Bisphenol a detection method based on cysteine-induced biocoo h surface polarization effect in situ

Abstract

The application provides a bisphenol A detection method based on in-situ cysteine-induced BiOCOOH surface polarization effect, and relates to the technical field of detection. The detection method is based on photoelectrochemistry, and establishes a novel PEC sensing platform which is environment-friendly, label-free, simple to operate and high in performance. The detection limit of the detection method for bisphenol A is as low as 1.4*10 ‑3 muM, the linear detection range is 0.01 muM-100 muM, and the demand for trace detection of bisphenol A can be met. Therefore, high-sensitivity and high-selectivity detection of bisphenol A is realized. A new idea is provided for novel application of PEC sensing.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and in particular to a method for detecting bisphenol A based on the in-situ induced surface polarization effect of BiOCOOH by cysteine. Background Technology

[0002] Bisphenol A (BPA), a widely used industrial raw material, is commonly found in plastic products and food packaging. It is a typical endocrine disruptor that can migrate into the environment and food chain, posing a potential threat to human health and potentially causing metabolic disorders, reproductive abnormalities, and even cancer. Therefore, establishing efficient and sensitive BPA detection methods is crucial for ensuring food safety and public health.

[0003] Currently, conventional methods for BPA detection mainly include chromatography (such as high-performance liquid chromatography and gas chromatography-mass spectrometry) and fluorescence immunoassay. While these methods offer good specificity, they generally suffer from limitations such as expensive equipment, complex operation, cumbersome sample pretreatment, or reliance on labels, making it difficult to meet the needs of rapid, low-cost on-site screening. Photoelectrochemical (PEC) biosensing technology has attracted considerable attention due to its advantages of simple equipment, low background signal, and high sensitivity. However, existing PEC sensing strategies still face challenges: on the one hand, many methods rely on halogen-containing photoelectric materials (such as BiOCl and BiOBr), whose potential ecotoxicity cannot be ignored; on the other hand, traditional signal amplification mechanisms (such as constructing heterojunctions) often require complex biomolecular labeling or immobilization steps, increasing operational costs and complexity. Furthermore, developing novel, environmentally friendly, high-performance semiconductor materials and exploring their novel sensing mechanisms is of great significance for promoting the development of the PEC analysis field. Therefore, there is an urgent need to develop a novel PEC sensing platform based on environmentally friendly materials, label-free operation, simple operation, and high performance to achieve highly sensitive and selective detection of BPA.

[0004] Therefore, this invention is proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a bisphenol A detection method based on the in-situ cysteine-induced surface polarization effect of BiOCOOH. This detection method, based on photoelectrochemistry, establishes a novel environmentally friendly, label-free, easy-to-operate, and high-performance PEC sensing platform, achieving highly sensitive and selective detection of bisphenol A.

[0006] In order to achieve the objective of this invention, the following technical solution is adopted: This invention provides a method for detecting bisphenol A based on the in-situ induced surface polarization effect of cysteine ​​in BiOCOOH, comprising the following steps: S1. Synthesize nanoflower-like BiOCOOH and modify the surface of ITO electrode with the BiOCOOH to prepare BiOCOOH / ITO working electrode. At the same time, pretreat the bisphenol A aptamer and the probe partially complementary to the bisphenol A aptamer respectively. S2. The sample to be tested is incubated with the bisphenol A aptamer in a buffer solution for the first time. S3. Add a probe partially complementary to the bisphenol A aptamer to the reaction system of S2, and perform a second incubation. S4. Add Exonuclease I to the reaction system of S3 to carry out enzymatic digestion, then add cysteine ​​solution to the reaction system, mix well and then contact the BiOCOOH / ITO working electrode for reaction. S5. After the reaction is complete, wash the BiOCOOH / ITO working electrode. Under optimized conditions, detect the photocurrent signal of the BiOCOOH / ITO working electrode. Based on the change in the photocurrent signal, realize the quantitative detection of bisphenol A.

[0007] Furthermore, in step S4, the concentration of the cysteine ​​solution is 0.2 mM, and the reaction time with the BiOCOOH / ITO working electrode is 10 min.

[0008] Furthermore, in S2, the buffer solution is a mixture of 10 mM Tris-HCl, 100 mM NaCl, 1.0 mM EDTA, and 5.0 mM MgCl2, and the pH of the buffer solution is 8.0.

[0009] Furthermore, in S5, the optimized conditions are: using an Ag / AgCl reference electrode and Pt as the counter electrode; Use a buffer solution with a pH of 3.5; The applied voltage is 0 V, and the excitation light source wavelength is 370 nm-380 nm.

