An MSNs@Au-LAC@PDA-PPy composite material, a preparation method and application thereof

Abstract

The application discloses a kind of MSNs@Au-LAC@PDA-PPy composite material and its preparation method and application.It is mainly prepared by MSNs@Au, lacase LAC, polydopamine PDA and polypyrrole PPy composite;Preparation is dissolved in acetic acid-sodium acetate buffer lacase to prepare lacase solution, MSNs@Au is added into lacase solution and is ultrasonic, stirring, soaking and acetic acid-sodium acetate buffer is washed by centrifugation, then hydrochloric acid dopamine, pyrrole and Tris solution, initiator are added, stirring is used at specific temperature Distilled water is washed by centrifugation, finally freeze-drying treatment obtains final MSNs@Au-LAC@PDA-PPy composite material, and further preparation electrode is used for bisphenol A detection.The application prepares high-sensitivity electrochemical sensor, is used for the high-sensitivity detection of BPA in complex matrix, can realize the efficient fixation and protection of lacase.

Description

Technical Field

[0001] This invention relates to the field of electrochemical sensing and detection, and in particular to an MSNs@Au-LAC@PDA-PPy composite material, its preparation method, and its application in rapid detection of bisphenol A. Background Technology

[0002] BPA, an industrial chemical widely used in plastic products and food packaging, has attracted considerable attention due to its potential endocrine-disrupting effects. BPA can migrate into food through packaging materials, and long-term exposure may pose health risks to humans, such as affecting the reproductive system and increasing cancer risk. Therefore, developing rapid and sensitive BPA detection methods is crucial for food safety and public health. Electrochemical detection methods, with their advantages of high sensitivity, low cost, ease of operation, and applicability to on-site testing, have become an important research direction in the field of BPA detection.

[0003] To enhance the specificity of electrochemical sensors, biomolecules such as enzymes and antibodies are often introduced to improve their selectivity and catalytic activity. Laccase (LAC), a polycopper oxidase, is highly efficient at catalyzing the oxidation of phenolic compounds, thus showing promising potential for BPA detection. Laccase not only possesses high catalytic activity but also operates under mild conditions, making it suitable for constructing electrochemical sensing interfaces. However, the direct application of laccase in sensors faces several challenges, such as the susceptibility of enzyme activity to environmental influences, the complexity of the immobilization process, and poor stability. Summary of the Invention

[0004] To address the issues of laccase immobilization and stability, this invention proposes an MSNs@Au-LAC@PDA-PPy composite material, its preparation method, and its application in bisphenol A detection.

[0005] The technical solution adopted in this invention is: I. A composite material of MSNs@Au-LAC@PDA-PPy: It is mainly prepared by combining MSNs@Au, laccase LAC, polydopamine PDA and polypyrrole PPy.

[0006] II. A method for preparing MSNs@Au-LAC@PDA-PPy composite material, the preparation process of which is as follows: S1. Prepare laccase solution by dissolving laccase in acetate-sodium acetate buffer and sonicating for 10 min to form laccase solution. S2. Add MSNs@Au to the laccase solution and continue ultrasonic treatment for 10 min to obtain a mixture. Then, immerse the mixture in a freezer at 4 ℃ for a certain period of time. S3. Add the dopamine hydrochloride, pyrrole and Tris solution to the above mixture, and sonicate for 10 min to obtain a homogeneous solution; S4. At room temperature, add the initiator (NH4)2S2O8 to the homogeneous solution, stir at a specific temperature, and then centrifuge and wash with water to collect the preliminary sample product. S5. The preliminary sample product was freeze-dried for 48 h to obtain the final MSNs@Au-LAC@PDA-PPy composite material.

[0007] In S1: the acetate-sodium acetate buffer solution has deionized water as the solvent and acetic acid and sodium acetate as the solutes, with a pH of 4.5; the laccase solution has a mass-volume ratio of (10~50) mg to 10 mL of laccase and acetate-sodium acetate buffer solution.

[0008] In S2, the mass ratio of MSNs@Au to laccase in the laccase solution is 25:(10~50)mg, and the mixture is stirred and soaked at 4 ℃ for 6~48 h.

[0009] In step S3, 10 mL of a Tris solution with pH=8.5 is added to the solid mixture. The Tris solution has a pH of 8.5, and the mass-to-volume ratio of dopamine hydrochloride to pyrrole is 0.05 g:14 µL. Specifically, the mass-to-volume ratio of dopamine hydrochloride to Tris solution is 0.05 g:10 mL, and the mass-to-volume ratio of pyrrole to Tris solution is 14 µL:10 mL.

[0010] In S4, an initiator with pH=8.5 is prepared by dissolving (NH4)2S2O8 in Tris solution. The molar concentration of (NH4)2S2O8 is 0.5 M. After adding the initiator to the homogeneous solution, the mixture is stirred at 4 °C for 24 h.

[0011] III. An MSNs@Au-LAC@PDA-PPy-2 / GCE electrode: This electrode is fabricated using an MSNs@Au-LAC@PDA-PPy composite material. The preparation method of the above MSNs@Au-LAC@PDA-PPy-2 / GCE electrode is as follows: First, the glassy carbon electrode GCE was pretreated by spreading Al2O3 powders of different particle sizes, 0.3 µm and 0.05 µm, evenly on chamois leather, adding water to form a slurry, and then finely polishing the electrode. The glassy carbon electrode surface was dirty, so it was polished and cleaned with polishing powder (alumina) slurry of different particle sizes.

[0012] Subsequently, MSNs@Au-LAC@PDA-PPy materials were weighed and added to deionized water, and ultrasonically vibrated to form a homogeneous mixed solution; Finally, the uniformly mixed solution was dropped onto the finely polished glassy carbon electrode GCE surface and allowed to dry naturally at room temperature for 12 h to prepare the MSNs@Au-LAC@PDA-PPy-2 / GCE electrode.

