An anti-interference molecularly imprinted sensor, a preparation method thereof and application thereof in detection of pentachlorophenol

Abstract

The application provides an anti-interference molecular imprinting sensor and a preparation method thereof and application thereof in detection of pentachlorophenol. The preparation method of the application comprises the following steps: providing an Au NPs / GCE electrode; dropping hydroxyproline helical peptide PHHP on the surface of the Au NPs / GCE electrode, and incubating under infrared lamp irradiation 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; washing the surface of the electrode, and performing elution, and finally removing the PCP template molecule to obtain the anti-interference molecular imprinting sensor. The Au NPs coating is helpful to enhance the voltammetry response, and the optimized setting of the MIP layer provides a 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] This invention relates to the field of electrochemical sensing technology, and in particular to an anti-interference molecularly imprinted sensor, its preparation method, and its application in the detection of pentachlorophenol. Background Technology

[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. Monitoring and control of PCP is a crucial aspect of environmental protection and ecological risk assessment. However, traditional methods for detecting PCP in groundwater, such as liquid chromatography, while possessing high sensitivity and accuracy, often suffer from problems such as expensive equipment, complex operation, cumbersome pretreatment, and susceptibility to interference from other substances, limiting their widespread application in rapid on-site detection and environmental monitoring. Molecularly imprinted sensors are sensors with specific recognition functions prepared based on molecular imprinting technology. By preparing imprinted cavities that match the shape, size, and functional groups of the target molecule (such as PCP), highly selective recognition of the target molecule is achieved. These sensors possess advantages such as high selectivity, high affinity, good stability, and ease of preparation.

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

[0004] Therefore, this study aims to develop a method for preparing an interference-resistant molecularly imprinted 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 interference resistance of the imprinted cavity, a pentachlorophenol molecularly imprinted sensor with high selectivity, high affinity, good stability, and excellent interference resistance was prepared. This sensor will enable rapid and accurate detection of pentachlorophenol, providing strong technical support for environmental protection and ecological risk assessment. Summary of the Invention

[0005] To address the above technical problems, this invention provides an anti-interference molecularly imprinted sensor, its preparation method, and its application in the detection of pentachlorophenol. The anti-interference molecularly imprinted sensor for pentachlorophenol detection of this invention has the advantages of high specificity, strong stability, and good anti-interference capability.

[0006] The method for preparing the interference-resistant molecularly imprinted sensor of this invention includes four steps: preparation of gold nanoparticles / glassy carbon electrode (Au NPs / GCE), construction of peptide interference-resistant interface (PHHP / Au NPs / GCE), preparation of o-phenylenediamine molecularly imprinted sensitive membrane (MIP / PHHP / Au NPs / GCE), and PCP analysis and detection. This invention develops an ultrasensitive electrochemical sensor for detecting PCP in groundwater through modification with Au NPs, PHHP peptides, and a MIP thin film coating. The MIP film is prepared by electrochemical polymerization of o-phenylenediamine (O-PD) and PCP on a glassy carbon electrode, where O-PD serves as the functional monomer for MIP formation, and PCP serves as the template molecule. The Au NPs coating helps enhance the voltammetric response, and the optimized setting of the MIP layer provides a higher peak variation. The prepared electrochemical sensor for detecting PCP in water exhibits advantages such as high specificity, strong stability, and good interference resistance.

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

[0008] The first objective of this invention is to provide a method for preparing an interference-resistant molecularly imprinted sensor, comprising the following steps:

[0009] Provide an Au NPs / GCE electrode;

[0010] Hydroxyproline helical peptide PHHP was drop-coated onto the surface of the Au NPs / GCE electrode and incubated under infrared lamp irradiation to obtain a peptide-modified electrode interface.

[0011] An electrolyte containing polymer monomers and PCP molecularly imprinted templates was prepared, and electrochemical polymerization was carried out at the electrode interface modified with the peptides. The electrode surface was rinsed and eluted, and finally the PCP template molecules were removed to obtain the anti-interference molecularly imprinted 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 chamber is 20 to 100 minutes; the temperature of the constant temperature chamber is 25°C.

[0015] In some embodiments of the present invention, the molar ratio of polymer monomer to PCP molecularly imprinted 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 0 to +1.0V, CV polymerization cycle number 20 to 40, and polymerization scan rate 25 to 300mV / s.

[0019] The second objective of this invention is to provide an anti-interference molecularly imprinted sensor, prepared by the aforementioned method.

