Fe por-tpa-based colorimetric-photoelectrochemical dual-mode biosensor and application for detection of s. aureus

Abstract

This invention belongs to the field of biosensors and provides a fePor-tpa-based colorimetric-photoelectrochemical dual-mode biosensor for the detection of Staphylococcus aureus. Bulk fePor-tpa films and two-dimensional fePor-tpa films were synthesized on ITO under solvothermal conditions and in situ growth. FePor-tpa originates from the catalytic activity of porphyrin iron and exhibits excellent dual-enzyme performance. By cleverly utilizing the peroxidase of the large-volume fePor-tpa film in colorimetric mode to oxidize TMB, and utilizing the catalase of the 2D fePor-tpa film in PEC mode to generate O2, the invention provides a solution for the detection of Staphylococcus aureus. 2 We constructed colorimetric and PEC dual-mode biosensors for the sensitive and specific quantification of Staphylococcus aureus.

Description

Application of FEPor-TPA colorimetric-photoelectrochemical dual-mode biosensor for the detection of Staphylococcus aureus Technical Field

[0001] This invention belongs to the field of biosensors, and specifically relates to the construction of a colorimetric-photoelectrochemical dual-mode biosensor for the detection of Staphylococcus aureus based on fePor-tpa-like peroxidase, catalase-like, and photoelectrochemical properties. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Human health is constantly threatened by bacterial infections, especially Staphylococcus aureus, which can cause a variety of serious diseases. Therefore, exploring methods for detecting Staphylococcus aureus is crucial. Currently, there are many methods for quantifying Staphylococcus aureus, including plate counting, ELISA, molecular biological detection, and polymerase chain reaction (PCR). However, these methods are often limited by several disadvantages, such as expensive equipment, long incubation times, and complex sample extraction. Furthermore, these methods are all based on a single signal readout mode, and errors are inevitable due to inconsistent testing standards, different operating habits, and varying experimental environments. In recent years, dual-signal readout modes based on independent signal channels have proven to be an ideal solution for improving accuracy through inherent self-calibration capabilities.

[0004] Colorimetric methods allow for direct qualitative analysis on-site, but suffer from low sensitivity. Photoelectrochemical analysis, as an emerging analytical approach, holds broad application prospects in fields such as bioanalysis and environmental monitoring. PEC biosensors employ two completely independent energy forms, exhibiting high sensitivity and low background signal, primarily due to their perfect integration of the advantages of optical and electrochemical biosensors. The integration of colorimetric and PEC signals not only enables visual analysis but also enhances sensitivity. Photoactive materials play a crucial role in PEC systems, and electrodes are typically constructed using drop-coating. However, the instability and non-reproducibility of photoactive materials inevitably pose challenges to charge transfer and sensing applications. Therefore, exploring rational photoactive material and electrode preparation techniques is urgently needed.

[0005] Covalent organic polymers (COPs) are porous polymers with advantages such as large surface area, low skeletal density, high stability, tunable structure, and diverse synthetic strategies. Numerous COPs composed of different monomers have been studied, revealing their great potential in drug delivery, gas storage, sensing, and photodetectors. Among them, porphyrin-based COPs (p-COPs) composed of metalloporphyrin monomers have been shown to possess excellent peroxidase properties. In particular, p-COPs, which combine catalase and peroxidase properties, show great potential in biosensing analysis. Furthermore, the high light absorption capacity and strong π-π conjugated structure of porphyrin monomers endow p-COPs with unique optical and electrical properties, making them promising candidates for photoactive materials. The porous structure and high specific surface area provide a conductive pathway for electron transport, effectively reducing electron-hole recombination and significantly improving photoelectric conversion efficiency. Therefore, combining these with excellent dual-enzyme properties will provide significant opportunities for the design of p-COPs on PEC and colorimetric dual-readout mode platforms.

[0006] Realizing photoactive materials on electrodes is essential, and a common method is to directly drop bulk materials onto the electrode via coating. However, uncontrolled coating methods result in films with large particles and weak interactions, making them prone to peeling off the electrode and inevitably compromising their photoactive properties (PEC). Furthermore, the fabrication process typically requires high temperature, high pressure, and long time, severely limiting its application. To date, several surface-mediated schemes have been introduced to prepare two-dimensional (2D) layered films that can be grown on various substrates, such as indium-doped tin oxide (ITO), monolayer graphene, and even glass substrates. However, PEC performance still needs improvement. Summary of the Invention

