method used is DPV. In the DPV test, the potential range of the parameters is -0.2 to 0.6 V (vs Ag / AgCl), the potential amplitude is 4 mV, the potential increment is 50 mV, the sampling width is 50 ms, and the pulse period is 500 ms. Figure 8 As shown, these six types of interferences have little effect on the accurate detection of pentachlorophenol, and the recovery ratio is 0.95 to 1.15.

[0063] In summary, the prepared MIP / PHHP / Au NPs / GCE sensor has good anti-interference ability and specificity.

[0064] The advantage is that this experimental technology does not require complex and expensive equipment, has extremely low cost, and is simple and clear to operate.

[0065] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for preparing an anti-interference molecular imprinting sensor, characterized in that: The following steps are involved: Provide an Au NPs / GCE electrode; The hydroxyproline helical peptide PHHP was drop-coated on the surface of the Au NPs / GCE electrode and incubated under infrared light to obtain a peptide-modified electrode interface; preparing an electrolyte containing polymer monomers and PCP molecular imprinting templates, and performing electrochemical polymerization at the electrode interface modified by the polypeptide; The electrode surface is rinsed and eluted, and finally the PCP template molecules are removed to obtain the anti-interference molecular imprinting sensor.

2. The preparation method according to claim 1, characterized in that: The sequence of the hydroxyproline helical peptide PHHP is CPPP(HYP)8.

3. The preparation method according to claim 1, characterized in that: The concentration of the hydroxyproline helical peptide PHHP is 0.5-5 mM.

4. The preparation method according to claim 1, characterized in that: The incubation time in a 25°C incubator is 20 to 100 minutes.

5. The preparation method according to claim 1, characterized in that: The molar ratio of the polymer monomer to the PCP molecular imprinting template in the electrolyte is (1:1) to (10:1).

6. The preparation method according to claim 1, characterized in that: The polymer monomer is selected from o-phenylenediamine (o-PD).

7. The preparation method according to claim 1, characterized in that: The electrochemical polymerization adopts cyclic voltammetry (CV).

8. The preparation method according to claim 7, characterized in that: Cyclic voltammetry conditions: voltage of 0 to +1.0 V, CV polymerization cycles of 20 to 40, and polymerization scan rate of 25 to 300 mV / s.

9. An anti-interference molecular imprinting sensor, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the anti-interference molecular imprinting sensor according to claim 9 in detecting pentachlorophenol.

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