[0020] A third objective of this invention is to provide the application of the aforementioned anti-interference molecular imprint sensor in the detection of pentachlorophenol.

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

[0022] 1. This invention utilizes a glassy carbon electrode (GCE) as a carrier and employs a carefully designed anti-interference sensing interface and molecularly imprinted recognition technology to successfully develop an ultrasensitive electrochemical voltammetric sensor for pentachlorophenol (PCP) in groundwater. This sensor modifies the GCE electrode surface with Au NPs (gold nanoparticles), significantly enhancing the electrochemical signal. Simultaneously, PHHP peptides are used to construct the sensing interface, effectively preventing interference from other organic matter and ions in the groundwater. Furthermore, the introduction of a MIP (molecularly imprinted polymer) film provides highly specific recognition capabilities for PCP, and statistical optimization further improves the detection sensitivity. The sensor's performance in detecting PCP in groundwater is comparable to that of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), demonstrating its superior analytical capabilities. The entire sensor fabrication process comprises four key steps: preparation of gold nanoparticles / glassy carbon electrodes (Au NPs / GCE) S1, construction of the anti-interference sensing interface (PHHP / Au NPs / GCE) S2, assembly of the anti-interference molecularly imprinted sensor MIP / PHHP / Au NPs / GCE S3, and analysis and detection of pentachlorophenol (PCP) S4. These steps enable the sensor to possess significant advantages in detecting PCP in groundwater, including ease of operation, rapid response, high sensitivity, strong specificity, good stability, and superior anti-interference performance.

[0023] 2. In exploring the electrochemical characteristics of GCE, Au NPs / GCE, and PHHP / Au NPs / GCE, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were employed. Differential pulse voltammetry (DPV) was used to further analyze the electrochemical responses of Au NPs / GCE and PHHP / Au NPs / GCE in PCP removal and MIP / PHHP / Au NPs / GCE in PCP recognition. Furthermore, DPV helped investigate the electrical signal variation trend of PCP concentrations in the range of 1 nM to 10 μM, and corresponding calibration curves were plotted. The entire preparation process has extremely low requirements for experimental equipment and conditions, is relatively inexpensive, and is safe and easy to operate. The developed electrochemical sensors exhibit excellent current response, strong stability, high repeatability, and operability. By optimizing the electropolymerization process of MIP, we successfully reduced the potential passivation risk on the electrode surface and decreased the possibility of analyte saturation. These advantages make this sensor of great significance for on-site detection of PCP in various sample matrices, including surface water, seawater, wastewater, and sludge. Attached Figure Description

[0024] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

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

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

[0027] Figure 3 The differential pulse voltammetry diagrams of AuNPs / GCE, PHHP / AuNPs / GCE, MIP / PHHP / AuNPs / GCE (PCP removed) and MIP / PHHP / AuNPs / GCE (PCP identified) obtained in Embodiment 1 of the present invention are shown.

[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 are shown in the PCP concentration range of 1nM to 10μM.

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

[0030] Figure 6 This invention compares the spiked recovery rates of the MIP / PHHP / AuNPs / GCE and MIP / AuNPs / GCE sensors in real water samples in the comparative examples.

[0031] Figure 7 This invention compares the anti-interference performance of the MIP / PHHP / AuNPs / GCE and MIP / AuNPs / GCE sensors in humic acid and salt ion solutions.

[0032] Figure 8 This is for the specificity evaluation of the MIP / PHHP / AuNPs / GCE sensors in the comparative examples of this invention. Detailed Implementation

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

[0034] Example 1:

[0035] The present invention provides a method for preparing an anti-interference molecularly imprinted sensor for pentachlorophenol detection, comprising: S1, preparation of gold nanoparticle / glassy carbon electrode (Au NPs / GCE); S2, construction of peptide (PHHP) anti-interference interface (PHHP / Au NPs / GCE); and S3, preparation of o-phenylenediamine (o-PD) molecularly imprinted sensitive membrane.

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

[0037] I. Preparation of Gold Nanoparticle / Glassy Carbon Electrode (Au NPs / GCE) S1 The operation steps are as follows:

[0038] (1) Use 0.05μm alumina slurry as polishing material to polish the GCE electrode to a mirror surface, and then ultrasonically treat it in 75% anhydrous ethanol and ultrapure water for 10 min in sequence to remove impurities from the surface of the gold electrode.

[0039] (2) The GCE electrode was immersed in 5 mL of the prepared 0.5 M H2SO4 solution, and the electrode was cleaned by scanning with an electrochemical workstation for 400 s. It was then rinsed with deionized water and placed upright to dry at room temperature.

