Method for detecting and killing staphylococcus aureus based on hollow Au-TiO2 photoelectrochemical sensor

Abstract

The invention discloses a method for detecting and killing staphylococcus aureus based on a hollow Au-TiO2 photoelectrochemical sensor, which comprises the following steps: firstly, preparing a hollow Au-TiO2 nano-enzyme substrate, dropwise casting the hollow Au-TiO2 nano-enzyme substrate on an FTO (Fluorine-doped Tin Oxide) electrode, and calcining to obtain a hollow Au-TiO2 / FTO electrode; the preparation method comprises the following steps: coupling carboxyl-functionalized hollow Au-TiO2 and an amino-functionalized staphylococcus aureus aptamer by utilizing an amidation reaction to serve as a signal probe, and fixing the signal probe on the surface of a hollow Au-TiO2 / FTO (Fluorine-doped Tin Oxide) electrode; in the detection process, the aptamer captures staphylococcus aureus and is specifically combined with a signal probe, and an electrode reacts with H2O2 and 4-chloro-1-naphthol by utilizing the peroxidase-like activity of nano-enzyme, so that a sandwich type signal amplification photoelectrochemical sensor is constructed; meanwhile, H2O2 is catalytically decomposed to generate high-toxicity. OH to kill staphylococcus aureus, so that the synergistic effect of detection and sterilization is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensing, and particularly relates to a method for detecting and killing Staphylococcus aureus by an optoelectrochemical sensor based on hollow Au-TiO₂. Background Art

[0002] Bacterial infection is a public health issue of global concern, seriously affecting people's physical health and life safety, and should be regarded as an urgent priority for intervention in the global health community. Among them, Staphylococcus aureus, as a typical Gram-positive bacterium, is the most common foodborne pathogen. As an infamous pathogen, it can produce a variety of virulence factors, has strong environmental adaptability, can produce membrane-damaging toxins, toxins that interfere with receptor function but do not damage the membrane, and secreted enzymes, often contaminating foods such as milk, meat, fish, and canned foods, thus causing diseases such as food poisoning. Moreover, it is predicted that the number of deaths caused by wound infections alone caused by bacteria is increasing. A typical example of bacterial infection is a wound accompanied by severe infection and a slow healing process. In severe cases, complex infectious complications may occur, such as systemic inflammatory response syndrome, sepsis, etc., and even the risk of amputation may be faced. Therefore, timely monitoring of bacterial infection is of great significance for the management and treatment of corresponding diseases, while avoiding the abuse of antibiotics.

[0003] In recent years, traditional methods for detecting Staphylococcus aureus include plate counting method, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction technology (PCR), and high-performance liquid chromatography (HPLC), etc. However, these methods usually require a long detection time, expensive instruments, and cumbersome operations, and even false positives may occur. Therefore, establishing a detection method for Staphylococcus aureus with high sensitivity, high specificity, rapidity, and low cost, while ensuring accuracy, improving detection efficiency, and reducing detection costs, is of great significance for the control and prevention of bacterial infections and wound healing complications and for promoting the development of clinical diagnostics and therapeutics.

[0004] Photoelectrochemical (PEC) sensing is an emerging technology that uses light as the excitation energy to convert biological information into photocurrent for detecting biochemical information (such as the concentration of target substances) in organisms. It has advantages such as high sensitivity, low background signal, and simple equipment requirements, and is very suitable for the highly sensitive detection of foodborne pathogens. As is well known, heterojunction nanomaterials play an important role in constructing high-performance PEC biosensors. Therefore, designing and synthesizing novel and excellent heterojunction nanomaterials will be a challenge for researchers. TiO2 is a typical n-type semiconductor material. After long-term exploration by researchers, it has been found that TiO2 can be well applied in the field of PEC sensors due to its inherent properties such as photo-electroactivity, chemical and mechanical stability, good catalytic ability, low cost, non-toxic and harmless nature, and high biocompatibility. However, TiO2 has a relatively wide bandgap (3.0 - 3.3 eV), so it can only absorb 3 - 5% of the ultraviolet light in sunlight, which means that the light utilization rate of TiO2 is low, resulting in slow transport of photo-generated carriers. Moreover, single crystal phases lead to rapid recombination of photo-generated electron-hole pairs. These disadvantages limit the application of TiO2 in PEC, greatly reducing its efficiency and stability in detecting bacteria, and there are problems such as weak anti-interference ability and high false positive risk. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for detecting and killing Staphylococcus aureus using a photoelectrochemical sensor based on hollow Au-TiO2. This method can achieve sensitive detection of Staphylococcus aureus, improve the detection efficiency. In addition, it can effectively kill bacteria and realize the synergistic effect of detection and sterilization, which is of great significance for the early prevention and diagnosis of diseases and is expected to achieve the integration of disease detection and treatment.