[0013] The fine polishing of the electrode specifically involves: 1) Place the electrode in an ethanol solution and ultrasonically clean it for 1 min to remove surface impurities; 2) Perform electrochemical scanning of the electrode using CV until the curve remains stable to ensure that its performance meets the standards; 3) Clean the electrode again with deionized water using ultrasonic cleaning for 1 minute to thoroughly remove any residue; 4) Dry the electrode surface with nitrogen gas and clean it to complete the polishing process of the GCE glassy carbon electrode.

[0014] 8. The application of the MSNs@Au-LAC@PDA-PPy composite material of claim 1 or the MSNs@Au-LAC@PDA-PPy-2 / GCE electrode of claim 6, characterized in that: it is used in the rapid electrochemical detection of bisphenol A.

[0015] The specific bisphenol A detection method employs an electrochemical sensing approach: a three-electrode system is used, with the MSNs@Au-LAC@PDA-PPy-2 / GCE electrode as the working electrode, the Ag / AgCl electrode as the reference electrode, and a platinum sheet electrode as the counter electrode for detection. Peak current parameters were obtained by DPV electrochemical method, and the concentration of bisphenol A was obtained by processing based on the peak current parameters.

[0016] In practice, CV and EIS are used to measure electrochemical performance.

[0017] IV. A method for detecting bisphenol A using an MSNs@Au-LAC@PDA-PPy-2 / GCE electrode: A three-electrode working system is adopted, wherein the MSNs@Au-LAC@PDA-PPy-2 / GCE electrode is used as the working electrode, the Ag / AgCl electrode is used as the reference electrode, and the platinum sheet electrode is used as the counter electrode for detection; the DPV electrochemical method is used, the enrichment potential of bisphenol A is 0.2 V, and the enrichment time is 120 s; a standard curve of bisphenol A concentration and peak current is pre-fitted experimentally, and the concentration of bisphenol A to be measured is obtained using the standard curve.

[0018] This invention constructs a highly sensitive electrochemical sensor based on MSNs@Au-LAC@PDA-PPy composite material for the highly sensitive detection of BPA in complex matrices. Figure 1The porous structure of MSNs@Au was utilized to adsorb laccase solution onto the substrate. The washing process of laccase was controlled by mixing and stirring LAC and MSNs@Au. An encapsulation layer was formed by the in-situ copolymerization reaction of dopamine (DA) and pyrrole (Py), thereby achieving efficient immobilization and protection of laccase.

[0019] The beneficial effects of this invention are: Polydopamine (PDA) possesses biocompatibility, hydrophilicity, and film-forming properties; polypyrrole (PPy), as a conductive polymer, can enhance electron transfer rates and significantly improve the sensor's response sensitivity. By comparing the test results with those of uncoated polymer MSNs@Au-LAC, and by regulating the distribution of laccase, it is demonstrated that the copolymer of PDA and PPy constructed in this invention not only stabilizes the three-dimensional structure of laccase but also optimizes the charge transport path on the electrode surface through a synergistic effect.

[0020] This invention optimizes several key parameters of the sensor, including laccase impregnation time, laccase concentration, titration amount of electrode surface material, detection enrichment potential, enrichment time, and pH of the BPA electrochemical detection system, to determine the optimal detection parameters. The detection limit for BPA is as low as 1.6 nM (S / N=3). Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the fabrication and application of the MSNs@Au-LAC@PDA-PPy / GCE sensor for BPA detection; Figure 2 These are scanning electron microscope (SEM) and transmission electron microscope (TEM) images of the materials, where (ac) are SEM images of MSNs@Au-LAC, MSNs@Au-LAC@PDA-PPy-1, and MSNs@Au-LAC@PDA-PPy-2; and (df) are the corresponding TEM images of the above materials. Figure 3 These are high-resolution transmission electron microscopy (HRTEM) images and elemental distribution maps, where (ac) are HRTEM images of MSNs@Au-LAC, MSNs@Au-LAC@PDA-PPy-1, and MSNs@Au-LAC@PDA-PPy-2; (df) is an overlay of the elemental distribution maps for the above materials; and (gi) is the Cu elemental distribution map for the above materials. Figure 4These are material element distribution diagrams, including (ac) the C, N, and O element distribution diagrams for MSNs@Au-LAC; (df) the C, N, and O element distribution diagrams for MSNs@Au-LAC@PDA-PPy-1; and (gi) the C, N, and O element distribution diagrams for MSNs@Au-LAC@PDA-PPy-2. Figure 5 These are Fourier transform infrared (FTIR) and thermogravimetric analysis (TGA) plots, where (a) are the FTIR plots of MSNs@Au (M), MSNs@Au-LAC (ML), MSNs@Au-LAC@PDA-PPy-1 (MLP-1), and MSNs@Au-LAC@PDA-PPy-2 (MLP-2); and (b) are the TGA plots of MSNs@Au-LAC (ML), MSNs@Au-LAC@PDA-PPy-1 (MLP-1), and MSNs@Au-LAC@PDA-PPy-2 (MLP-2). Figure 6 These are CLSM images, including (a, b, c) dark-field, bright-field, and overlay images of MSNs@Au-LAC@PDA-PPy-1 and (d, e, f) MSNs@Au-LAC@PDA-PPy-2. Figure 7 These are electrochemical characterization diagrams of the materials, including (a) CV curves of GCE, MSNs@Au / GCE, MSNs@Au-LAC / GCE, MSNs@Au-LAC@PDA-PPy-1 / GCE, and MSNs@Au-LAC@PDA-PPy-2 / GCE; and (b) EIS curves of the modified electrodes mentioned above. Figure 8 This is a comparison chart of the feasibility verification and material properties of BPA testing, where (a) are the DPV curves of MSNs@Au-LAC@PDA-PPy-2 / GCE in solutions without and containing 40 µM BPA; and (b) are the DPV curves of GCE, MSNs@Au / GCE, MSNs@Au-LAC / GCE, MSNs@Au-LAC@PDA-PPy-1 / GCE, and MSNs@Au-LAC@PDA-PPy-2 / GCE in solutions containing 40 µM BPA. Figure 9 The graph shows the relationship between DPV curves and response values ​​at different pH values, where (a) DPV curves of MSNs@Au-LAC@PDA-PPy-2 / GCE in PBS containing 40 µM BPA at different pH values; (b) the relationship between peak current and pH; and (c) the relationship between peak potential and pH. Figure 10The graph shows the relationship between CV scan rate and peak response value, where (a) CV curves of MSNs@Au-LAC@PDA-PPy-2 / GCE at different scan rates in PBS containing 40 µM BPA; (b) the relationship between peak current and scan rate; and (c) the relationship between peak potential and the natural logarithm of scan rate. Figure 11 This is a schematic diagram of the electrochemical oxidation principle of bisphenol A; Figure 12 This is a condition optimization diagram, in which (a) the laccase impregnation time is optimized; (b) the laccase concentration is optimized; (c) the amount of material modification is optimized; (d) the enrichment potential is optimized; and (e) the enrichment time is optimized. Figure 13 This is a standard curve diagram of the BPA electrochemical sensor, where (a) the DPV curves of the MSNs@Au-LAC@PDA-PPy-2 / GCE sensor for different concentrations of BPA; and (b) the linear relationship between the oxidation peak current and the BPA concentration.