[0007] To address the aforementioned issues, this invention provides a colorimetric-electrochemical dual-mode biosensor for detecting Staphylococcus aureus based on fePor-tpa-like peroxidases, catalase-like peroxidases, and photoelectrochemical properties. Previously, the inventors prepared 2D COP films (D-TA COP films) on ITO using a simple in-situ growth strategy, demonstrating strong adhesion between the material and the electrode. Therefore, this in-situ growth strategy can serve as an ideal method for realizing photoactive materials on electrodes. Based on this, this invention synthesized bulk fePor-tpa films and two-dimensional fePor-tpa films on ITO using solvothermal conditions and in-situ growth, respectively. FePor-tpa originates from the catalytic activity of porphyrin iron and possesses excellent dual-enzyme performance. By cleverly utilizing the peroxidase of the large-volume fePor-tpa film to oxidize TMB in colorimetric mode and utilizing the catalase of the 2D fePor-tpa film to generate O2 in PEC mode, colorimetric and PEC dual-mode biosensors were constructed for the sensitive and specific quantification of Staphylococcus aureus. This invention demonstrates innovative insights into the flexible design and utilization of various enzyme properties in the field of biosensing using nanocomposites.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a GOx-Ab2-Au-COP probe for detecting Staphylococcus aureus using a dual-mode strategy, wherein the probe is prepared by a method comprising:

[0010] Fe-COP was synthesized using Fe-ATPP and terephthalaldehyde (TPA) as raw materials.

[0011] A mixture of Fe-ATPP and TPA was dropped onto ITO. After 10–20 minutes, the ITO was placed in dichloromethane, removed, and dried to obtain an Fe-COP film.

[0012] Fe-COP was added to water and sonicated for 15-20 min. Then chloroauric acid was added and stirred for 5-6 h. Finally, sodium borohydride was added and stirred for 1-2 h. After centrifugation, Au-COP was obtained, washed with water, and dried.

[0013] First, add Au-COP to water to obtain Au-COP dispersion, then add Ab2 and stir for 4-6 hours; then add glucose oxidase solution and stir for 10-12 hours; finally add BSA and stir for 1-2 hours, then centrifuge to obtain GOx-Ab2-Au-COP probe.

[0014] A second aspect of the present invention provides an immunosensor for detecting Staphylococcus aureus using a dual-mode strategy, comprising: the aforementioned GOx-Ab2-Au-COP probe, specifically comprising:

[0015] First, add BSA to a 48-well microplate and carry out the reaction. After the reaction is complete, wash the plate several times with PBS.

[0016] Meanwhile, vancomycin was added to a MES buffer solution containing EDC and NHS to activate COOH. Then, the vancomycin solution was dropped into the plate and incubated overnight at 4°C to allow it to covalently link with the residual BSA through an amidation reaction. The plate was then washed with PBS.

[0017] Add the Staphylococcus aureus to be tested and react at 37°C for 1–1.2 h;

[0018] After washing with PBS, the probe GOx-Ab2-Au-COP was added to the plate and reacted for 2-3 hours.

[0019] After washing with PBS, add oxygenated glucose solution. After 1-1.2 hours, take 1 mL of the solution from the system and transfer it to PEC for testing.

[0020] Then add TMB solution and mix it with the remaining solution in the wells for 20-25 minutes. Then measure the absorbance value at 652 nm in each well using a microplate reader.

[0021] Beneficial effects of the present invention

[0022] (1) In this invention, bulk fepor-tpa films and two-dimensional fepor-tpa films were synthesized on ITO under solvothermal conditions and in situ growth, respectively. Fepor-tpa originates from the catalytic activity of porphyrin iron and has excellent dual-enzyme performance. By cleverly utilizing the peroxidase of the large-volume fepor-tpa film to oxidize TMB in colorimetric mode and utilizing the catalase of the 2D fepor-tpa film to generate O2 in PEC mode, colorimetric and PEC dual-mode biosensors were constructed for the sensitive and specific quantification of Staphylococcus aureus.