[0006] To achieve the above-mentioned invention purpose, the technical solution adopted by the present invention is as follows:

[0007] A method for detecting and killing Staphylococcus aureus using a photoelectrochemical sensor based on hollow Au-TiO2, comprising the following steps:

[0008] S1. Prepare hollow Au-TiO2 nanozyme;

[0009] S1-1. Prepare hollow TiO2 nanospheres by a hydrothermal method using titanium tetrafluoride as the precursor;

[0010] S1-2. In-situ reduce HAuCl4·3H2O on the hollow TiO2 nanospheres prepared in step S1-1 to prepare hollow Au-TiO2 nanozyme;

[0011] S2. Construct a photoelectrochemical sensor substrate to detect and kill Staphylococcus aureus;

[0012] S2-1. The hollow Au-TiO₂ nanozyme was uniformly coated on the cleaned FTO electrode by the drop-coating method, dried and then calcined to obtain the hollow Au-TiO₂ / FTO electrode, which was used as the photoelectrochemical sensor substrate;

[0013] S2-2. The hollow Au-TiO₂ nanozyme prepared in step S1 was subjected to carboxyl functionalization, and the Staphylococcus aureus aptamer was subjected to amino functionalization. Then, the carboxyl-functionalized hollow Au-TiO₂ nanozyme was coupled with the amino-functionalized Staphylococcus aureus aptamer to prepare the hollow Au-TiO₂@Apt signal probe;

[0014] S2-3. The amino-functionalized Staphylococcus aureus aptamer was immobilized on the surface of the hollow Au-TiO₂ / FTO electrode prepared in step S2-1 by amidation reaction to obtain the Apt / TGA / hollow Au-TiO₂ / FTO electrode;

[0015] S2-4. When Staphylococcus aureus was detected on the surface of the Apt / TGA / hollow Au-TiO₂ / FTO electrode obtained in step S2-3, the amino-functionalized Staphylococcus aureus aptamer immobilized on the electrode specifically recognized and bound to Staphylococcus aureus, and then specifically bound to the hollow Au-TiO₂@Apt signal probe prepared in step S2-2 to construct a "sandwich" structure, resulting in a decrease in the photoelectric signal;

[0016] S2-5. The electrode obtained in step S2-4 was reacted with H₂O₂ and 4-chloro-1-naphthol to form a purple precipitate, which blocked the active sites of the sensor substrate, further reducing the photoelectric signal. At the same time, H₂O₂ was catalytically decomposed to generate highly toxic ·OH to kill Staphylococcus aureus.

[0017] Furthermore, the specific steps of step S1-1 are as follows:

[0018] a. PVP and TiF₄ were respectively dissolved in deionized water to prepare a PVP aqueous solution and a TiF₄ aqueous solution;

[0019] b. The prepared PVP aqueous solution and TiF₄ aqueous solution were mixed with acidified absolute ethanol, and then transferred to a liner and left to stand at room temperature for a period of time;

[0020] c. The liner was placed in an autoclave and heated for a period of time;

[0021] d. The reaction product was cooled to room temperature, then centrifuged, and the precipitate obtained after centrifugation was washed with deionized water and ethanol. After removing the supernatant, it was dried to obtain hollow TiO₂ nanospheres.

[0022] Further, the specific steps of step S1-2 are as follows: Disperse the hollow TiO2 nanospheres prepared in step S1-1 in deionized water, then add HAuCl4 and stir at room temperature to obtain a mixed solution; dropwise add an ice aqueous solution of NaBH4 to the mixed solution; centrifuge to remove the supernatant to obtain a precipitate, which is the hollow Au-TiO2 nanozyme, and redisperse it in deionized water for standby.

[0023] Further, the specific steps of step S2-1 are as follows:

[0024] a. Wash the FTO electrode successively with acetone, absolute ethanol and deionized water, dry it, and fix the electrode with insulating tape to form an electrode working area with a certain area;

[0025] b. Ultrasonically disperse the hollow Au-TiO2 nanozyme prepared in step S1-2 in deionized water, then evenly coat it on the FTO electrode, dry it and calcine it in an air atmosphere for a period of time, and then cool it to room temperature to obtain a hollow Au-TiO2 / FTO electrode.