[0022] Figure 14 The stability and selectivity assessment of MSNs@Au-LAC@PDA-PPy-2 / GCE includes (a) reproducibility of the sensing electrode material; (b) stability of the sensing electrode material; (c) anti-interference capability of the sensing electrode material; and (d) selectivity of the sensing electrode material. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] The embodiments of the present invention are as follows: 1. Material preparation The following materials were prepared: a substrate material MSNs@Au loaded with laccase; a material MSNs@Au-LAC@PDA-PPy-1 in which laccase is encapsulated inside MSNs@Au; and a material MSNs@Au-LAC@PDA-PPy-2 in which laccase is simultaneously distributed inside both MSNs@Au and PDA-PPy polymers.

[0025] 1.1) Preparation of MSNs@Au Using the template method, at 35 °C, 55 mL of deionized water was added to a round-bottom flask, followed by the addition of 160 mg of CTAB and stirring. Once the solution became clear, 26 mL of anhydrous ethanol was added. After 5 min, 8 mL of colloidal gold was added and stirred, followed by 1 mL of ammonia. Then, 1 mL of TEOS was added dropwise to the mixture to allow for complete reaction. After 3 h of reaction, the precipitate was collected by centrifugation and dried overnight in a 60 °C oven. The material MSNs@Au was obtained by calcination in a muffle furnace at 550 °C for 6 h.

[0026] 1.2) Preparation of MSNs@Au-LAC@PDA-PPy-1 To prepare the laccase solution, 30 mg of laccase was dissolved in 10 mL of acetate-sodium acetate buffer (pH=4.5) and sonicated for 10 min. Then, 25 mg of MSNs@Au was added to the laccase solution, and sonication was continued for another 10 min to obtain a mixture. The mixture was stirred and soaked at 4 °C for 24 h. The mixture was then washed by centrifugation with acetate-sodium acetate buffer (pH=4.5).

[0027] Then, 0.05 g of dopamine hydrochloride, 14 μL of pyrrole, and 10 mL of Tris solution were added to the above-mentioned centrifuged and washed solid mixture, and the mixture was sonicated for 10 min to obtain a homogeneous solution. Next, at room temperature, 1.05 g of (NH4)2S2O8 was dissolved in 10 mL of Tris (pH=8.5) solution to prepare an initiator, and the initiator was added to the above mixture. After stirring at 4 °C for 24 h, the mixture was centrifuged and washed with water, and the preliminary sample product was collected.

[0028] Finally, the preliminary sample product was freeze-dried for 48 h to obtain the final material MSNs@Au-LAC@PDA-PPy-1.

[0029] 1.3) Preparation of MSNs@Au-LAC@PDA-PPy-2 for comparison: The synthesis method of MSNs@Au-LAC@PDA-PPy-2 is similar to that of MSNs@Au-LAC@PDA-PPy-1. The main difference is that after laccase is soaked with MSNs@Au for 24 hours, centrifugation and washing with acetate-sodium acetate buffer are not performed. Subsequent steps are carried out directly to finally synthesize MSNs@Au-LAC@PDA-PPy-2.

[0030] 1.4) Preparation of modified electrodes MSNs@Au, MSNs@Au-LAC, MSNs@Au-LAC@PDA-PPy-1, and MSNs@Au-LAC@PDA-PPy-2 were each processed as follows to obtain their respective GCE electrodes: Weigh 2 mg of electrode material, add 2 mL of deionized water and sonicate to form a homogeneous mixture.

[0031] Using a pipette, 6 µL of the homogeneous mixture was added dropwise to the cleaned GCE surface and allowed to air dry at room temperature. After 12 h, the modified electrode was formed.

[0032] The preparation method for the required modified electrode is similar.

[0033] 2. Testing and Characterization: 2.1) Characterization of material structure and morphology The morphology and structure of the MSNs@Au-LAC@PDA-PPy composite material were systematically characterized using SEM and TEM. SEM images ( Figure 2 The image (ac) shows the surface characteristics of different composite materials. Among them, MSNs@Au-LAC ( Figure 2 a) Relatively smooth. Figure 2 b and Figure 2 c represents SEM images of MSNs@Au-LAC@PDA-PPy-1 and MSNs@Au-LAC@PDA-PPy-2, respectively. Both composite materials exhibit distinct chain-like structures around the particles, which is attributed to the formation of the polydopamine-polypyrrole (PDA-PPy) encapsulation layer. TEM images ( Figure 2 The df) further reveals the internal structure of the composite material. Figure 2 e and Figure 2 f are TEM images of MSNs@Au-LAC@PDA-PPy-1 and MSNs@Au-LAC@PDA-PPy-2, respectively, showing that the PDA-PPy chain structure encapsulates the MSNs@Au-LAC particles, forming an encapsulation layer.