[0010] Furthermore, the buffer solution is Tris-HCl with a concentration of 0.1 mM.

[0011] Furthermore, in S1, the specific steps of the pretreatment include: heating the bisphenol A aptamer and the probe partially complementary to the bisphenol A aptamer at 95°C for 5 min, followed by natural cooling.

[0012] Furthermore, the reaction conditions for the first incubation are: incubation at 35℃-38℃ for 60 min-80 min.

[0013] Furthermore, the reaction conditions for the first incubation are: incubation at 37°C for 70 min.

[0014] Furthermore, the reaction conditions for the second incubation are: maintaining the same reaction temperature as the first incubation and continuing incubation for 50 min-70 min.

[0015] Furthermore, the incubation time is 60 minutes.

[0016] Furthermore, in step S4, the time for adding Exonuclease I for enzymatic digestion is 40 min.

[0017] Furthermore, in step S1, the specific steps for synthesizing nanoflower-like BiOCOOH are as follows: S101. Dissolve bismuth nitrate pentahydrate in N,N-dimethylformamide and stir until a homogeneous solution is formed. Then add deionized water and stir until a stable milky white suspension is formed. S102. Add citric acid solution to the suspension and continue stirring to obtain a mixed solution. Then, transfer the mixed solution to a high-pressure reactor lined with polytetrafluoroethylene and maintain it at 120°C for 12 hours to obtain a white product. Wash four times each with deionized water and anhydrous ethanol, and dry under vacuum at 80°C for 10 hours to obtain a dried product.

[0018] Furthermore, in step S1, the specific method for preparing the BiOCOOH / ITO working electrode is as follows: The BiOCOOH was ultrasonically dispersed in ultrapure water and then deposited onto pre-cleaned indium tin oxide (ITO) and dried at room temperature.

[0019] The present invention also provides a detection kit for implementing the above detection method, the kit comprising: (a) BiOCOOH / ITO working electrode; (b) Bisphenol A aptamer; (c) A nucleic acid probe partially complementary to the bisphenol A aptamer; (d) Exonuclease I; (e) Cysteine ​​solution; (f) Buffer solution.

[0020] Furthermore, the concentration of the bisphenol A aptamer is 4.0 μM; the concentration of the nucleic acid probe partially complementary to the bisphenol A aptamer is 4.0 μM; the concentration of the exonuclease I is 10 U / μL; and the concentration of the cysteine ​​solution is 0.2 mM.

[0021] Furthermore, the buffer solution is a mixture of 10 mM Tris-HCl, 100 mM NaCl, 1.0 mM EDTA, and 5.0 mM MgCl2, and the pH of the buffer solution is 8.0.

[0022] The present invention also provides the above-mentioned photoelectrochemical detection method for bisphenol A, or the application of the above-mentioned detection kit in the detection of bisphenol A in textiles.

[0023] The present invention has the following technical effects: (1) The method achieves high sensitivity and wide linear range for the detection of bisphenol A, with a detection limit as low as 1.4 × 10⁻⁶. -3 With a linear detection range of 0.01 μM to 100 μM, it can meet the needs for trace detection of bisphenol A.

[0024] (2) The detection method achieves excellent selectivity and reproducibility. Based on the binding strategy of specific recognition of aptamers and competition for enzyme cleavage, the sensor exhibits excellent anti-interference ability against a variety of common ions, proteins and antibiotics. The relative standard deviation (RSD) was only 0.62% when tested with 15 independent electrodes, indicating that the method has good reproducibility.

[0025] (3) The core photoelectric material BiOCOOH of this detection method is halogen-free and environmentally friendly. The detection process does not require any labeling or fixation of biomolecules, which simplifies the operation process, reduces costs, and facilitates high-throughput analysis.

[0026] (4) For the first time, the surface polarization effect (SPE) induced by cysteine ​​on the BiOCOOH surface was used as a signal amplification mechanism, providing a new idea for the novel application of PEC sensing. Attached Figure Description