[0023] (2) The preparation method of the present invention is simple, practical and easy to promote. Attached Figure Description

[0024] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. Exemplary embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0025] Figure 1. (A) Infrared spectra of Fe-ATPP, tpa, and fepor-tpa COP and thin films; (B) TEM image of fepor-tpa COP; SEM (C) top view and (D) cross view of fepor-tpa thin film; (E) AFM image of FePor-TPA thin film; (F) TEM image of Au-COP;

[0026] Figure 2. (A) Color changes and absorbance fluctuations in each group: TMB+H2O2+fepor-tpa(a), TMB+H2O2(b), TMB+fepor-tpa(c) and TMB(d); (B) Changes in fepor-tpa mimic enzyme activity with pH; (C) UV absorption of H2O2(a) and fepor-tpa+H2O2(b); (D) Dissolved oxygen of fepor-tpa+H2O2(a) and H2O2(b);

[0027] Figure 3(A) Photoelectric response of fepor-tpa COP film; (B) Response of fepor-tpa COP film in N2(a), air(b) and O2(c) saturated solutions; (C) Response of fepor-tpa COP film in N2 saturated buffer solution (a), response of fepor-tpa COP film after adding H2O2 to N2 saturated buffer solution (b); (D) Schematic diagram of the predicted response mechanism;

[0028] Figure 4. Optimization of key experimental parameters: (A) pH value of buffer solution; (B) concentration of TMB; (C) reaction time of glucose; (D) performance of electrode in buffer solution containing H2O2 at different reaction times;

[0029] Figure 5. Linear relationships: (A) photoelectric response; (B) linear relationship of photoelectric response; (C) ultraviolet response; (D) linear relationship of ultraviolet response; and the inhibitory effect on the performance of the colorimetric platform (D);

[0030] Figure 6. Selectivity of electrochemical (A) and colorimetric (B) sensing platforms: (a) 0 U / mL Dam, (b) 50 U / mL BSA, (c) 50 U / mL M.SssI, (d) 50 U / mL Dam. 5-fluorouracil against Dam on electrochemical platform (C);

[0031] Figure 7. Schematic diagram of material synthesis for Scheme 1;

[0032] Figure 8. Schematic diagram of scheme 2. Dual-mode strategy for Staphylococcus aureus detection. Detailed Implementation

[0033] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] A GOx-Ab2-Au-COP probe for dual-mode strategy detection of Staphylococcus aureus, the preparation method of the probe includes:

[0035] Fe-COP was synthesized using Fe-ATPP and terephthalaldehyde (TPA) as raw materials.

[0036] A mixture of Fe-ATPP and TPA was dropped onto ITO. After 10–20 minutes, the ITO was placed in dichloromethane, removed, and dried to obtain an Fe-COP film.

[0037] Fe-COP was added to water and sonicated for 15-20 min. Then chloroauric acid was added and stirred for 5-6 h. Finally, sodium borohydride was added and stirred for 1-2 h. After centrifugation, Au-COP was obtained, washed with water, and dried.

[0038] First, add Au-COP to water to obtain Au-COP dispersion, then add Ab2 and stir for 4-6 hours; then add glucose oxidase solution and stir for 10-12 hours; finally add BSA and stir for 1-2 hours, then centrifuge to obtain probe GOx-Ab2-Au-COP.

[0039] In some embodiments, during the synthesis of Fe-COP, the molar ratio of Fe-ATPP to TPA is 0.1:2 to 3.

[0040] In some embodiments, during the preparation of Fe-COP thin films, the mass ratio of Fe-ATPP to TPA is 1:1 to 1.2.

[0041] In some embodiments, the mass ratio of Fe-COP, chloroauric acid, and sodium borohydride is 1:1:0.1 to 0.2.

[0042] In some embodiments, the mass ratio of Au-COP, Ab2 to glucose oxidase, and BSA is 10:0.01 to 0.02:2:2.

[0043] An immunosensor for detecting Staphylococcus aureus using a dual-mode strategy, comprising:

[0044] First, add BSA to a 48-well microplate and carry out the reaction. After the reaction is complete, wash the plate several times with PBS.

[0045] Meanwhile, vancomycin was added to a MES buffer solution containing EDC and NHS to activate COOH. Then, the vancomycin solution was dropped into the plate and incubated overnight at 4°C to allow it to covalently link with the residual BSA through an amidation reaction. The plate was then washed with PBS.

[0046] Add the Staphylococcus aureus to be tested and react at 37°C for 1–1.2 h;

[0047] After washing with PBS, the probe GOx-Ab2-Au-COP was added to the plate and reacted for 2-3 hours.

[0048] After washing with PBS, add oxygenated glucose solution. After 1-1.2 hours, take 1 mL of the solution from the system and transfer it to PEC for testing.

[0049] Then add TMB solution and mix it with the remaining solution in the wells for 20-25 minutes. Then measure the absorbance value at 652 nm in each well using a microplate reader.

[0050] In some embodiments, the amount of the GOx-Ab2-Au-COP probe added is 300–350 μL / well.

[0051] In some embodiments, the concentration of Staphylococcus aureus is 10. 1 ~10 8 CFU mL -1 .