[0034] Figure 3 The HRTEM images of MSNs@Au-LAC, MSNs@Au-LAC@PDA-PPy-1, and MSNs@Au-LAC@PDA-PPy-2 are shown. Figure 3 ac) and element distribution map ( Figure 3 di). Used to analyze laccase loading. In MSNs@Au-LAC ( Figure 3 In the (a, d, g) HRTEM images, the mesoporous structure and rough surface of the material are shown. Cu elemental distribution map ( Figure 3g) showed that laccase was evenly distributed on the surface of the spheres, but the loading was low. For MSNs@Au-LAC@PDA-PPy-1 ( Figure 3 (b, e, h), HRTEM images show that a polymer layer has formed on the material surface. Elemental overlay diagram ( Figure 3 e) and Cu element distribution map ( Figure 3 h) shows that Cu is mainly distributed around the mesoporous spheres, which may be due to the coating effect of the polymer layer, allowing more laccase to be immobilized on the spheres. MSNs@Au-LAC@PDA-PPy-2 (prepared under unwashed conditions) Figure 3 In the (c, f, i) HRTEM images, the presence of a polymer layer is shown. Elemental overlay diagram ( Figure 3 f) and Cu element distribution map ( Figure 3 i) indicates that Cu is distributed throughout the entire interface (including the polymer layer and the interior of the spheres), and the distribution range is wider, suggesting that the loading of laccase increases with the introduction of the polymer layer and the application of unwashed conditions.

[0035] like Figure 4 As shown, the C, N, and O elemental distributions were characterized for MSNs@Au-LAC, MSNs@Au-LAC@PDA-PPy-1, and MSNs@Au-LAC@PDA-PPy-2. The elemental distribution in MSNs@Au-LAC (…) Figure 4 In MSNs@Au material, C and N elements derived from laccase are uniformly distributed on the surface, confirming that laccase is uniformly loaded on the MSNs@Au material surface. This is compared to MSNs@Au-LAC@PDA-PPy (ac). Figure 4 di) and MSNs@Au-LAC ( Figure 4 ac), Figure 4 The distribution range of C and N elements in (di) expanded and their intensity increased, confirming that PDA-PPy has been successfully coated onto the MSNs@Au-LAC surface. Further comparison... Figure 4 (df) and Figure 4 (gi) discovered that MSNs@Au-LAC@PDA-PPy-2 ( Figure 4 The uniformity of C and N element distribution in MSNs@Au-LAC@PDA-PPy-2 is more prominent, which directly reflects the better uniformity of polymer coating and the more ideal encapsulation effect. This indicates that in the MSNs@Au-LAC@PDA-PPy-2 system, the polymer layer coats the substrate more uniformly and densely.

[0036] Figure 5 a shows the FTIR spectra of different samples. For MSNs@Au(M), at 1081 cm⁻¹ -1A distinct Si-O-Si tensile vibration peak was observed at 806 cm⁻¹, indicating the presence of a silica framework. Furthermore, a peak at 806 cm⁻¹... -1 The Si-OH bending vibration peak at 3427 cm⁻¹ indicates the presence of hydroxyl groups on the material surface, which is beneficial for subsequent enzyme immobilization. -1 At this point, the OH stretching vibration peak may originate from the absorption of water or hydroxyl groups. For MSNs@Au-LAC (ML), 1627 cm⁻¹ -1 The peak value at 1467 cm⁻¹ is enhanced, belonging to the C=O vibration peak (protein amide I band) and the peak value at 1467 cm⁻¹. -1 The presence of NH vibration peaks (protein amide II band) indicates that laccase (LAC) has been successfully immobilized onto the MSNs@Au surface. For MSNs@Au@PDA-PPy (MLP), a new peak at 1720 cm⁻¹ was observed. -1 The C=C skeletal vibration peak (from polypyrrole) at 3215 cm⁻¹ -1 The NH vibration peak at the location also indicates that polydopamine (PDA) and polypyrrole (PPy) were successfully coated on the material surface. For MSNs@Au-LAC@PDA-PPy (MLP-1 & MLP-2), the increased C=O and NH vibration peaks (laccase), C=C and CN vibration peaks (from polypyrrole), and the enlarged OH stretching vibration peak (from PDA) indicate that the final composite material was successfully synthesized.

[0037] Figure 5 b shows the TGA curves for different samples. The three samples exhibited similar weight loss curves. The weight loss ratios differed among the three samples due to the laccase loading and the coating of the conductive polymer. In the first stage, from 0 °C to approximately 200 °C, all materials showed slight weight loss, which is generally related to the evaporation of adsorbed or surface-bound water. MSNs@Au-LAC, without an additional polymer layer, showed the best thermal stability after the loaded laccase was completely thermally decomposed. In contrast, MSNs@Au-LAC@PDA-PPy-1 and MSNs@Au-LAC@PDA-PPy-2 showed poorer thermal stability due to the thermal decomposition of laccase, polydopamine, and polypyrrole. MSNs@Au-LAC@PDA-PPy-2, in particular, showed a larger weight loss, which may indicate that it immobilized more laccase.

[0038] like Figure 6As shown, the CLSM images reveal the microstructure and component distribution of the sample. Fluorescence signals indicate a relatively uniform distribution of the target components within the material, and the overlapping areas in the images from different channels suggest high co-localization of the components. This result demonstrates that the fluorescent label or dye has been successfully introduced and well dispersed within the material matrix. Furthermore, the uniformity of fluorescence intensity and signal stability further confirm the integrity and dispersion of the sample structure, laying a reliable foundation for subsequent functional testing. Notably, compared to MSNs@Au-LAC@PDA-PPy-1, MSNs@Au-LAC@PDA-PPy-2, lacking a washing step, may contain more unwashed laccase, resulting in a higher enzyme content immobilized in the composite material. Overall, the CLSM analysis results provide intuitive and effective evidence for the immobilization and spatial distribution of key components in the sample, validating the uniformity and reliability of the prepared material at the microscopic level.

[0039] 2.2) Electrochemical characterization of electrodes with different modifications 2.2.1) Electrochemical characterization of different modified electrodes in potassium ferrocyanide solution To characterize the electrochemical performance of different modified electrodes, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used.