[0027] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 Characterization of the prepared BiOCOOH, where A and B are SEM images of BiOCOOH; C is an XRD image of BiOCOOH; Figure 2The photocurrent test results of the BiOCOOH / ITO electrode with 0.1 mM Cys are as follows: A shows the photocurrent test results obtained by changing the applied voltage; B shows the photocurrent test results at different pH values; C shows the photocurrent test results obtained by changing the reaction time; D shows the photocurrent test results obtained by changing the excitation wavelength; E shows the photocurrent response of the BiOCOOH / ITO electrode under different Cys concentrations (0, 0.1, 0.25, 0.5, 1.0, 5.0, 10, 50, and 100 µM); F ... with different Cys concentrations (0, 0.1, 0.25, 0.5, 1.0, 5.0, 10, 50, and 100 µM). A linear relationship curve between I (i.e., the increase in photocurrent before and after the reaction with Cys) and the logarithm of the Cys concentration; Figure 3 : FT-IR and Ramman spectra of BiOCOOH / ITO electrodes, where A is the FT-IR spectrum of BiOCOOH, BiOCOOH / Cys and Cys; B is the Ramman spectrum of BiOCOOH, BiOCOOH / Cys and Cys. Figure 4 : UV-vis DRS and Tauc plots of the reaction system, where A is the UV-vis DRS of BiOCOOH and BiOCOOH / Cys; B is the Tauc plot of BiOCOOH and BiOCOOH / Cys; C is the linear sweep voltammetric curve of the conduction band (CB) of BiOCOOH and BiOCOOH / Cys; D is the linear sweep voltammetric curve of the valence band (VB) of BiOCOOH and BiOCOOH / Cys. Figure 5 XPS analysis was used to analyze the surface elemental composition and interactions between constituent elements of BiOCOOH. A is the overall XPS plot of BiOCOOH and BiOCOOH / Cys; B is the high-resolution spectrum of C1s, C is Bi 4f, and D is N 1s. Figure 6 The reason why Cys enhances the photocurrent of BiOCOOH, where A is the surface potential curve of BiOCOOH and B is the surface potential curve of BiOCOOH / Cys; Figure 7 A shows the photocurrent response of the BiOCOOH modified electrode to different components (including Cys and reactions with dNMP followed by Cys) (left panel) and biological systems containing / without BPA (right panel); B shows the gel electrophoresis diagrams of the products generated in different biological reaction systems; Lane 1: Products in the biological reaction system where the BPA target is present, where the aptamer binds to CP to form a small amount of double-stranded DNA; Lane 2: Products in the biological reaction system where the BPA target is present, where a larger amount of double-stranded DNA is generated; Figure 8A shows the photocurrent response curve; B shows the linear response curves of different concentrations of bisphenol A (a to j: 0, 0.01, 0.1, 1.0, 5.0, 10, 25, 50, 100 μM); C evaluates the selectivity of the detection platform by comparing the response of bisphenol A (25 μM) with other interfering substances; D evaluates the repeatability of this method for detecting 25 μM bisphenol A by detecting 15 independently prepared electrodes under the same experimental conditions. Figure 9 : To optimize the incubation time for the biological reaction in BPA detection; Figure 10 : A schematic diagram of the mechanism for PEC detection of BPA. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0030] In a first aspect, the present invention provides a method for detecting bisphenol A based on the in-situ induced surface polarization effect of cysteine ​​in BiOCOOH, comprising the following steps: S1. Synthesize nanoflower-like BiOCOOH and modify the surface of ITO electrode with the BiOCOOH to prepare BiOCOOH / ITO working electrode. At the same time, pretreat the bisphenol A aptamer and the probe partially complementary to the bisphenol A aptamer respectively. S2. The sample to be tested is incubated with the bisphenol A aptamer in a buffer solution for the first time. S3. Add a probe partially complementary to the bisphenol A aptamer to the reaction system of S2, and perform a second incubation. S4. Add Exonuclease I to the reaction system of S3 to carry out enzymatic digestion, then add cysteine ​​solution to the reaction system, mix well and then contact the BiOCOOH / ITO working electrode for reaction. S5. After the reaction is complete, wash the BiOCOOH / ITO working electrode. Under optimized conditions, detect the photocurrent signal of the BiOCOOH / ITO working electrode. Based on the change in the photocurrent signal, realize the quantitative detection of bisphenol A.

[0031] This method is a highly sensitive, highly selective, label-free, and immobilization-free photoelectrochemical (PEC) detection method for bisphenol A (BPA) based on the surface polarization effect (SPE) induced by cysteine ​​(Cys) in BiOCOOH semiconductor material, combined with aptamer recognition and enzyme cleavage signal amplification strategies. Cysteine ​​reacts with the BiOCOOH surface through its thiol group (-SH) with the BiOCOOH surface. 3+ Coordination forms a polarized electric field on the material surface, significantly improving the separation efficiency of photogenerated electron-hole pairs, thereby enhancing the photocurrent signal.