[0052] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0053] Example 1: Construction of Probes and Sensors

[0054] 1. Materials, reagents and equipment

[0055] Chloroauric acid (HAuCl4·4H2O), lactic acid, nitrobenzene, p-nitrobenzaldehyde, hydrochloric acid, ammonia, ferric chloride, terephthalic acid, acetic acid, ethanol, and dichloromethane were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd., China. H2O2, dimethylformamide (DMF), acetone, 1,2-dichlorobenzene, and n-butanol were all from Beijing Chemical Co., Ltd. (Beijing, China). Pyrrole and vancomycin hydrochloride (Van) were obtained from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). 3,3',5,5'-Tetramethylbenzidine (TMB) and bovine serum albumin (BSA) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Rabbit anti-Staphylococcus aureus / Streptococcus roximately var. lutea antibody (bs-4582R) (Ab2) was from Bissin Antibody Biochemical Technology Co., Ltd. (Beijing). 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Chengdu Best Reagent Co., Ltd. 2-(N-morpholino)-ethylsulfonic acid (MES) was purchased from Shanghai Shangen Chemical Technology Co., Ltd. A self-prepared phosphate-buffered saline (PBS, 0.01M, pH=7.4), composed of 100mg KCl, 720mg Na2HPO4·12H2O, 120mg KH2PO4, and 4g NaCl, was placed in a 500mL ultrapure water indium tin oxide (ITO) glass container and purchased from Zhuhai Kaiwo Optoelectronic Technology Co., Ltd. (China).

[0056] The morphology and structure of the materials were characterized using transmission electron microscopy (TEM, HT-7700, Hitachi, Japan) and Schottky field emission scanning electron microscopy (FESEM, ZEISS Gemini SEM 300). Powder X-ray diffraction (PXRD) was performed using an in-situ X-ray diffractometer (Smartlab 9KW, Rigaku, Japan). UV-Vis absorption spectra were recorded on a spectrometer (EU-2800DS, Onlab, China). All electrochemical measurements were performed on a CHI 760D electrochemical workstation (Shanghai Chenhua Instruments, China) using a three-electrode system, with a glassy carbon electrode (GCE, Φ = 3 mm) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire as the auxiliary electrode.

[0057] 2. Synthesis of Fe-ATPP

[0058] To synthesize Fe-ATPP, ATPP was first prepared using a method reported in the literature. Lactic acid (7 mL) and nitrobenzene (25 mL) were mixed. A nitrobenzene solution (12 mL) containing p-nitrobenzaldehyde (4.16 g, 27.5 mmol) and pyridine (1.84 g, 27.5 mmol) was added at 150 °C. The reaction was allowed to proceed for 2 h. After cooling to below 60 °C, 15 mL of methanol was added, and the mixture was stirred for 30 min and then kept undisturbed for 8 h. TNPP was obtained by filtration, washed with methanol, and dried at 60 °C. To reduce TNPP to ATPP, 550 mg of TNPP was dissolved in 75 mL of hydrochloric acid, and 7 mL of hydrochloric acid dissolved in SnCl2·2H2O was added. The mixture was added dropwise at room temperature for 10 min, reacted at 70 °C for 30 min, and then cooled in a cold water bath. The filtered solid was dispersed in 70 mL of ultrapure water. After adjusting the pH of the system to 9 with ammonia, ATPP was obtained by centrifugation, dried at 60 °C, and purified by Soxhlet extraction with chloroform.

[0059] Fe-ATPP preparation: Add ATPP (300 mg) and ferric chloride (250 mg) to DMF (30 mL). Stir at 160 °C for 4.5 h. Remove DMF and wash with water. Finally, centrifuge and dry to obtain Fe-ATPP.

[0060] 3. Synthesis of Fe-COP (fepor–tpa)

[0061] In Pyrex tubes, Fe-ATPP (32 mg, 0.044 mmol) and terephthalaldehyde (TPA, 11.8 mg, 0.88 mmol) were added to 2.2 mL of a mixture of acetic acid, 1,2-dichlorobenzene, and n-butanol, and incubated for 15 min. After three freeze-evacuation-thawing cycles, the tubes were sealed and heated at 120 °C for 72 h. The Fe-COP obtained by centrifugation was washed with THF and acetone and dried at 60 °C.