[0026] Further, the specific steps of step S2-2 are as follows:

[0027] a. First, add mercaptoacetic acid, EDC and NHS to the PBS solution containing the hollow Au-TiO2 nanozyme to obtain a mixed solution;

[0028] b. Add the Staphylococcus aureus aptamer to the mixed solution obtained in step a and incubate it at room temperature for a period of time to obtain a mixture;

[0029] c. Disperse the mixture obtained in step b in a PBS solution containing BSA to remove non-specific binding sites, and incubate it at room temperature for a period of time; then wash it and disperse it in PBS and store it at 4°C to prepare a hollow Au-TiO2@Apt signal probe.

[0030] Further, the specific steps of step S2-3 are as follows:

[0031] a. Spin-coat a layer of mercaptoacetic acid on the surface of the hollow Au-TiO2 / FTO electrode obtained in step S2-1, incubate it, and the mercapto group of mercaptoacetic acid self-assembles on the surface of the hollow Au-TiO2 / FTO electrode through the action of Au-S chemical bonds to form a TGA / hollow Au-TiO2 / FTO electrode;

[0032] b. Add EDC and NHS to the TGA / hollow Au-TiO₂ / FTO electrode obtained in step a, activate the carboxyl group of mercaptoacetic acid at room temperature, wash away the excess EDC and NHS, and then drop the Staphylococcus aureus aptamer onto the TGA / hollow Au-TiO₂ / FTO electrode to form the Apt / TGA / hollow Au-TiO₂ / FTO electrode;

[0033] c. After incubating at 4 °C for a period of time, wash the Apt / TGA / hollow Au-TiO₂ / FTO with PBS to remove the physically adsorbed aptamer; finally, drop the BSA solution onto the Apt / TGA / hollow Au-TiO₂ / FTO electrode and incubate for a period of time to block the non-specific binding sites.

[0034] Furthermore, the specific steps of step S2-4 are as follows: After washing the electrode with PBS, when Staphylococcus aureus is detected on the surface of the Apt / TGA / hollow Au-TiO₂ / FTO electrode, the aptamer on the Apt / TGA / hollow Au-TiO₂ / FTO electrode specifically recognizes and binds to Staphylococcus aureus, and then the Apt / TGA / hollow Au-TiO₂ / FTO electrode is placed in the solution containing the hollow Au-TiO₂@Apt signal probe prepared in step S2-2 and incubated at 37 °C for a period of time to construct a "sandwich" structure, resulting in a decrease in the optoelectronic signal.

[0035] Furthermore, the specific steps of step S2-5 are as follows: Incubate the electrode obtained in step S2-4 with a 4-chloro-1-naphthol solution containing H₂O₂ at room temperature to trigger the mimetic enzyme-catalyzed precipitation reaction. The generated precipitate blocks the active sites of the sensor substrate. At the same time, H₂O₂ is catalytically decomposed to produce highly toxic ·OH; finally, prepare an electrolyte solution by mixing PBS and ascorbic acid, measure the photocurrent under a xenon lamp and an electrochemical workstation, and there will be a significant decrease in the optoelectronic signal. Observe the bacterial morphology on the electrode with a scanning electron microscope. The surface of the bacteria after detection will become wrinkled and then collapse due to the action of ·OH, indicating that ·OH kills Staphylococcus aureus.