[0032] When BPA is present, it specifically binds to its aptamer, forming a BPA-Apt complex (single-stranded DNA structure). Upon addition of a probe (CP) partially complementary to the aptamer, the unbound BPA-binding free aptamer binds to the CP to form double-stranded DNA (dsDNA). The addition of Exonuclease I (Exo I), an enzyme that specifically cleaves single-stranded DNA but not double-stranded DNA, results in the enzymatic cleavage of both the BPA-Apt complex (single-stranded) and the free CP (single-stranded), releasing a large amount of free nucleotides.

[0033] These free nucleotides compete with subsequently added cysteine ​​residues for binding to the BiOCOOH surface, inhibiting Cys-induced SPE and resulting in a weakened photocurrent signal. When BPA is absent, the aptamer completely binds to CP to form dsDNA, Exo I cannot cleave it, and no free nucleotides are produced. Cys can bind normally to BiOCOOH, generating SPE and enhancing the photocurrent signal.

[0034] In some embodiments, in step S4, the concentration of the cysteine ​​solution is 0.2 mM, and the reaction time with the BiOCOOH / ITO working electrode is 10 min.

[0035] In some embodiments, in S2, the buffer solution is a mixture of 10 mM Tris-HCl, 100 mM NaCl, 1.0 mM EDTA, and 5.0 mM MgCl2, and the pH of the buffer solution is 8.0.

[0036] In some embodiments, the optimized condition in S5 is: using an Ag / AgCl reference electrode and Pt as the counter electrode; Use a buffer solution with a pH of 3.5; The applied voltage is 0 V, and the excitation light source wavelength is 370 nm-380 nm.

[0037] In some embodiments, the buffer solution is Tris-HCl with a concentration of 0.1 mM.

[0038] In some embodiments, the specific steps of the pretreatment in S1 include: heating the bisphenol A aptamer and a probe partially complementary to the bisphenol A aptamer at 95°C for 5 min, followed by natural cooling.

[0039] In some embodiments, the reaction conditions for the first incubation are: incubation at 35°C-38°C for 60 min-80 min.

[0040] In some embodiments, the reaction conditions for the first incubation are: incubation at 37°C for 70 min.

[0041] In some embodiments, the reaction conditions for the second incubation are: maintaining the same reaction temperature as the first incubation and continuing incubation for 50 min-70 min.

[0042] In some embodiments, the incubation time is 60 min.

[0043] In some embodiments, in step S4, the time for adding Exonuclease I for enzymatic digestion is 40 min.

[0044] In some embodiments, the specific steps for synthesizing nanoflower-like BiOCOOH in step S1 are as follows: S101. Dissolve bismuth nitrate pentahydrate in N,N-dimethylformamide and stir until a homogeneous solution is formed. Then add deionized water and stir until a stable milky white suspension is formed. S102. Add citric acid solution to the suspension and continue stirring to obtain a mixed solution. Transfer the mixed solution to a high-pressure reactor with polytetrafluoroethylene lining and maintain it at 120°C for 12 hours to obtain a white product. Wash the product four times each with deionized water and anhydrous ethanol, and dry it under vacuum at 80°C for 10 hours to obtain a dried product.

[0045] In some embodiments, the specific method for preparing the BiOCOOH / ITO working electrode in step S1 is as follows: A BiOCOOH suspension was dropped onto a pre-cleaned ITO electrode, with an active area of ​​0.5 × 0.5 cm². 2 Dry at room temperature.

[0046] Secondly, this application also provides a detection kit for implementing the above-described detection method, the kit comprising: (a) BiOCOOH / ITO working electrode; (b) Bisphenol A aptamer; (c) A nucleic acid probe partially complementary to the bisphenol A aptamer; (d) Exonuclease I; (e) Cysteine ​​solution; (f) Buffer solution.

[0047] In some embodiments, the concentration of the bisphenol A aptamer is 4.0 μM; the concentration of the nucleic acid probe partially complementary to the bisphenol A aptamer is 4.0 μM; the concentration of the exonuclease I is 10 U / μL; and the concentration of the cysteine ​​solution is 0.2 mM.

[0048] In some embodiments, the buffer solution is a mixture of 10 mM Tris-HCl, 100 mM NaCl, 1.0 mM EDTA, and 5.0 mM MgCl2, and the pH of the buffer solution is 8.0.

[0049] The following is a detailed explanation using specific embodiments: Example 1: Detection of Bisphenol A in Textiles Bismuth nitrate (Bi(NO3)3·5H2O), bisphenol A (BPA), citric acid (CA), N,N-dimethylformamide (DMF), tris(hydroxymethyl)aminomethane (Tris), and ethanol (EtOH) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). BPA aptamers, partially complementary probes of BPA aptamers (hereinafter referred to as CP), Exonuclease I (hereinafter referred to as Exo I), and Exonuclease III (hereinafter referred to as Exo III) were purchased from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China).