[0062] Preparation of 4Fe-COP thin films (Fepor–TPA thin films)

[0063] The preparation of Fe-COP films was based on previous reports with some modifications. Fe-ATPP (1 mg) and TPA (1 mg) were added to a mixture of tetrahydrofuran (THF), ethanol, and acetic acid, respectively. Equal amounts of Fe-ATPP and TPA were placed together. Then, 20 μL of this mixture was dropped onto ITO (1 cm × 2 cm). After about 10 minutes, the ITO was placed in dichloromethane. Finally, the ITO was removed and dried to obtain the film.

[0064] 5. Probe Preparation

[0065] First, Au-COP is synthesized. Specifically, Fe-COP (10 mg) is added to 10 ml of water and incubated for 15 minutes, followed by the addition of 30 μL of chloroauric acid (10 mg / mL).-1 Stir for 5 hours, then add 200 μL of sodium borohydride (1 mg / mL). -1 Stir for 1 hour, centrifuge to obtain gold-coated Au-COP, wash with water and dry.

[0066] When preparing the GOx-Ab2-Au-COP probe, first add 10 mg of Au-COP to 10 mL of water to obtain an Au-COP dispersion (10 mL, 1 mg / mL). -1 Next, add 100 μL of Ab2 (0.1 mg / mL) and stir for 4 hours. Then add 2 mL of glucose oxidase solution (1 mg / mL). -1 Stir for 10 hours. Finally, add 200 μL of BSA (1%), stir for 1 hour, centrifuge to obtain the probe, and wash with water.

[0067] 6. Constructing a dual-mode sensor

[0068] The Staphylococcus aureus dual-mode immunosensor, as shown in Protocol 2, initially involves adding 600 μL (5%) BSA to a 48-well microplate and reacting at 4°C for 24 h. After the reaction, the plate is washed three times with PBS (10 mM, pH 7.4). Simultaneously, 100 mg vancomycin is added to 15 mL of MES buffer (pH 6.0) containing 300 mg EDC and 15 mg NHS to activate the COOH. Then, 400 μL of vancomycin solution is added to each well and the plate is incubated overnight at 4°C to allow it to covalently bind to the remaining BSA via amidation. After the vancomycin-specific phase is bound, the plate is washed with PBS to remove unbound portions. A series of concentrations of Staphylococcus aureus (10¹–10⁻⁶) are then used. 8 Add 400 μL of CFU (mL-1) to the plate and react at 37°C for 1 h. After washing with PBS, add the probe GOX-Ab2-Au-COP (300 μL / well) to the plate and react for 2 h. After washing with PBS, add 1.2 mL of oxygenated glucose solution (1 mg / mL). After 1 h, transfer 1 mL of the solution to PEC and test using catalase on a 2D fepor-tpa membrane in PEC mode. Then add TMB solution (0.1 mM) and mix with the remaining 200 μL of solution in the wells for 20 min. Then measure the absorbance at 652 nm in each well using a microplate reader.

[0069] Example 2: Related Performance Tests

[0070] A dual-mode strategy was used to detect Staphylococcus aureus, and the relevant test results are as follows: 1. Dual-mode strategy for detecting Staphylococcus aureus

[0071] Many reagents have been developed for the capture of Staphylococcus aureus. Vancomycin (Van), an antibiotic for treating bacterial infections, has numerous binding sites with the d-alanyl-d-alanine of Gram-positive bacteria. Due to its good stability, low cost, and commercial availability, Van was chosen for the capture of Staphylococcus aureus in this invention. However, Van alone lacks specificity in identifying Staphylococcus aureus. Fortunately, studies have found that Staphylococcus aureus is rich in protein A, which has specific recognition ability against rabbit anti-Staphylococcus aureus antibody (bs-4582R). Therefore, bs-4582R was selected as another recognition agent (Ab2) in this biosensor, significantly improving specificity.

[0072] Scheme 2 details the principle of the photochemical and colorimetric dual-mode biosensor. On one hand, the prepared fepor-tpa COP nanocomposite material possesses a large surface area and high porosity, making it not only an ideal cargo carrier but also exhibiting excellent peroxide-like activity due to the catalytic activity of porphyrin iron. Considering these superior properties, the prepared fepor-tpa COP nanocomposite material was selected as a substrate to load GOx and Au NPs, constructing a GOx-Ab2-Au-COP probe. In this probe, gold nanoparticles are grown in situ on the Fe-COP surface through the reduction of NaBH4. The connection between Ab2 and gold nanoparticles is achieved through the Au-NH2 reaction, and the insertion of GOx into the pores of fepor-tpa COP is achieved through host-guest interactions. Utilizing the high porosity of fepor-tpa COP, abundant GOx molecules are introduced, enabling signal amplification from one-to-one to one-to-many. Furthermore, gold nanoparticles can accelerate electron transport, improving the sensitivity of the biosensor. Therefore, the prepared GOx-Ab2-Au-COP probe can not only identify targets but also significantly amplify signals. On the other hand, the two-dimensional (2D) fepor-tpa COP film prepared by in-situ growth exhibits high porosity and an ordered layered structure, thus demonstrating excellent photochemical performance. Simultaneously, the excellent catalytic performance of porphyrin iron in the fepor-tpa COP film makes it exhibit a supersensitive response to H2O2, and the in-situ growth characteristic greatly improves its stability. Therefore, fepor-tpa COP film is used as a photoactive material to construct a PEC platform.