[0040] In a solution containing 5 mM [Fe(CN)6] 3- / 4- CV measurements were performed in 0.1 M PBS solution (pH=7.0) containing 0.1 M KCl, with a voltage scan range of -0.2 to 0.6 V; in 0.1 M PBS solution containing BPA, the voltage range for CV measurements was 0.2 to 0.8 V, with a scan rate of 50 mV / s. Furthermore, differential pulse voltammetry (DPV) was used to measure the voltage in BPA solution from 0.2 to 0.7 V with an amplitude of 50 mV. In the electrochemical tests, a three-electrode system was used, with the electrode prepared according to this invention as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum sheet electrode as the counter electrode.

[0041] This experiment used CV and EIS measurements to explore [Fe(CN)6]. 3- / 4- Performance of electrodes with different modifications in solution. Figure 7 a represents the CV curves of various materials in the redox probe solution, with different modified electrodes in [Fe(CN)6]. 3- / 4-The oxidation peak current intensity in the solution shows the following order: MSNs@Au > MSNs@Au-LAC@PDA-PPy-1 > MSNs@Au-LAC@PDA-PPy-2 > MSNs@Au-LAC > GCE. Among them, the MSNs@Au electrode (89.5 µA) exhibits the strongest oxidation peak current, mainly attributed to the excellent conductivity and electrocatalytic activity of gold nanoparticles. Subsequently, the immobilization of laccase (LAC) and the encapsulation of polydopamine-polypyrrole (PDA-PPy) change the microenvironment of the electrode surface to some extent: on the one hand, as a protein, laccase has certain insulating properties, which may increase the mass transfer and charge transfer resistance; on the other hand, the conductivity of the copolymer PDA-PPy compensates for the non-conductive effect brought by laccase to some extent and provides additional electrochemically active sites. After the two balance each other, MSNs@Au-LAC@PDA-PPy-1 (81.1 µA) and MSNs@Au-LAC@PDA-PPy-2 (72.0 µA) still maintain relatively high oxidation peak currents, but are slightly lower than the pure MSNs@Au electrode.

[0042] To further study the electron transfer characteristics of different modified electrodes, the Nyquist plots of each electrode were characterized by EIS ( Figure 7 b). The results show that the semicircle radius (R ct ) increases in turn as: MSNs@Au < MSNs@Au-LAC@PDA-PPy-1 < MSNs@Au-LAC@PDA-PPy-2 < MSNs@Au-LAC < GCE. The MSNs@Au electrode has the smallest semicircle, indicating that its charge transfer impedance is the lowest, which is consistent with its high oxidation peak current shown in CV. After introducing laccase, due to the insulation of the protein, the charge transfer resistance increases, but the conductive properties of the copolymer PDA-PPy offset this adverse effect to some extent, making the R ct of MSNs@Au-LAC@PDA-PPy-1 and MSNs@Au-LAC@PDA-PPy-2 still significantly lower than that of the unmodified electrode and MSNs@Au-LAC with only encapsulated laccase. In summary, the results of CV and EIS both show that the synergistic effect of gold nanoparticles, laccase and PDA-PPy can achieve a balance between conductivity and biocatalysis, providing a feasible optimization strategy for the high performance of electrochemical sensors.

[0043] 2.2.2) Electrochemical behavior characterization of bisphenol A on different modified electrodes Figure 8This figure shows the DPV curves of MSNs@Au-LAC@PDA-PPy-2 / GCE in 0.1 M PBS (pH=7) solution, in the absence of BPA and in the presence of 40 μM BPA. The black curve represents the blank PBS solution, where almost no obvious oxidation peak signal was observed, indicating that no significant oxidation reaction occurred on the electrode surface in the absence of BPA. In contrast, the red curve corresponds to the solution containing 40 μM BPA, where a significant oxidation peak appeared at approximately 0.55 V, indicating that MSNs@Au-LAC@PDA-PPy-2 / GCE exhibits excellent electrochemical response to BPA. This result confirms the feasibility of MSNs@Au-LAC@PDA-PPy-2 / GCE modified electrodes in the electrochemical detection of BPA.

[0044] Figure 8 b shows the DPV curves of different modified electrodes detecting 40 μM BPA in 0.1 M PBS (pH=7). The experimental results show that MSNs@Au-LAC@PDA-PPy-2 / GCE exhibits the highest oxidation peak current (4.97 µA), significantly outperforming other modified electrodes (such as GCE, MSNs@Au / GCE, MSNs@Au-LAC / GCE, MSNs@Au-LAC@PDA-PPy-1 / GCE, etc.). This phenomenon further confirms the superior performance of the MSNs@Au-LAC@PDA-PPy-2 / GCE modified layer. Specifically, the three-dimensional porous structure of MSNs effectively promotes the diffusion and adsorption of BPA molecules, while the high conductivity and catalytic activity of Au NPs significantly accelerate the oxidation kinetics of BPA. Furthermore, the introduction of laccase (LAC) further enhances the electrode sensitivity through its highly efficient catalytic activity and selectivity for phenolic compounds. Meanwhile, the high conductivity and adsorption capacity of polydopamine-polypyrrole (PDA-PPy) significantly improved the stability and reaction efficiency of the electrode. It is possible that MSNs@Au-LAC@PDA-PPy-2 / GCE immobilized more laccase with higher activity, thereby enhancing its catalytic oxidation of BPA.