[0050] Textile samples were used for actual sample analysis. The textiles were cut into pieces smaller than 5 mm × 5 mm and mixed thoroughly. 1 g of the mixture, accurate to 0.01 g, was weighed and placed in a glass extraction bottle containing 30 mL of methanol. The bottle was then tightened. The extraction bottle was placed in an ultrasonic cleaner and extracted at 35°C for 40 min. After cooling to room temperature, the extract was filtered through a 0.45 μm tetrafluoroethylene membrane.

[0051] Different concentrations (0, 0.01, 0.1, 1.0, 5.0, 10, 25, 50, 100 μM) of BPA standard solution were added to 10.0 μL of filtrate. The biological reaction and PEC determination were then carried out according to the experimental procedure described above, and the recovery rate was calculated.

[0052] 1. Synthesis of nanoflower-like BiOCOOH Weigh 0.86 mmol of bismuth nitrate pentahydrate (Bi(NO3)3·5H2O) and dissolve it in 5 mL of N,N-dimethylformamide (DMF). Stir on a magnetic stirrer until the solute is completely dissolved to form a homogeneous solution. Then add 20 mL of deionized water to the solution and stir continuously at room temperature for 30 min to obtain a stable milky white suspension.

[0053] Next, 5 mL of 10 mmol / L citric acid solution was added to the suspension and the mixture was stirred for 30 min to obtain a mixed solution. The mixed solution was transferred to a 50 mL autoclave lined with polytetrafluoroethylene and kept at 120 °C for 12 h to obtain a white product. The product was washed four times each with deionized water and anhydrous ethanol, and dried under vacuum at 80 °C for 10 h to obtain a dried product, which was named BiOC(BiOCOOH).

[0054] 2. Preparation of BiOCOOH / ITO working electrode 30 μL of the synthesized BiOC suspension (2 mg / mL, ultrasonically dispersed in ultrapure water) was deposited onto a pre-cleaned indium tin oxide (ITO) electrode, with an active area of ​​0.5 × 0.5 cm². 2 The modified electrode was then dried at room temperature for 5 hours.

[0055] 3. Detection of bisphenol A in the sample. 6.0 μL of BPA aptamer (4.0 μM) was mixed with 32.0 μL of reaction buffer containing different concentrations of bisphenol A (10 mM Tris-HCl, 100 mM NaCl, 1.0 mM EDTA, 5.0 mM MgCl2, pH=8.0) and incubated at 37°C for 70 minutes. Then, 6.0 μL of CP (4.0 μM) was added and the reaction continued for 60 minutes. Next, 6.0 μL of Exo I (10 U / μL) was added and the reaction continued for 40 minutes. Finally, 50 μL of 0.2 mM cysteine ​​(Cys) was added to the reaction mixture and mixed thoroughly. The BiOCOOH / ITO electrode was then immersed in the mixture and reacted for 10 minutes. After washing the BiOCOOH / ITO working electrode with ultrapure water, the photocurrent generated was recorded using a CHI 800C electrochemical workstation in a Tris-HCl buffer solution with an applied voltage of 0 V and a pH of 3.5, under an LED light source (wavelength 370-380 nm). The BiOCOOH / ITO electrode was used as the working electrode, and the Pt and Ag / AgCl electrodes were the counter and reference electrodes, respectively.

[0056] Experimental Example 1: Characterization of BiOCOOH The morphology and composition of BiOCOOH were analyzed using scanning electron microscopy (SEM) and X-ray diffraction (XRD), such as... Figure 1 A and Figure 1 As shown in Figure B, BiOCOOH is assembled from 2D nanosheets into nanoflowers with diameters ranging from 3.0 to 5.0 μm. (The text repeats itself here.) Figure 1 The XRD pattern in C shows that for BiOCOOH, distinct diffraction peaks are observed at 24.2°, 29.6°, 32.6°, 46.9°, 53.05°, and 56.6°, corresponding to the (101), (102), (110), (200), (211), and (212) planes of BiOCOOH. These results are in excellent agreement with standard BiOCOOH diffraction data (PDF#00-09-0787), confirming the crystallinity and phase purity of the synthesized material.