[0036] The present invention develops hollow TiO2 with uniform size and large specific surface area, and through in-situ reduction, Au NPs are grown on its surface to form hollow Au-TiO2 nanozymes, and a multifunctional photoelectrochemical sensing platform is constructed with this as the sensor substrate. This sensor substrate utilizes the Schottky barrier and the local surface plasmon resonance effect of Au NPs to synergistically improve the mobility of photo-generated carriers, enabling it to have excellent photoelectric conversion efficiency. In addition, this sensor substrate has strong peroxidase-like activity, so its catalytic reaction can be used to achieve photoelectric signal amplification. On the one hand, the aptamer-modified hollow Au-TiO2 weakens the photocurrent of the immunosensor by hindering the transfer and diffusion of electrons to the surface of the photoelectrode, generating a steric hindrance effect; on the other hand, in the presence of H2O2, the hollow Au-TiO2 can catalyze the oxidation of 4-chloro-1-naphthol on the FTO electrode to form an insulating precipitate, further reducing the photocurrent signal, indicating the detection of Staphylococcus aureus. At the same time, H2O2 is catalytically decomposed to generate highly toxic ·OH. Therefore, after detecting foodborne pathogenic bacteria, it can quickly and completely kill the bacteria, achieving highly sensitive detection and effective killing of Staphylococcus aureus in food.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] The present invention constructs a multifunctional photoelectrochemical sensing platform with hollow Au-TiO2 nanozymes as the substrate to achieve highly sensitive detection and effective killing of Staphylococcus aureus in food. The preparation method of the present invention is simple, low-cost, and has high biocompatibility. Based on the immobilization of Staphylococcus aureus on the surface of the FTO electrode, the excellent peroxidase-like activity of the hollow Au-TiO2@Apt signal probe quickly converts the active substance into a precipitate, hindering the active sites of the sensor substrate, and magnifying the photocurrent signal of the captured Staphylococcus aureus by several times. The detection range is as wide as 10-10 8 CFU / mL, and the detection limit is as low as 1.09 CFU / mL; the hollow Au-TiO2@Apt signal probe in the photoelectrochemical sensing platform of the present invention can catalyze H2O2 into highly toxic ·OH, thereby killing Staphylococcus aureus, and the sterilization rate reaches 97.06%. It provides a new idea for the field of bacterial sensing and has broad application prospects. Description of the Drawings

[0039] Figure 1 It is the flow chart (b) of preparing hollow Au-TiO2 nanozymes (a) and constructing a multifunctional photoelectrochemical biosensing platform in the embodiment of the present invention;

[0040] Figure 2 It is the SEM image (a) and TEM image (b) of the hollow Au-TiO2 nanozymes prepared in the embodiment of the present invention;

[0041] Figure 3 These are the photoelectrochemical performance effect diagrams of the hollow TiO2 nanospheres and hollow Au-TiO2 nanozymes prepared in the embodiments of the present invention;

[0042] Figure 4 These are the peroxidase-like activity effect diagrams of the hollow Au-TiO2 nanozymes prepared in the embodiments of the present invention;

[0043] Figure 5 These are the result diagrams of detecting different concentrations of Staphylococcus aureus by the photoelectrochemical biosensing platform based on hollow Au-TiO2 prepared in the embodiments of the present invention. Among them, (a) is the photoelectric response diagram of the developed photoelectrochemical biosensor to different concentrations of Staphylococcus aureus (10-10 8 CFU / mL), and (b) is the linear relationship diagram between the logarithm of the Staphylococcus aureus concentration and the photocurrent signal;

[0044] Figure 6 These are the clearance effect diagrams of the photoelectrochemical biosensing platform based on hollow Au-TiO2 prepared in the embodiments of the present invention for Staphylococcus aureus;

[0045] Figure 7 These are the SEM diagrams of the photoelectrochemical biosensing platform based on hollow Au-TiO2 prepared in the embodiments of the present invention after killing Staphylococcus aureus. Detailed implementation manners

[0046] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0047] The raw materials and reagents used in the following examples are all commercially available from regular suppliers unless otherwise specified.

[0048] Example

[0049] As Figure 1 shown, a method for detecting and killing Staphylococcus aureus by a photoelectrochemical sensor based on hollow Au-TiO2 includes the following steps:

[0050] S1. Prepare hollow Au-TiO2 nanozyme as a biosensing substrate. The schematic diagram is shown in Figure 1 (a)

[0051] S1-1. Dissolve 9.75 mg of PVP and 12.39 mg of TiF4 in 4 mL and 2.5 mL of deionized water respectively to prepare an aqueous PVP solution and an aqueous TiF4 solution. Secondly, mix the above-prepared aqueous PVP solution and aqueous TiF4 solution with 27.6 mL of anhydrous ethanol acidified by HCl (0.1 M 250 μL), and then transfer it to a polytetrafluoroethylene liner and let it stand at room temperature for 1 h. Then, place the liner into a stainless steel autoclave and heat it at 180 °C for 3 hours. Finally, cool the reaction product to room temperature, wash it 3 times with deionized water and ethanol by centrifugation, remove the supernatant, and place it in an oven at 60 °C to dry to obtain pure hollow TiO2 nanospheres in the form of a milky white powder;