[0045] 2.3) Laccase Immobilization and Enzyme Activity Determination in MSNs@Au-LAC@PDA-PPy (1) Laccase fixation test Quantitative analysis of laccase (LAC) fixation was performed using the Bradford protein assay. First, Bradford stock solution was prepared: 100 mg of Coomassie Brilliant Blue G-250 dye was dissolved in 50 mL of 95% ethanol, and 100 mL of 85% phosphate was slowly added. The mixture was stirred until the dye was completely dissolved, and then diluted to 1 L of deionized water. The solution was stored at 4 °C in the dark. Subsequently, LAC standard solutions with gradient concentrations (0 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL) were prepared using acetate-sodium acetate buffer (pH 4.5). During the experiment, 0.3 mL of the sample was mixed with 3.0 mL of Bradford stock solution, and after development at 25 °C in the dark for 10 min, the absorbance at 595 nm was measured using a UV-Vis spectrophotometer. Each sample was measured in triplicate, and the average value was taken. A standard curve was plotted based on the known LAC concentration and absorbance to establish their functional relationship. Under the same conditions, the absorbance of the enzyme solution before and after LAC fixation was measured using Bradford stock solution, and the amount of LAC fixed was calculated using the formula.

[0046] L=((C0-C1)×V) / m In the formula, C0 and C1 are the initial concentration of the laccase solution before immobilization and the residual laccase concentration in the supernatant after immobilization, respectively (unit: mg / mL), V is the volume of the laccase solution (mL), and m is the mass of the immobilized carrier (mg).

[0047] (2) Assay of laccase activity Laccase activity was determined by the ABTS oxidation colorimetric method. The experimental procedure was as follows: 0.1 mL of free laccase solution or an equal volume of immobilized laccase sample was mixed with 1 mL of ABTS substrate solution (1 mM, dissolved in pH 4.5 acetate-sodium acetate buffer) in a 2 mL centrifuge tube. The mixture was incubated at 35 °C for 5 min, and then immediately terminated by incubating on ice. After centrifugation (8000 rpm, 2 min), the supernatant was collected and transferred to a cuvette. The absorbance change (ΔOD) at 420 nm was measured using a UV-Vis spectrophotometer. 420 Enzyme activity (A, U / g) is calculated using the formula: A=(∆OD 420 ×V t ×10 6 ) / (ε×t×m) In the formula, V t The total volume of the reaction system is ε (mL), and ε is the molar extinction coefficient of ABTS (36000 M). -1 ·cm -1t represents the reaction time (min), and m represents the mass of laccase (mg). The activity of immobilized laccase was calculated by comparing the residual enzyme activity in the supernatant before and after immobilization to determine the loading efficiency. All experiments were performed in triplicate, and the data were averaged to ensure reproducibility.

[0048] The test results are shown in Tables 1 and 2. Based on the experimental data in Tables 1 and 2, the MSNs@Au-LAC@PDA-PPy-2 material exhibits superior performance compared to MSNs@Au-LAC@PDA-PPy-1 in both laccase immobilization amount and enzyme activity. Specifically, the laccase immobilization amount of MSNs@Au-LAC@PDA-PPy-2 is 77.78 mg / g, with an immobilization rate of 90.74%, significantly higher than that of MSNs@Au-LAC@PDA-PPy-1 (69.84 mg / g and 81.48%). Furthermore, the laccase activity of MSNs@Au-LAC@PDA-PPy-2 is 63.56 U / g, with a relative activity of 78%, also higher than that of MSNs@Au-LAC@PDA-PPy-1 (57.85 U / g and 71%). These results indicate that the MSNs@Au-LAC@PDA-PPy-2 material outperforms MSNs@Au-LAC@PDA-PPy-1 in both laccase immobilization efficiency and catalytic activity. This difference may be attributed to the following reasons: First, the MSNs@Au-LAC@PDA-PPy-2 material may possess a more optimized surface structure or chemical properties, allowing for more effective immobilization of laccase on the support, thereby increasing the immobilization amount and rate. Second, the laccase in the MSNs@Au-LAC@PDA-PPy-2 material may retain higher activity, possibly due to more effective protection of the enzyme structure during immobilization, or a more favorable microenvironment for laccase from the support material.

[0049] Table 1. Immobilization amount of laccase in MSNs@Au-LAC@PDA-PPy Based on previous electrochemical data, MSNs@Au-LAC@PDA-PPy-2 / GCE exhibited higher oxidation peak current and superior electrochemical performance in BPA detection, which is closely related to the improved laccase immobilization and activity. The efficient immobilization and maintained activity of laccase significantly enhanced the electrode's catalytic oxidation ability for BPA, thereby improving the sensitivity and stability of detection. Therefore, the superior performance of MSNs@Au-LAC@PDA-PPy-2 / GCE in BPA electrochemical detection is due not only to its three-dimensional pore structure and the high conductivity of Au NPs, but also to the efficient immobilization and high activity of laccase. These results further confirm the potential of MSNs@Au-LAC@PDA-PPy-2 / GCE as a highly efficient electrode material for BPA detection.

[0050] Table 2. Laccase activity in MSNs@Au-LAC@PDA-PPy 2.4) Effect of electrolyte pH on BPA detection Figure 9 The electrochemical behavior of MSNs@Au-LAC@PDA-PPy-2 / GCE in 0.1 M PBS solutions at different pH values ​​for detecting 40 μM BPA is demonstrated. Figure 9 As shown in Figure a, the oxidation peak current of BPA increases accordingly as the solution pH gradually rises from acidic to neutral, indicating that protons play an important role in the reaction between the electrode surface and BPA molecules. However, when the pH further increases, the oxidation peak current begins to decrease, possibly because the negatively charged electrode surface electrostatically repels the deprotonated BPA anions, inhibiting the adsorption and oxidation of BPA on the electrode surface. Experimental results show that the oxidation peak current reaches its maximum at pH 7.0. At this pH, the solution pH is lower than the pKa of BPA (9.73), and BPA mainly exists in molecular form, making it easier to adsorb on the electrode surface and achieve the best electrochemical response. Figure 9 As shown in b, the oxidation peak current of BPA shows a trend of first increasing and then decreasing with pH value, reaching its maximum value near pH 7.0, which further verifies that the best detection sensitivity can be obtained under this condition.

[0051] at the same time, Figure 9 c shows E pa The relationship with pH value. As pH value increases, E... pa Gradually shifting towards the negative; within the pH range of 5.0-9.0, E pa It shows a good linear relationship with pH value (E pa =-0.064pH+0.972, R 2=0.999). Among them, each unit change in pH value resulted in a peak potential shift of approximately 64 mV, close to the theoretical Nernst slope (59 mV / pH), indicating that the oxidation reaction of BPA on the MSNs@Au-LAC@PDA-PPy-2 / GCE electrode also involves an equal amount of proton and electron transfer processes.