[0073] In the immunoassay section, BSA was first immobilized on a 48-well plate via nonspecific adsorption. Vancomycin was then introduced as a nonspecific trap and anchored by the BSA to capture Staphylococcus aureus. After adding a certain amount of GOx-Ab2-Au-COP probe, Ab2 specifically identified Staphylococcus aureus, forming a sandwich pattern with the number of bacteria proportional to the number of Staphylococcus aureus. After washing away unlinked probes from the suspension, a quantitative glucose solution was introduced and incubated for 1 hour. Under the catalysis of GOx, glucose was oxidized by O2, simultaneously generating H2O2. A portion of the solution in the plate was transferred to a photoelectric platform, and the remaining mixture in the plate was added to TMB for colorimetric analysis. As the number of Staphylococcus aureus increased, more GOx was introduced, resulting in more H2O2 production. Therefore, a "signal-on" dual-modal detection platform was constructed.

[0074] 2. Material Characterization

[0075] In this invention, Fe-ATPP and TPA monomers (Scheme X) are synthesized via an aldehyde-amine condensation reaction to form fepor-tpa COP and its thin film. The FT-IR spectra of Fe-ATPP, TPA, fepor-tpa COP, and their thin film are shown in Figure 1A. The TPA spectrum is at 1680 cm⁻¹. -1 The peak is produced by the C=O vibration, at 2760 cm⁻¹. -1 A strong aldehyde CH vibration is observed at this location. The NH peak in Fe-ATPP appears at 3219 cm⁻¹. -1 and 3341cm -1 Location. fepor-tpa at 1620cm -1 A strong peak appeared at the C=N junction, while the aldehyde peak of TPA and the amino peak of Fe-ATPP dropped sharply, confirming the occurrence of the condensation reaction. TEM images of feport-tpa (Figure 1, B) show a sheet-like morphology, and vertical SEM views (Figure 1, C) show a continuous but uneven film. Cross-sectional SEM (Figure 1, D) shows no clear boundary between the film and the electrode, confirming that the film is ultrathin and that the interaction between the film and the electrode is strong. AFM images show that the film height fluctuates within 10 nm (Figure 1, E), with an Rq of 3.99 nm, further indicating that the film is ultrathin. The gold nanoparticles grown in situ on feport-tpa are approximately 2 nm in size and uniformly distributed (Figure 1, F).

[0076] Peroxidase-like and catalase-like activities of 3fepor-tpa

[0077] The oxidation of TMB was catalyzed with the aid of H2O2, and a classic colorimetric experiment was conducted. Four experimental groups were designed. Figure 2A shows the color changes and absorbance fluctuations of each group. It can be clearly seen that only the TMB + H2O2 + fepor-tpa COP showed a color change, and the absorption peak intensity at 652 nm was significantly increased, indicating that fepor-tpa COP has peroxidase-mimicking properties. In addition, it was found that the highest activity was found at around 3 (Figure 2B).

[0078] Since catalase (CAT) catalyzes the decomposition of H2O2 to generate O2, the CAT performance of febor-tpa COP was investigated by measuring the change in the absorption peak of H2O2 at 240 nm and the dissolved O2 generated in the system. As shown in Figure 2C, the absorption peak of H2O2 in febor-tpa COP+H2O2 is much lower than that in the pure H2O2 system. Furthermore, many bubbles are visible in febor-tpa COP+H2O2, while very few bubbles are observed in pure H2O2. Because the peroxidase behavior of febor-tpa consumes hydrogen peroxide, a dissolved oxygen meter was used to record changes in dissolved oxygen to ensure the elimination of the peroxidase effect. As shown in Figure 2D, dissolved oxygen in the pure H2O2 system increases to some extent with the passage of response time, but the increase rate is much lower than that in the febor-tpa COP+H2O2 system. Therefore, the above results clearly demonstrate that febor-tpa possesses CAT-mimicking enzyme performance.