[0052] S1-2. Disperse the hollow TiO2 nanospheres (10 mg) prepared in step S1-1 in 20 mL of deionized water. Secondly, add HAuCl4·3H2O (10 mM, 100 μL) and stir at room temperature for 1 min to obtain a mixed solution. Then, dropwise add the prepared ice-cold aqueous solution of NaBH4 (1 M, 50 μL) to this mixed solution. Finally, place the mixture in a centrifuge tube and centrifuge at 10000 rpm for 10 min to remove the supernatant to obtain a precipitate, which is the hollow Au-TiO2 nanozyme, and redisperse it in deionized water for standby;

[0053] S2. Construct a photoelectrochemical sensor substrate to detect and kill Staphylococcus aureus. The schematic diagram is shown in Figure 1 (b)

[0054] S2-1. Wash the FTO electrode successively with acetone, anhydrous ethanol and deionized water, dry it, and fix the electrode with insulating tape to form a 1.0 cm 2 working area of the electrode. Ultrasonically disperse the hollow Au-TiO2 nanozyme prepared in step S1-2 in deionized water, and then evenly coat it on the FTO electrode and dry it at 37 °C. After drying, calcine it at 400 °C in an air atmosphere at a heating rate of 5 °C / min for 1.5 h, and then cool it to room temperature at a rate of 5 °C / min to construct a hollow Au-TiO2 / FTO electrode as the photoelectrochemical sensor substrate;

[0055] S2-2: First, add 0.1 M mercaptoacetic acid, EDC, and NHS to a PBS solution (5 mL, pH = 7.4) containing 1.0 mg of hollow Au-TiO₂ nanozyme to obtain a mixed solution; then add 100 μL of Staphylococcus aureus aptamer (0.1 mM) to the mixed solution and incubate for 6 hours at room temperature to obtain a mixture; then disperse the mixture in a PBS solution (1 mL) containing 1% BSA to remove non-specific binding sites and incubate for 1 h at room temperature; finally, wash and then disperse in 1 mL of PBS and store at 4 °C to prepare the hollow Au-TiO₂@Apt signal probe;

[0056] S2-3: Spin-coat a layer of mercaptoacetic acid (0.1 M, 30 μL) on the surface of the hollow Au-TiO₂ / FTO electrode obtained in step S2-1, incubate for 1 h, so that the mercapto group of mercaptoacetic acid can self-assemble on the surface of this electrode through the action of Au-S chemical bonds to form a TGA / hollow Au-TiO₂ / FTO electrode; at the same time, add 0.1 M, 20 μL of EDC and NHS to activate the carboxyl group of TGA at room temperature for 1 h. After washing off the excess EDC and NHS, drop 30 μL of Staphylococcus aureus aptamer onto the TGA / hollow Au-TiO₂ / FTO electrode to form an Apt / TGA / hollow Au-TiO₂ / FTO electrode; after incubating for 12 h at 4 °C, wash the Apt / TGA / hollow Au-TiO₂ / FTO electrode with PBS to remove the physically adsorbed aptamer; to block non-specific binding sites, drop 20 μL of 1% BSA solution onto this electrode and incubate for 1 h;

[0057] S2-4: When different concentrations of Staphylococcus aureus are detected on the surface of the Apt / TGA / hollow Au-TiO₂ / FTO electrode, the aptamer on the Apt / TGA / hollow Au-TiO₂ / FTO electrode specifically recognizes and binds to Staphylococcus aureus, and then place the Apt / TGA / hollow Au-TiO₂ / FTO electrode in a solution containing the hollow Au-TiO₂@Apt signal probe prepared in step S2-2 and incubate for a period of time at 37 °C to construct a "sandwich" structure, resulting in a decrease in the optoelectronic signal;

[0058] S2-5. Incubate the electrode obtained in step S2-4 with a 10 mM 4-chloro-1-naphthol solution (containing 5 mM H2O2) at room temperature for 10 min to trigger the mimetic enzyme-catalyzed precipitation reaction. The generated precipitate blocks the active sites of the sensor substrate. Meanwhile, H2O2 is catalytically decomposed to produce highly toxic ·OH. Finally, prepare an electrolyte solution by mixing 50 mL of PBS and 0.8806 g of AA (0.1 M). Measure the photocurrent at 0 V under a xenon lamp (λ>400 nm) and an electrochemical workstation. The result will show a significant decrease in the photoelectric signal. Observe the bacterial morphology on the electrode with a scanning electron microscope. The surface of the bacteria after detection will become wrinkled and then collapse due to the action of ·OH, indicating that ·OH kills Staphylococcus aureus.