[0052] 2.5) Effect of electrode scan rate on BPA detection Figure 10 The electrochemical behavior of MSNs@Au-LAC@PDA-PPy-2 / GCE on 40 μM BPA at different scan rates was demonstrated. Figure 10 a shows the CV curves in 0.1 M PBS solution containing 40 μM BPA, with scan rates ranging from 50 mV / s to 250 mV / s. As the scan rate increases, both the oxidation peak and oxidation peak potential of the CV curve gradually increase. Figure 10 b shows the relationship between the BPA oxidation peak current and the scan rate after identifying 40 μM BPA. It can be seen that the BPA oxidation peak current gradually increases with increasing scan rate and exhibits a good linear correlation with the scan rate in the range of 50 mV / s to 250 mV / s, indicating that the oxidation process of BPA on the MSNs@Au-LAC@PDA-PPy-2 / GCE surface is a typical adsorption-controlled process.

[0053] also, Figure 10 c shows the E of BPA pa The relationship between the peak potential and the logarithm of the scan rate is shown. Experimental results indicate that the oxidation peak potential shifts positively with increasing scan rate, and the linear regression equation is E0. pa =0.03199lnv+0.4720 (R 2 =0.997). For the adsorption-controlled, completely irreversible electrode process, the number of electrons transferred during the BPA oxidation process was found to be approximately 2, further verifying that the BPA oxidation reaction involves the transfer of two electrons. According to literature reports, the BPA oxidation reaction involves the redox reaction of two phenolic hydroxyl groups on the benzene ring, occurring in an irreversible process at a positive potential, where each phenolic hydroxyl group involves a reaction of two electrons and two protons. The electrochemical catalytic process of this reaction and its corresponding reaction equation are as follows: Figure 11 As shown.

[0054] 2.6) Optimization of electrode material preparation and detection conditions To obtain a modified electrode with optimal sensitivity and selectivity in BPA detection, this invention systematically investigated the effects of several key parameters on the electrochemical response during the preparation and testing of the synthetic material MSNs@Au-LAC@PDA-PPy-2 using the DPV method. These parameters include: the laccase impregnation time in the MSNs@Au material solution, the amount of laccase used (i.e., the laccase solution concentration), the drop volume of the material dispersion, and the enrichment potential and enrichment time in electrochemical detection.

[0055] The immersion times were set to 12, 24, 36, 48, and 60 hours. Materials from each group were then applied to the GCE surface, and DPV was used to detect BPA to assess performance differences. Subsequently, the laccase addition amount was optimized. Laccase addition amounts of 10, 20, 30, 40, and 50 mg were set, and materials from each group were coated onto the GCE surface. The oxidation current response to BPA was detected using the DPV method to compare the effect of different laccase addition amounts on sensor performance.

[0056] Relevant experimental results are as follows Figure 12 As shown in (ae).

[0057] like Figure 12 As shown in a, 24 h was determined to be the optimal immersion time, and this was used in subsequent experiments.

[0058] The effect of laccase dosage on the response signal of the modified electrode was further investigated while keeping the impregnation time constant. Figure 12 As shown in b, 30 mg was selected as the optimal condition for subsequent experiments.

[0059] Next, the effect of the material dispersion droplet volume on the electrochemical response of the modified electrode was investigated. For example... Figure 12 As shown in Figure c, 6 µL was selected as the optimal volume for the dispersion droplet coating.

[0060] Figure 12 Figure d shows the change in the sensor's response current under different enrichment potentials. Subsequent experiments selected 0.2 V as the enrichment potential to optimize the detection performance.

[0061] like Figure 12 As shown in Figure e, based on the enrichment time corresponding to the peak value in the figure, 120 s is the optimal enrichment time. This enrichment time is determined as the optimal value and used in subsequent experiments.

[0062] 2.7) Establishment of a standard curve for bisphenol A detection using the MSNs@Au-LAC@PDA-PPy-2 sensor The electrochemical response of the MSNs@Au-LAC@PDA-PPy-2 sensor at different concentrations of BPA was investigated using DPV. Figure 13As shown in Figure a, the oxidation peak current of the sensor increases significantly as the BPA concentration increases from 0.005 μM to 40 μM. In the low concentration range (0.005–1 μM), the current response rises rapidly. When the BPA concentration increases to the range of 1 μM to 40 μM, the current response continues to increase, but the slope decreases. This may be due to changes in the oxidation reaction kinetics of BPA at high concentrations or the occupancy of active sites on the electrode surface, leading to a limited reaction rate.

[0063] Figure 13 b shows the linear relationship between the oxidation peak current and BPA concentration. In the two concentration ranges of 0.005–1 μM and 1–40 μM, the oxidation peak current and BPA concentration exhibit a good linear relationship. The specific linear regression equations are as follows: In the 0.005–1 μM range: I p =0.5468C+0.5411(R) 2 =0.998), in the range of 1-40 μM: I p =0.09343C+0.9214(R 2 =0.996), a high correlation coefficient value further verifies the reliability of the linear relationship. Based on the above linear relationship, the detection limit (LOD) of the sensor for BPA was calculated to be 1.6 nM (S / N=3), indicating that the sensor has high sensitivity and low detection limit. 2.7) Reproducibility, stability, anti-interference ability, and selectivity of MSNs@Au-LAC@PDA-PPy-2 for detecting BPA. To evaluate the performance of the MSNs@Au-LAC@PDA-PPy-2 sensing electrode material, we conducted a detailed study on its reproducibility, stability, anti-interference, and selectivity. First, by measuring the current response of five electrodes prepared using the same method to 40 μM BPA under optimal conditions, the results showed that the RSD of these five electrodes was 0.67%, indicating that the sensor possesses excellent reproducibility. Figure 14 a). The stability of the sensor was further investigated. The prepared electrodes were stored in a 4 ℃ refrigerator for 9 days, and the recognition ability for the same concentration of BPA was measured every 2 days. The results showed that the response values ​​of MSNs@Au-LAC@PDA-PPy-2, MSNs@Au-LAC@PDA-PPy-1, MSNs@Au-LAC, and MSNs@Au decreased by 8.32%, 10.96%, 15.53%, and 9.42% respectively over 9 days, indicating that polymer encapsulation improved the stability of the material in detecting BPA. Figure 14 b).