[0079] 4. Photoelectric properties of fepor-tpa

[0080] To investigate the photoelectric properties of the fepor-tpa COP film, the photocurrent signal of the fepor-tpa COP film was recorded under Xe lamp illumination without bias voltage. As shown in Figure 3A, the fepor-tpa COP film is sensitive to light, generating a cathode photocurrent of approximately 0.8 μA. After 250 s of switching on and off, the response did not decrease, indicating that the signal of the fepor-tpa COP film is very stable.

[0081] Since conventional cathodic photocurrents readily respond to O2, the signal changes of the fepor-tpa COP membrane in a buffer solution saturated with N2 and O2 were investigated. As shown in Figure 3B, after deoxygenation of the buffer solution, the signal of the fepor-tpa COP membrane decreased to -0.25 μA, while it increased to -1.1 μA after oxygen saturation. These phenomena indicate that the cathodic photocurrent of the fepor-tpa COP film is O2-dependent. Furthermore, the addition of H2O2 to the deoxygenated buffer solution revealed a strong H2O2-controlled photocurrent signal response in the fepor-tpa COP membrane (Figure 3C).

[0082] The above results clearly demonstrate that the fepor-tpa membrane not only exhibits strong O2-controlled signal changes but also possesses H2O2-dependent characteristics. For the O2-dependent cathodophotocurrent principle, oxygen is typically the electron acceptor, but H2O2-controlled phenomena are not common. According to previous reports, the H2O2-dependent cathodophotocurrent mechanism can be attributed to the enzyme-mimicking activity within the electrode material. Considering the excellent peroxidase and catalase-mimicking enzyme activities of the fepor-tpa membrane, Figure 3D summarizes the principle of strong H2O2-controlled signal changes. Under light irradiation, fepor-tpa generates charge carriers, with charge transferring from the HOMO to the LUMO, resulting in photoinduced electron-hole pairs. When H2O2 is present, on the one hand, under the action of the catalase-mimicking enzyme properties of fepor-tpa, H2O2 is converted into O2, thereby allowing electrons in the fepor-tpa LUMO to be harvested by O2, forming O2. 2·- On the other hand, due to fepor-tpa's inherent peroxidase-mimicking activity, its LUMO electrons are captured by H2O2 to generate ·OH. This dual-transfer method effectively avoids electron-hole pair recombination, thereby greatly enhancing the photocurrent signal.

[0083] 5. Optimization of Experimental Conditions

[0084] Target bacteria 10 7 CFU mL -1 To optimize the sensing performance, parameters such as buffer pH, TMB concentration, and glucose reaction time were optimized. During detection, the buffer solution's pH is a key parameter, affecting the photocurrent by influencing the catalase activity of the Feport-TPA membrane. As shown in Figure 4A, the signal initially rises and then falls from pH 3 to 6, indicating that pH 6 is the optimal pH for the buffer solution. The required TMB concentration for the biosensor is another parameter. Figure 4B shows that the signal gradually increases with increasing TMB concentration; however, the rate of increase slows down when TMB reaches 10 mM, thus selecting 10 mM as the optimal TMB concentration. The glucose reaction time was also investigated. As shown in Figure 4C, the signal gradually increases when the reaction time is extended from 10 to 70 min, then essentially stops rising after 60 min. To save time while ensuring efficient reaction, 60 min was chosen as the most suitable reaction time. In addition, the performance of the electrode in a buffer solution containing H2O2 at different reaction times was compared to maximize the CAT-mimicking enzyme performance of the fepor-tpa membrane. As shown in Figure 4D, the signal rises significantly before 2 minutes, then rises slowly and eventually stops rising. Therefore, the electrode performance is fully utilized when the reaction time in the H2O2 buffer solution is 2 minutes.

[0085] Analytical performance of 6 dual-mode sensors

[0086] Photocurrent was measured under optimal conditions after adding different amounts of target bacteria. As shown in Figure 5A, the higher the concentration of target bacteria, the stronger the signal. The concentration was 10 CFU / mL. -1 ~108 CFU / mL -1 The regression showed a good linear relationship within the range (B in Figure 5), with the corresponding regression equation being ΔI = -0.054 - 0.121lg C (R² = 0.9901), and the calculated LOD was 8.73 CFU / mL. -1 (S / N = 3).