[0057] Experimental Example 2: Effect of Cys on the photocurrent of BiOCOOH / ITO electrode The synthesized BiOCOOH nanoflowers were used as photoresponsive materials and modified on indium tin oxide (ITO) electrodes, referred to here as BiOCOOH / ITO, with the aim of transmitting cathode photocurrent signals. Figure 2 As shown in Figure A, experimental results indicate that a significant enhancement of photocurrent was observed when the BiOCOOH / ITO electrode was reacted with a Cys aqueous solution. Furthermore, the optimal PEC response parameters of the BiOCOOH / ITO electrode to Cys were investigated under specific conditions: excitation wavelength 370-380 nm. Figure 2 D) A buffer solution with a pH of 3.5 ( Figure 2 B) Apply voltage 0 V ( Figure 2 E) The reaction time is 10 min ( Figure 2 C). Under these conditions, the linear range of the photocurrent response of Cys is 0.1–100 μM ( Figure 2 F). By utilizing the observed effect of Cys concentration on the photocurrent of the BiOCOOH / ITO electrode, a PEC bioassay platform with Cys as the signal molecule was established.

[0058] Experimental Example 3: Principle Verification of the Influence of Cys on the Photocurrent of BiOCOOH / ITO Electrode To reveal the structural changes after Cys coordinates with the BiOC surface, a series of characterization methods were employed, including XRD, Fourier transform infrared spectroscopy (FT–IR), Raman spectroscopy, ultraviolet-visible diffuse reflectance spectroscopy (UV–Vis DRS), and X-ray photoelectron spectroscopy (XPS). Figure 3The diffraction results of A show that the position, intensity, and full width at half maximum (FWHM) of the BiOC / Cys diffraction peaks are not significantly different from those of the original BiOC. Therefore, it can be inferred that the bonding of Cys to the BiOC surface did not affect its bulk crystal structure.

[0059] Characterization results using FT-IR and Raman spectroscopy confirmed the surface coordination between cysteine ​​(Cys) and BiOCOOH. FT-IR analysis results showed ( Figure 3 A) Pure BiOC at 1561 cm⁻¹ -1 and 1380 cm -1 The characteristic peak at that location belongs to COO The asymmetric and symmetric stretching vibrations, and 741 cm -1 The absorption peak at 3000-3500 cm⁻¹ corresponds to the bending vibration of -OH. After the introduction of Cys, the BiOC / Cys complex exhibits absorption peaks at 3000-3500 cm⁻¹. -1 The interval is 3192 cm. -1 The broad peak can be attributed to -NH3. + The stretching vibration. At 1479 cm. -1 The characteristic peak of NH bending vibration appears, and it is also at 691 cm⁻¹. -1 The presence of a CS stretching vibration peak indicates the presence of the -SH group. Further verification using Raman spectroscopy revealed ( Figure 3 B), 651 cm in the complex -1 CC skeleton stretching vibration at 792 cm -1 CH2 rocking vibration, 953 cm -1 SH bending vibration, 1039 cm -1 NH3 rocking vibration and 1363cm -1 The characteristic peaks of Cys, such as the NH3 bending vibration, were effectively identified. These spectroscopic evidences collectively indicate that Cys has successfully coordinated with the BiOCOOH surface.

[0060] The optical absorption properties of BiOCOOH before and after its reaction with Cys were evaluated by analyzing its UV-vis diffuse reflectance (UV-vis DRS). Figure 4 As shown in Figure A, after BiOCOOH reacts with Cys, the absorption sideband undergoes a red shift, and the absorption intensity is significantly enhanced. This change is attributed to the formation of a polarized electric field on the material surface. Based on the UV-vis DRS results, a Tauc plot with a Y-axis index of 1 / 2 was plotted. Figure 4(B) This demonstrates that BiOCOOH is a direct bandgap semiconductor. The direct bandgap (Eg) of BiOCOOH and BiOCOOH / Cys, obtained by the tangent method, are 3.48 eV and 3.39 eV, respectively. The red shift of the absorption band edge and the narrowing of the bandgap in BiOCOOH / Cys are due to the formation of a surface complex on the BiOCOOH surface by Cys through coordination, resulting in a unique absorption band that affects the material's light-harvesting ability and photoinduced electron transitions.

[0061] The band structures of BiOCOOH and BiOCOOH / Cys were obtained by linear sweep voltammetry (LSV), such as... Figure 4 As shown in CD. Relative to the saturated Ag / AgCl reference electrode, the conduction band (CB) potentials of BiOCOOH and BiOCOOH / Cys are -1.23 V and -1.21 V, respectively, while the valence band (VB) potentials are +2.25 V and +2.18 V, respectively.