[0059] Characterize the morphology of the hollow Au-TiO2 nanozyme prepared in step S1-2 of this example: Observe the morphology of the hollow Au-TiO2 nanozyme using a scanning electron microscope and a transmission electron microscope. The results are shown in Figure 2 (a) and Figure 2 (b). As can be seen from Figure 2 (a), the hollow TiO2 presents a typical spherical structure, and ultra-small gold nanoparticles are evenly distributed on the hollow TiO2 nanospheres, indicating the successful preparation of the hollow Au-TiO2 nanozyme. As can be seen from Figure 2 (b), the image shows a rough surface and an obvious core-shell hollow structure inside. The color of the periphery of the material is darker, black, and the color of the central part is lighter, gray. Gold nanoparticles can also be found on the hollow nanospheres. The size of the gold nanoparticles is about 5.0 nm.

[0060] Test the optoelectronic properties of the hollow Au-TiO2 nanozyme prepared in this example: Uniformly coat the hollow Au-TiO2 nanozyme on an FTO electrode with an area of 1.0 cm 2 by the drop-casting method and dry it at 37 °C. After drying, calcine it at 400 °C for 1.5 h in an air atmosphere at a heating rate of 5 °C / min, and then cool it to room temperature at a rate of 5 °C / min. Use 0.1 M ascorbic acid as the electrolyte and measure its photocurrent with an electrochemical workstation under irradiation of a xenon lamp (λ>400 nm) and a voltage of 0 V. The result is shown in Figure 3 , when Au NPs grow on the hollow TiO2, compared with pure hollow TiO2, the optoelectronic signal is significantly improved and very stable, reaching 354 μA.

[0061] The peroxidase-like activity of the prepared hollow Au-TiO₂ nanozyme was tested: in the presence of H₂O₂, the peroxidase activity of hollow Au-TiO₂ was determined using the common nanozyme activity indicator TMB. A solution containing H₂O₂ (5 mM), TMB (10 mM), and acetate buffer (pH = 4.0) was incubated with hollow Au-TiO₂ at 37 °C for 5 min. The ultraviolet characteristic absorbance reflecting the oxidation product of TMB (oxTMB) was measured at 652 nm. The results are shown in Figure 4 , and the TMB + hollow Au-TiO₂, TMB + hollow TiO₂ + H₂O₂, and TMB + H₂O₂ systems showed almost no catalytic activity. Compared with the TMB + hollow TiO₂@Apt + H₂O₂ system, the absorbance of the TMB + hollow Au-TiO₂ + H₂O₂ and TMB + hollow Au-TiO₂@Apt + H₂O₂ systems increased significantly, TMB was oxidized to oxTMB, presenting a more distinct bright blue color, indicating that hollow Au-TiO₂ has strong catalytic activity.

[0062] Figure 5 Figure Figure 5 (a) shows that the photocurrent response decreases with the logarithmic increase of the target S. aureus concentration, because a sandwich structure and a steric hindrance effect mediated by nanozymes are constructed on the sensor. From Figure 5 (b), it can be seen that there is a linear relationship between the photocurrent in the concentration range of 10 to 10 8 CFU / mL, and the linear regression equation is y = -18.63 log C + 235.96, with a correlation coefficient (R 2 ) of 0.9943, and the detection limit (LOD) is as low as 1.09 CFU / mL (σ / S = 3).

[0063] The results of the photocatalytic inactivation of S. aureus by the hollow Au-TiO₂-based photoelectrochemical sensor were verified by the spread plate method: S. aureus bacteria were cultured in LB liquid medium under a constant temperature shaker for 16 h to obtain a seed culture. Then, the bacterial suspension was diluted to 1×10 4 CFU mL -1。Then, the constructed photoelectrochemical sensor was immersed in the diluted bacterial suspension for 30 min. After that, an equal volume of the bacterial suspension was divided into two groups. One group was added with H2O as the control group, and the other group was added with hollow Au-TiO2@Apt + H2O2. After incubation for 3 h, the bacterial solution (100 μL) was evenly spread on the LB agar plate. After incubation at 37 °C for 12 h, the single colonies on the LB solid medium were counted. The results are shown in Figure 6 。 Figure 6 The experimental results show that the photoelectrochemical sensor based on hollow Au-TiO2 constructed in this example can effectively kill bacteria compared with the control group.