[0087] The target bacteria can also be determined by colorimetric methods. As expected, when the target bacteria were measured from 10 CFU / mL... -1 Increased to 108 CFU / mL -1 The characteristic peak absorbance value increased (C in Figure 5). The calibration curve fitting formula ΔA = 0.249 + 0.046lg C, the correlation index was 0.9987, and the LOD was 5.91 CF mL. -1 (D in Figure 5).

[0088] 7. Selectivity of detection

[0089] To investigate the selectivity of the constructed sensor, experiments were conducted using *E. coli* k12, *Acinetobacter jouhnsonii*, and *E. coli* Dhα. As shown in Figure 6, in both the PEC and colorimetric experiments, only the system containing the target bacteria exhibited high signal intensity, indicating the excellent selectivity of the dual-mode sensor of this invention.

[0090] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A GOx-Ab2-Au-COP probe for detecting Staphylococcus aureus using a colorimetric-electrochemical dual-mode strategy, characterized in that, The preparation method of the probe includes: synthesizing Fe-COP using Fe-ATPP and terephthalaldehyde (TPA) as raw materials; adding Fe-ATPP and terephthalaldehyde to a mixture of acetic acid, 1,2-dichlorobenzene, and n-butanol, sonicating for 15 min, performing three freeze-vacuum-thaw cycles, sealing, and heating at 120°C for 72 hours to obtain Fe-COP; adding Fe-COP to water, sonicating for 15-20 min, adding chloroauric acid, stirring for 5-6 h, finally adding sodium borohydride and stirring for 1-2 h, centrifuging to obtain Au-COP, washing with water, and drying; first adding Au-COP to water to obtain Au-COP dispersion, then adding Ab2 and stirring for 4-6 h; then adding glucose oxidase solution and stirring for 10-12 h; finally adding BSA, stirring for 1-2 h, and centrifuging to obtain the probe GOx-Ab2-Au-COP, which is the final product.

2. The GOx-Ab2-Au-COP probe for detecting Staphylococcus aureus using a colorimetric-electrochemical dual-mode strategy as described in claim 1, characterized in that, During the synthesis of Fe-COP, the molar ratio of Fe-ATPP to TPA is 0.1:2~3.

3. The GOx-Ab2-Au-COP probe for detecting Staphylococcus aureus using a colorimetric-electrochemical dual-mode strategy as described in claim 1, characterized in that, The mass ratio of Fe-COP, chloroauric acid, and sodium borohydride is 1:1:0.1~0.

2.

4. The GOx-Ab2-Au-COP probe for detecting Staphylococcus aureus using a colorimetric-electrochemical dual-mode strategy as described in claim 1, characterized in that, The mass ratio of Au-COP, Ab2 to glucose oxidase and BSA is 10:0.01~0.02:2:

2.

5. An immunosensor for detecting Staphylococcus aureus using a colorimetric-electrochemical dual-mode strategy, characterized in that, include: The GOx-Ab2-Au-COP probe according to any one of claims 1-4.

6. The immunosensor for detecting Staphylococcus aureus using a colorimetric-photoelectrochemical dual-mode strategy as described in claim 5, characterized in that, include: First, add BSA to a 48-well microplate and react. After the reaction is complete, wash the plate several times with PBS. At the same time, add vancomycin to a MES buffer solution containing EDC and NHS to activate COOH. Then, drop the vancomycin solution into the plate and incubate overnight at 4°C to allow it to covalently link with the residual BSA through an amidation reaction. Finally, wash the plate with PBS. Add the Staphylococcus aureus to be tested and react at 37℃ for 1-1.2 h. After washing with PBS, add the probe GOx-Ab2-Au-COP to the plate and react for 2-3 h. After washing with PBS, add oxygenated glucose solution and react for 1-1.2 h. Then, take 1 mL of the solution from the system and transfer it to PEC for testing. Then add TMB solution and mix it with the remaining solution in the wells for 20-25 min. Then, measure the absorbance value at 652 nm in each well using a microplate reader.

7. The immunosensor for detecting Staphylococcus aureus using a colorimetric-photoelectrochemical dual-mode strategy as described in claim 5, characterized in that, The amount of the GOx-Ab2-Au-COP probe added is 300~350 μL / well.

8. The immunosensor for detecting Staphylococcus aureus using a colorimetric-electrochemical dual-mode strategy as described in claim 5, characterized in that, The concentration of Staphylococcus aureus was 10. 1 ~10 8 CFU mL -1 .

9. The use of the GOx-Ab2-Au-COP probe according to any one of claims 1-4 in the colorimetric-photoelectrochemical dual-mode non-diagnostic detection of Staphylococcus aureus.

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