[0062] Detailed analysis of the surface elemental composition and interactions between constituent elements of BiOCOOH was performed using X-ray photoelectron spectroscopy (XPS). The results showed that both samples contained Bi, O, and C elements. Figure 5 A). For example Figure 5 As shown in Figure B, two peaks can be fitted in the high-resolution C1s spectrum. The peak at 284.9 eV is generally attributed to non-fixed carbon, while the weaker peak at 288.0 eV corresponds to the carboxyl carbon. Figure 5 The Bi 4f spectrum shown in Figure C is a Bi 4f spectrum of BiOCOOH. 7 / 2 and Bi 4f 5 / 2 Compared to the characteristic peaks of the original material (at 159.0 and 164.3 eV, respectively), the peaks corresponding to BiOCOOH / Cys shift to lower binding energies (i.e., at 158.8 and 164.1 eV). This is due to the reaction of the -SH group of Cys with Bi(III), resulting in a polarized electric field on the material surface. In addition, the BiOCOOH / Cys sample shows differences in its high-resolution N1s spectrum (…). Figure 5 A new characteristic peak appears at 399.3 eV in D), which is attributed to the CN bond. This is the result of surface complexation between Cys and BiOCOOH.

[0063] Experimental Example 4: The reason why Cys enhances the photocurrent of BiOCOOH The surface potentials of BiOCOOH and BiOCOOH / Cys were recorded to reveal the effect of the polarization electric field. Figure 6As shown in AB, the surface potentials of BiOCOOH and BiOCOOH / Cys are 10 and 142 mV, respectively. The increase in the surface potential of BiOCOOH / Cys due to the coordination of the polar molecule Cys leads to an asymmetric distribution of surface charge on BiOCOOH, resulting in a localized surface polarization electric field and a significant enhancement of the photocurrent.

[0064] Experimental Example 5: Determination of PEC of BPA like Figure 10 As shown, when BPA is present in the system, Apt specifically binds to BPA to form a BPA-Apt complex. The subsequently added probe CP (complementary probe) can only bind to unbound free Apt, forming double-stranded DNA (dsDNA). Since Exo I can only digest single-stranded DNA (ssDNA) and cannot act on dsDNA, the BPA-Apt complex (ssDNA structure) and free CP (ssDNA structure) in the system are digested by Exo I, producing a large number of free nucleotides. These free nucleotides compete with the subsequently added Cys for coordination on the BiOCOOH surface, resulting in a significant decrease in the photocurrent signal. Conversely, when BPA is absent, Apt does not form a BPA-Apt complex but completely binds to CP to form dsDNA. Since dsDNA cannot be digested by Exo I, preventing nucleotide production, the added Cys can bind to the BiOCOOH surface, significantly enhancing the photocurrent signal.

[0065] Specific detection of BPA can be achieved by analyzing changes in photocurrent. To evaluate the feasibility of BPA detection, gel electrophoresis was used to validate the biological reactions in the system. Figure 7 As shown in Figure B, the gel electrophoresis results indicate that in the presence of BPA, a large amount of Apt and probe CP are hydrolyzed into nucleotides, with a small portion forming dsDNA, resulting in a faint DNA band in the first lane. Conversely, in the absence of BPA, the large amount of dsDNA formed by the binding of Apt and probe CP cannot be completely hydrolyzed into nucleotides, leading to a distinct DNA band in the second lane. Furthermore, the photocurrent measurement results also confirmed the feasibility of the detection strategy. To obtain optimal detection performance, we optimized the BPA incubation time, such as... Figure 9 As shown, the photocurrent signal gradually weakens with increasing reaction time and stabilizes after 70 minutes. Therefore, the optimal incubation time is 70 minutes. To study the sensor's detection performance, as... Figure 8 As shown in Figure A, the photocurrent signal gradually increases with the increase of BPA concentration.