[0040] (3) Add 0.5 mL of chloroauric acid stock solution to 8.5 mL of deionized water. First, pulse the solution for 15 s at an initial potential of 0.8 V using chronoamperometry (CA) to form Au nuclei. Then, use cyclic voltammetry (CV) to scan 50 times at a speed of 50 mV / s in the range of -0.4 to +0.3 V to obtain gold nanoparticle / glassy carbon electrode (Au NPs / GCE).

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

[0042] Hydroxyproline helical peptide (PHHP) at concentrations of 0.5, 1, 2, 3, 4, and 5 mM was drop-coated onto the surface of a gold nanoparticle / 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] III. Preparation of Molecularly Imprinted Sensitive Membrane (MIP / PHHP / Au NPs / GCE) S3 The operation steps are as follows:

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

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

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

[0047] IV. Structural Characterization: In 5 mM [Fe(CN)6] 3- / 4- The electrochemical behavior of PHHP / Au NPs / GCE, Au NPs / GCE, and GCE was characterized using electrochemical impedance spectroscopy and cyclic voltammetry in (0.1 M KCl) solution. The results are as follows: Figure 1 As shown in the EIS diagram, with the deposition of Au NPs on the electrode surface, the electron transfer capability of Au NPs / GCE is enhanced, and its charge transfer resistance (Rc) decreases.ct The electrochemical impedance spectroscopy (EIS) was significantly smaller than that of the gas ionization chemiluminescence (GCE), and the cyclic voltammetry (CV) current increased. With the modification of the PHHP peptide on the electrode surface, the electrochemical impedance spectroscopy (EIS) value increased significantly, and the current value in the CV plot decreased significantly. The polymerization of the molecularly imprinted membrane was characterized by cyclic voltammetry, and the results are as follows: Figure 2 As shown, the conductivity of the electrode decreases with the increase of the number of scans, indicating that a polymer layer has formed on the electrode surface.

[0048] V. The S4 operation steps for PCP analysis and detection are as follows:

[0049] The prepared MIP / PHHP / Au NPs / GCE sensor was placed in 5 mM [Fe(CN)6] using differential pulse voltammetry (DPV). 3- / 4- Measurements were performed in a mixture with 0.1M KCl, utilizing the signal change of the potassium ferricyanide probe before and after PCP incubation to quantitatively analyze the PCP concentration in the water sample. The electrochemical measurement process included pre-washing, incubation, and detection. During pre-washing, the MIP / PHHP / Au NPs / GCE electrode surfaces were gently rinsed with deionized water to clean them. During incubation, the sample was incubated for 90 minutes in 0.5 mL of a PCP solution (pH = 6.0) containing a specific concentration. Detection was performed using a DPV potassium ferricyanide solution.

[0050] Differential pulse voltammetry (DPV) was used to characterize the electrochemical responses of Au NPs / GCE, PHHP / Au NPs / GCE in PCP removal, and MIP / PHHP / Au NPs / GCE in PCP recognition. The results are as follows: Figure 3 As shown, a significant current response occurs around 0.220V. Due to the PCP recognition hindering electron transfer in the MIP cavity, the current value decreases significantly.

[0051] Example 2:

[0052] As an improvement to the previous embodiment, the preparation S3 of MIP / PHHP / Au NPs / GCE did not follow the traditional laboratory method of incubation in PCP solution. Instead, a more practical approach was adopted, in which the samples were directly immersed in standard samples for up to 90 minutes. Subsequently, DPV tests were performed on these samples after immersion in standard samples according to established steps and procedures.

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

[0054] Using the above technical solution, differential pulse voltammograms and calibration curves were obtained in the PCP concentration range of 1 nM to 10 μM, as shown in the following results. Figure 4 As shown, the peak position is approximately at 0.220 V, gradually decreasing with increasing PCP concentration. The logarithmic calibration curve shows that the developed sensor exhibits high sensitivity to PCP in the range of 1 nM to 10 μM, with a correlation coefficient R0. 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 absence of step S2 (peptide modification), the preparation steps were the same as for MIP / PHHP / Au NPs / GCE. These samples were then immersed in standard samples for 90 minutes, and DPV testing was performed on them according to the established procedures.

[0057] The DPV test parameters had 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. The differential pulse voltammograms and calibration curves within the 1 nM to 1 nM PCP concentration range are shown in the following figures. Figure 5 As shown, the peak position is approximately at 0.220 V, gradually decreasing with increasing PCP concentration. The logarithmic calibration curve shows that the developed sensor exhibits high sensitivity to PCP in the range of 1 nM to 1 μM, with a correlation coefficient R0. 2 The value was 0.9922. Compared with Example 2, the peptide-modified sensor has a wider detection range for PCP.