[0064] To evaluate the sensor's anti-interference capability, we added 100 times the concentration of Na to a solution containing 40 μM BPA. + Ca2+ Mg 2+ K + and CO3 2- DPV detection was performed using 50-fold concentrations of p-nitrophenol (CL), sucrose, and glucose. The results showed that the response current variation of these interfering substances to BPA was within 3% of that under interference-free conditions, indicating that the sensor has good anti-interference capabilities. Figure 14 c). Furthermore, we investigated the sensor's selectivity. By comparing the DPV responses in solutions containing various interfering substances (without BPA) and solutions containing BPA, we found that the sensor exhibits good selectivity for BPA. Within the range of 0.2 V to 0.7 V, only CL and BPA showed peaks, and the peak positions differed significantly, having no impact on the interpretation of the BPA response value. Figure 14 d). In summary, the MSNs@Au-LAC@PDA-PPy-2 sensor electrode material exhibits excellent performance in reproducibility, stability, anti-interference, and selectivity, and has promising application prospects.

[0065] Through the aforementioned experiments and tests, this invention successfully constructed an electrochemical sensor (MSNs@Au-LAC@PDA-PPy-2 / GCE) by synergistic modification of MSNs@Au nanomaterials with laccase (LAC), polydopamine (PDA), and polypyrrole (PPy), achieving highly sensitive and selective detection of BPA. Its morphology, composition, and interfacial electron transfer characteristics were verified using various techniques. The sensor exhibits good reproducibility, stability, and anti-interference capabilities, with actual sample spike recoveries ranging from 96.08% to 111.53% and RSDs from 0.05% to 7.90%, highly consistent with HPLC results (recovery deviation <15%). The MSNs@Au-LAC@PDA-PPy-2 sensor is easy to operate, has a rapid response, and is low in cost, providing an efficient solution for the on-site detection of trace BPA in food and environmental samples, and possesses significant practical application value.

[0066] The above specific embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

[0067] The above description is only a preferred embodiment of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the claims of this patent application are included in the scope of this patent application.

Claims

1. A composite material of MSNs@Au-LAC@PDA-PPy, characterized in that: It is mainly prepared by combining MSNs@Au, laccase LAC, polydopamine PDA and polypyrrole PPy.

2. A method for preparing the MSNs@Au-LAC@PDA-PPy composite material according to claim 1, characterized in that: The preparation process is as follows: S1. Prepare laccase solution by dissolving laccase in acetate-sodium acetate buffer and sonicating it to form laccase solution; S2. Add MSNs@Au to the laccase solution, continue ultrasonic treatment to obtain a mixture, and soak at 4 ℃ for a certain time; S3. Add dopamine hydrochloride, pyrrole and Tris solution to the above mixture, and sonicate to obtain a homogeneous solution; S4. At room temperature, add the initiator to the homogeneous solution, stir at a specific temperature, and then centrifuge and wash with water to collect the preliminary sample product. S5. The preliminary sample product is freeze-dried to obtain the final MSNs@Au-LAC@PDA-PPy composite material.

3. The preparation method of the MSNs@Au-LAC@PDA-PPy composite material according to claim 2, characterized in that: In S1: the acetate-sodium acetate buffer solution has deionized water as the solvent and acetic acid and sodium acetate as the solutes, with a pH of 4.5; the laccase solution has a mass-volume ratio of (10~50) mg to 10 mL of laccase and acetate-sodium acetate buffer solution.

4. The preparation method of the MSNs@Au-LAC@PDA-PPy composite material according to claim 2, characterized in that: In S2, the mass ratio of MSNs@Au to laccase in the laccase solution is 25:(10~50), and the mixture is stirred and soaked at 4 °C for 6~48 h.

5. The method for preparing the MSNs@Au-LAC@PDA-PPy composite material according to claim 2, characterized in that: In S3, the pH of the Tris solution is 8.5, and the mass-to-volume ratio of dopamine hydrochloride to pyrrole is 0.05 g:14 µL.

6. The method for preparing the MSNs@Au-LAC@PDA-PPy composite material according to claim 2, characterized in that: In S4, an initiator with pH=8.5 was prepared by dissolving (NH4)2S2O8 in Tris solution. The molar concentration of (NH4)2S2O8 was 0.5M. After adding the initiator to the homogeneous solution, the mixture was stirred at 4 °C for 24 h.

7. An MSNs@Au-LAC@PDA-PPy-2 / GCE electrode, characterized in that: It is made using the MSNs@Au-LAC@PDA-PPy composite material of claim 1 or the MSNs@Au-LAC@PDA-PPy composite material obtained by any of the preparation methods of claims 2-6.

8. The application of the MSNs@Au-LAC@PDA-PPy composite material of claim 1 or the MSNs@Au-LAC@PDA-PPy-2 / GCE electrode of claim 6, characterized in that: Application in rapid electrochemical detection of bisphenol A.

9. A method for detecting bisphenol A using the MSNs@Au-LAC@PDA-PPy composite material of claim 1 or the MSNs@Au-LAC@PDA-PPy-2 / GCE electrode of claim 6, characterized in that: A three-electrode working system was adopted, with the MSNs@Au-LAC@PDA-PPy-2 / GCE electrode as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum sheet electrode as the counter electrode for detection. The DPV electrochemical method was used, with an enrichment potential of 0.2 V and an enrichment time of 120 s. A standard curve was pre-fitted to match the concentration of bisphenol A and the peak current, and the concentration of the bisphenol A to be tested was obtained using the standard curve.

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