[0064] The results of killing Staphylococcus aureus by the photoelectrochemical sensor based on hollow Au-TiO2 were verified by scanning electron microscopy. The results are shown in Figure 7 。In an environment of pure water or only the presence of Hollow TiO2, Staphylococcus aureus can still maintain a relatively smooth spherical shape and retain a complete bacterial membrane, indicating that these two systems have little obvious toxicity to the two bacteria. Since H2O2 itself has the function of oxidative disinfection and sterilization, even using a low concentration of H2O2 will cause some wrinkles and damage to the cell wall of the bacteria. However, the SEM images of bacteria exposed to hollow Au-TiO2@Apt + H2O2 show that these two bacterial strains suffered the most serious damage, indicating that the photoelectrochemical sensor can kill the bacteria after detecting them.

Claims

1. A method for detecting and killing Staphylococcus aureus based on a photoelectrochemical sensor of hollow Au-TiO2, characterized in that: The following steps are involved: S1, preparation of hollow Au-TiO2 nanozymes; S1-1, preparing hollow TiO2 nanospheres by hydrothermal method using titanium tetrafluoride as precursor; S1-2, in situ reducing HAuCl4·3H2O on the hollow TiO2 nanospheres prepared in step S1-1 to prepare hollow Au-TiO2 nanozymes; S2, constructing a photoelectrochemical sensor substrate to detect and kill Staphylococcus aureus; S2-1, using the drop coating method to evenly coat the hollow Au-TiO2 nanozyme on the cleaned FTO electrode, drying and calcining to obtain the hollow Au-TiO2 / FTO electrode, which is used as the photoelectrochemical sensor substrate; S2-2, performing carboxyl functionalization treatment on the hollow Au-TiO2 nanozyme prepared in step S1, performing amino treatment on the Staphylococcus aureus aptamer, and then coupling the carboxyl functionalized hollow Au-TiO2 nanozyme with the amino functionalized Staphylococcus aureus aptamer to prepare a hollow Au-TiO2@Apt signal probe; S2-3, using an amidation reaction to immobilize the amino-functionalized Staphylococcus aureus aptamer on the surface of the hollow Au-TiO2 / FTO electrode prepared in step S2-1 to prepare an Apt / TGA / hollow Au-TiO2 / FTO electrode; S2-4. When Staphylococcus aureus is detected on the surface of the Apt / TGA / hollow Au-TiO2 / FTO electrode obtained in step S2-3, the amino-functionalized Staphylococcus aureus aptamer fixed on the electrode specifically recognizes and binds to Staphylococcus aureus, and then specifically binds to the hollow Au-TiO2@Apt signal probe prepared in step S2-2, thereby constructing a "sandwich" structure to reduce the photoelectric signal; S2-5. The electrode obtained in step S2-4 is reacted with H2O2 and 4-chloro-1-naphthol to generate a purple precipitate. The precipitate blocks the active sites of the sensor substrate, further reducing the photoelectric signal. At the same time, H2O2 is catalytically decomposed to produce highly toxic ·OH to kill Staphylococcus aureus.

2. The method for detecting and killing Staphylococcus aureus based on a photoelectrochemical sensor of hollow Au-TiO2 according to claim 1, characterized in that: The specific steps of step S1-1 are: a. Dissolve PVP and TiF4 in deionized water to prepare PVP aqueous solution and TiF4 aqueous solution respectively; b. Mix the prepared PVP aqueous solution and TiF4 aqueous solution with the acidified anhydrous ethanol, transfer them to the liner, and let them stand at room temperature for a period of time; c. Place the liner into the autoclave and heat to react for a period of time; d. The reaction product was cooled to room temperature and then centrifuged. The precipitate obtained after centrifugation was washed with deionized water and ethanol. The supernatant was removed and then dried to obtain hollow TiO2 nanospheres.

3. The method for detecting and killing Staphylococcus aureus based on a photoelectrochemical sensor of hollow Au-TiO2 according to claim 1, characterized in that: The specific steps of step S1-2 are: disperse the hollow TiO2 nanospheres prepared in step S1-1 in deionized water, then add HAuCl4 and stir at room temperature to obtain a mixed solution; add NaBH4 ice water solution drop by drop into the mixed solution; centrifuge to remove the supernatant to obtain a precipitate, which is the hollow Au-TiO2 nanozyme, and redisperse it in deionized water for later use.