[0058] Subsequently, PCP concentrations of 10, 20, 50, and 100 nM were determined using the standard spiking method in complex water quality samples from groundwater (1#, 2#, 3#) and surface water (4#). PCP was detected using MIP / PHHP / Au NPs / GCE and MIP / Au NPs / GCE sensors, respectively, and the results are as follows: Figure 6 As shown, the average recoveries of samples detected using MIP / PHHP / Au NPs / GCE ranged from 96.4% to 104.4%, with relative standard deviations (RSDs) of 1.33% to 4.13%; the average recoveries of samples detected using MIP / Au NPs / GCE ranged from 103.15% to 168.90%, with RSDs of 2.31% to 5.12%. This indicates that the introduction of peptides can prevent sensor interference when detecting pentachlorophenol in complex water samples, thus contributing to high-precision detection of pentachlorophenol.

[0059] Anti-interference and specificity

[0060] Based on the comparative examples, 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 conducted.

[0061] The antifouling performance of the MIP / Au NPs / GCE and MIP / PHHP / Au NPs / GCE electrodes was tested using humic acid solutions of different concentrations (1, 10, 25, 50, 100 ppm) and salt ion solutions (Na₂SO₄, CaCl₂, KCl) (10, 50, 100 mM). The anti-interference capability of the electrodes was evaluated using the current retention rate, calculated as a percentage (I / I₀ × 100%), where I₀ represents the peak DPV current measured before immersion in different solutions, and I represents the same peak DPV current after immersion in the relevant solutions for 90 minutes. For the DPV test, the potential range was -0.2 to 0.6 V (vs Ag / AgCl), the potential amplitude was 4 mV, the potential increment was 50 mV, the sampling width was 50 ms, and the pulse period was 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] The specificity of the MIP / PHHP / AuNPs / GCE sensor was tested by comparing its detection results with those of salt ions (Na2SO4, CaCl2, FeCl3, KCl, K2SO4), pentachlorophenol analogs (4-chlorophenol (CP), 2,4-dichlorophenol (DCP), 2,4,5-trichlorophenol (TCP)), humic acid (HA), antibiotics (TCH), endocrine disruptors (BPA), or surfactants (SAA) in the presence of 10 μM PCP (at the same concentration as PCP / 10-fold excess / 100-fold excess). Specificity was assessed using the recovery ratio, calculated as a ratio (c / c0 × 100%), where c0 represents the actual PCP concentration in solution, and c represents the PCP concentration detected by the MIP / PHHP / AuNPs / GCE sensor using the DPV assay. The DPV test parameters have a potential range of -0.2 to 0.6V (vs Ag / AgCl), a potential amplitude of 4mV, a potential increment of 50mV, a sampling width of 50ms, and a pulse period of 500ms. Figure 8 As shown, these six types of interference have almost no impact on the accurate detection of pentachlorophenol, with recovery rates ranging from 0.95 to 1.15.

[0063] In summary, the fabricated MIP / PHHP / Au NPs / GCE sensor exhibits good anti-interference capability and specificity.

[0064] The advantages are that this experimental technique does not require complex and expensive equipment, has extremely low cost, and is simple and straightforward to operate.

[0065] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing an anti-interference molecularly imprinted sensor, characterized in that, Includes the following steps: Provide an Au NPs / GCE electrode; Hydroxyproline helical peptide PHHP was drop-coated onto the surface of the Au NPs / GCE electrode and incubated under infrared lamp irradiation to obtain a peptide-modified electrode interface. An electrolyte containing polymer monomers and PCP molecularly imprinted templates was prepared, and electrochemical polymerization was carried out at the electrode interface modified with the peptide. The electrode surface is rinsed and eluted, and finally the PCP template molecules are removed to obtain the anti-interference molecular imprint 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 constant temperature incubator at 25℃ is 20 to 100 minutes.

5. The preparation method according to claim 1, characterized in that, The molar ratio of polymer monomer to PCP molecularly imprinted 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 was performed using cyclic voltammetry (CV).

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

9. An anti-interference molecular imprint sensor, characterized in that, Prepared by the preparation method described in any one of claims 1 to 8.

10. The application of the anti-interference molecular imprint sensor according to claim 9 in the detection of pentachlorophenol.

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