[0066] Furthermore, within the detection range of (0.01~100 μM), the photocurrent signal showed a linear correlation with the logarithm of the BPA concentration. Figure 8 B). The linear equation is ΔI = 190.60Log[BPA] + 760.73, with a correlation coefficient of 0.98 and a detection limit of 1.4 nmol / L (S / N = 3). Compared with other methods, this sensor exhibits better detection performance. Furthermore, we investigated several potential interfering substances, including Na. + Mg 2+ Zn 2+ The specificity of the sensor was assessed using glucose (Glu), lysozyme (Lys), trypsin, antibiotics such as chloramphenicol (CAP), kanamycin (Kana), and exonucleases (ExoⅠ and ExoⅢ). Figure 8 C) In the presence of BPA, the photocurrent signal of the sensor is significantly higher than that in the presence of interfering substances. This demonstrates the excellent selectivity of the sensor. Figure 8 As shown in Figure D, to evaluate the reproducibility of the sensor, 100 μM BPA was analyzed using 15 independent BiOCOOH / ITO electrodes under the same experimental conditions. The results showed that the relative standard deviation (RSD) of multiple tests was 0.62%, thus demonstrating the excellent reproducibility performance of the sensor.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting bisphenol A based on the in-situ cysteine-induced surface polarization effect of BiOCOOH, characterized in that, Includes the following steps: S1. Synthesize nanoflower-like BiOCOOH and modify the surface of ITO electrode with the BiOCOOH to prepare BiOCOOH / ITO working electrode. At the same time, pretreat the bisphenol A aptamer and the probe partially complementary to the bisphenol A aptamer respectively. S2. The sample to be tested is incubated with the bisphenol A aptamer in a buffer solution for the first time. S3. Add a probe partially complementary to the bisphenol A aptamer to the reaction system of S2, and perform a second incubation. S4. Add Exonuclease I to the reaction system of S3 to carry out enzymatic digestion, then add cysteine ​​solution to the reaction system, mix well and then contact the BiOCOOH / ITO working electrode for reaction. S5. After the reaction is complete, wash the BiOCOOH / ITO working electrode. Under optimized conditions, detect the photocurrent signal of the BiOCOOH / ITO working electrode. Based on the change in the photocurrent signal, realize the quantitative detection of bisphenol A.

2. The bisphenol A detection method based on the cysteine-induced BiOCOOH surface polarization effect according to claim 1, characterized in that, In step S4, the concentration of the cysteine ​​solution is 0.2 mM, and the reaction time with the BiOCOOH / ITO working electrode is 10 min.

3. The bisphenol A detection method based on the cysteine-induced BiOCOOH surface polarization effect according to claim 1, characterized in that, In S2, the buffer solution is a mixture of 10 mM Tris-HCl, 100 mM NaCl, 1.0 mM EDTA, and 5.0 mM MgCl2, and the pH of the buffer solution is 8.

0.

4. The bisphenol A detection method based on the in-situ cysteine-induced BiOCOOH surface polarization effect according to claim 1, characterized in that, In S5, the optimized conditions are: using an Ag / AgCl reference electrode and a Pt electrode as the counter electrode; Use a buffer solution with a pH of 3.5; The applied voltage is 0 V, and the excitation light source wavelength is 370 nm-380 nm.

5. The bisphenol A detection method based on the cysteine-induced BiOCOOH surface polarization effect according to claim 4, characterized in that, The buffer solution is Tris-HCl with a concentration of 0.1 mM.

6. The bisphenol A detection method based on the cysteine-induced BiOCOOH surface polarization effect according to claim 1, characterized in that, In step S1, the specific pretreatment steps include: heating the bisphenol A aptamer and a probe partially complementary to the bisphenol A aptamer at 95°C for 5 min, followed by natural cooling.

7. The method for detecting bisphenol A based on the in-situ cysteine-induced surface polarization effect of BiOCOOH according to claim 1, characterized in that, In step S1, the specific steps for synthesizing nanoflower-like BiOCOOH are as follows: S101. Dissolve bismuth nitrate pentahydrate in N,N-dimethylformamide and stir until a homogeneous solution is formed. Then add deionized water and stir until a stable milky white suspension is formed. S102. Add citric acid solution to the suspension and continue stirring to obtain a mixed solution. Then, transfer the mixed solution to a high-pressure reactor lined with polytetrafluoroethylene and keep it at 120°C for 12 hours to obtain a white product. Wash it four times each with deionized water and anhydrous ethanol, and dry it under vacuum at 80°C for 10 hours to obtain a dried product.

8. The method for detecting bisphenol A based on the in-situ cysteine-induced surface polarization effect of BiOCOOH according to claim 1, characterized in that, In step S1, the specific method for preparing the BiOCOOH / ITO working electrode is as follows: A BiOCOOH suspension was dropped onto a pre-cleaned ITO electrode, with an active area of ​​0.5 × 0.5 cm². 2 Dry at room temperature.

9. A test kit for implementing the method according to any one of claims 1-8, characterized in that, The kit includes: (a) BiOCOOH / ITO working electrode; (b) Bisphenol A aptamer; (c) A nucleic acid probe partially complementary to the bisphenol A aptamer; (d) Exonuclease I; (e) Cysteine ​​solution; (f) Buffer solution.

10. The application of the bisphenol A detection method based on the cysteine-induced BiOCOOH surface polarization effect as described in any one of claims 1-8 and the detection kit as described in claim 9 in the detection of bisphenol A in textiles.

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