4. The method for detecting and killing Staphylococcus aureus based on a photoelectrochemical sensor of hollow Au-TiO2 according to claim 1, characterized in that: The specific steps of step S2-1 are: a. Wash the FTO electrode with acetone, anhydrous ethanol and deionized water in turn, dry it, and fix the electrode with insulating tape to form an electrode working area of ​​a certain area; b. Ultrasonicate the hollow Au-TiO2 nanozyme dispersed in deionized water prepared in step S1-2, evenly coat it on the FTO electrode, dry it, calcine it in air atmosphere for a period of time, and then cool it to room temperature to obtain a hollow Au-TiO2 / FTO electrode.

5. The method for detecting and killing Staphylococcus aureus based on a photoelectrochemical sensor of hollow Au-TiO2 according to claim 1, characterized in that: The specific steps of step S2-2 are: a. First, add thioglycolic acid, EDC and NHS into the PBS solution containing the hollow Au-TiO2 nanozyme to obtain a mixed solution; b. adding the Staphylococcus aureus aptamer to the mixed solution obtained in step a, and incubating at room temperature for a period of time to obtain a mixture; c. dispersing the mixture obtained in step b in a PBS solution containing BSA to remove non-specific binding sites, and incubating at room temperature for a period of time; Then it was washed, dispersed in PBS and stored at 4 °C to prepare the hollow Au-TiO2@Apt signal probe.

6. The method for detecting and killing Staphylococcus aureus based on a hollow Au-TiO2 photoelectrochemical sensor according to claim 1, characterized in that: The specific steps of step S2-3 are: a. Spin-coat a layer of thioglycolic acid on the surface of the hollow Au-TiO2 / FTO electrode obtained in step S2-1, and incubate. The thiol groups of thioglycolic acid self-assemble on the surface of the hollow Au-TiO2 / FTO electrode through the action of Au-S chemical bonds to form a TGA / hollow Au-TiO2 / FTO electrode; b. Add EDC and NHS to the TGA / hollow Au-TiO2 / FTO electrode obtained in step a, activate the carboxyl group of thioglycolic acid at room temperature, wash away excess EDC and NHS, and then drop the Staphylococcus aureus aptamer onto the TGA / hollow Au-TiO2 / FTO electrode to form an Apt / TGA / hollow Au-TiO2 / FTO electrode; c. After incubation at 4°C for a period of time, Apt / TGA / hollow Au-TiO2 / FTO was washed with PBS to remove the physically adsorbed aptamer; finally, BSA solution was dropped on the Apt / TGA / hollow Au-TiO2 / FTO electrode and incubated for a period of time to block nonspecific binding sites.

7. The method for detecting and killing Staphylococcus aureus based on a hollow Au-TiO2 photoelectrochemical sensor according to claim 1, characterized in that: The specific steps of step S2-4 are: after washing the electrode with PBS, when Staphylococcus aureus is detected on the surface of the Apt / TGA / hollow Au-TiO2 / FTO electrode, the aptamer on the Apt / TGA / hollow Au-TiO2 / FTO electrode specifically recognizes and binds to Staphylococcus aureus, and then the Apt / TGA / hollow Au-TiO2 / FTO electrode is placed in a solution containing the hollow Au-TiO2@Apt signal probe prepared in step S2-2, and incubated at 37°C for a period of time to construct a "sandwich" structure to reduce the photoelectric signal.

8. The method for detecting and killing Staphylococcus aureus based on a photoelectrochemical sensor of hollow Au-TiO2 according to claim 1, characterized in that: The specific steps of step S2-5 are: incubating the electrode obtained in step S2-4 with a 4-chloro-1-naphthol solution containing H2O2 at room temperature to trigger a simulated enzyme-catalyzed precipitation reaction, and the generated precipitate blocks the active sites of the sensor substrate. At the same time, H2O2 is catalytically decomposed to produce highly toxic ·OH; finally, PBS and ascorbic acid are mixed to prepare an electrolyte solution, and the photocurrent is measured under a xenon lamp and an electrochemical workstation. The result is that the photoelectric signal is significantly reduced, and the bacterial morphology on the electrode is observed using a scanning electron microscope. After detection, the bacterial surface becomes wrinkled due to the action of ·OH, and then collapses, indicating that ·OH kills Staphylococcus aureus.

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