Self-powered three-mode sensor based on double photoelectrodes, preparation method and application

Abstract

The invention discloses a preparation method and multifunctional application of a self-powered three-mode sensor based on double photoelectrodes, and belongs to the field of food-borne pathogenic bacterium detection. CuI / CuSCN is used as a photoelectric cathode material, NTW is used as a photoelectric anode material, the Fermi level difference between the two electrodes is utilized, bias voltage and an oxidation-reduction medium do not need to be additionally arranged, and the photoelectric sensor has the advantages of high light energy utilization rate, portability and anti-interference capability. Au NPs is further modified on the surface of the photoanode material, and an aptamer is connected through an Au-S bond to serve as a recognition element, so that a novel dual-photoelectrode self-energized aptamer sensor is constructed. Besides, photoelectrochemistry, electrochemistry and fluorescence three-mode sensing and photocatalytic sterilization technologies are introduced into the self-powered system, so that the detection accuracy and sensitivity are improved, and a feasible scheme is provided for detecting and killing food-borne pathogenic bacteria at the same time.

Description

Technical Field

[0001] The present invention belongs to the field of detection of foodborne pathogenic bacteria, and particularly relates to a preparation method and multifunctional application of a self-powered triple-mode sensor based on a dual-photoelectrode. Background Art

[0002] With the continuous increase in people's dependence on processed foods, the number of global foodborne diseases has been increasing year by year. There are dozens of foodborne pathogenic bacteria, among which the most common are Salmonella, Staphylococcus aureus, Campylobacter jejuni, Escherichia coli, Vibrio parahaemolyticus, Listeria monocytogenes, etc. Among them, food poisoning caused by Staphylococcus aureus is a foodborne disease caused by exotoxins or endotoxins produced in food. In addition, during the metabolism of foodborne pathogenic bacteria themselves, the metabolites produced will contaminate food, causing food spoilage and deterioration, thus endangering the health of consumers and directly or indirectly leading to illness. Therefore, the application of accurate and rapid detection and killing technologies to control the contamination of foodborne pathogenic bacteria has very important public health significance.

[0003] Traditional detection methods for foodborne pathogenic bacteria include the plate colony counting method. However, this method is time-consuming and cumbersome in operation and cannot meet the requirements of rapid detection. In addition, there are immunological detection methods based on the combination of antigens and antibodies, such as traditional enzyme-linked immunosorbent assay, fluorescence immunoassay, colloidal gold immunoassay, etc. These methods are simple in operation and short in detection time, but they are costly and low in sensitivity. As a highly sensitive, simple, rapid and low-cost detection method, the nanobiosensor is considered an alternative to traditional methods. The electrochemical biosensor has many advantages, such as fast response speed, convenient operation, low cost and high sensitivity. The optoelectrochemical biosensor is a new type of biosensor that combines optoelectronic materials and electrochemical technology and is an important direction for the development of future analysis technologies. As an innovative technology, the self-powered optoelectrochemical sensor has attracted extensive attention due to its incomparable characteristics, namely simple instrument, rapid response ability, superior sensing performance and environmental friendliness. The self-powered optoelectrochemical sensor is based on the fact that photo-generated electrons can spontaneously transfer from the photoanode to the photocathode through an external circuit due to the potential difference existing between the photoanode and the photocathode. As one of the important components in the self-powered optoelectrochemical sensor, the photoanode determines how much energy can be generated per unit time. In addition, the photocathode is a key factor for performance output. However, as a traditional photocathode, Pt has limitations such as slow migration of photo-excited electrons, low light absorption rate and high cost. Therefore, finding a photocathode to replace Pt and forming a dual-photoelectrode self-powered optoelectrochemical sensor provides new ideas for the preparation of high-efficiency sensors. The fluorescence sensor uses the property that a substance emits light with a longer wavelength after absorbing light of a specific wavelength for detection and has advantages such as high sensitivity, high selectivity, rapid response and strong anti-interference ability. Although these technologies have made remarkable developments, they mainly rely on single signal output and the accuracy may be interfered by external conditions. Compared with single-response systems, multi-mode sensors not only have higher sensitivity but also can avoid false positives and improve accuracy. However, when using these technologies for the detection of foodborne pathogenic bacteria, the problem of secondary contamination may be faced. Summary of the Invention

[0004] The object of the present invention is to provide a preparation method and multifunctional application of a self-powered triple-mode sensor based on a dual-photoelectrode aiming at the problems existing in the prior art. Using CuI / CuSCN as the photocathode material, N-Nb 2 CT x / TpPa-1 / m-WO 3-x(NTW) was used as the photoanode material, and gold nanoparticles (Au NPs) were modified on the surface of the photoanode material. The aptamer was connected by Au-S bonds as the recognition element to construct a novel dual-photoelectrode self-powered aptasensor. In addition, photoelectrochemical, electrochemical, and fluorescence triple-mode sensing and photocatalytic sterilization technologies were introduced into the self-powered system to achieve accurate detection and rapid killing of Staphylococcus aureus.

[0005] The complete technical solution of the present invention includes:

[0006] A self-powered triple-mode sensor based on a dual-photoelectrode, the sensor includes photoelectrochemical, electrochemical, and fluorescence three sensing modes, and the dual-photoelectrodes are a photocathode and a photoanode; the photocathode material is CuI / CuSCN, and the photoanode material is NTW.

[0007] Further, the photoanode material is N-Nb 2 CT x / TpPa-1 / m-WO 3-x , and the surface is modified with gold nanoparticles (Au NPs), and the aptamer is connected by Au-S bonds as the recognition element, where the aptamer is used as the capture unit, the gold nanoparticles are used as the connection unit, and the photoanode material is used as the killing unit.

[0008] Further, there is a Fermi level difference between the photoanode and the photocathode, and no external bias voltage and redox medium are required.

[0009] Further, for the photocathode material CuI / CuSCN, in the XRD pattern of CuI, diffraction peaks appear at 2θ values of 25.5°, 29.5°, 42.2°, 50.0°, 52.4°, 61.3°, 67.5°, and 69.5°, corresponding to (111), (200), (220), (311), (222), (400), (331), and (420) respectively; CuSCN has obvious diffraction peaks at 16.1° and 28.8°, corresponding to the 003 crystal plane and the 012 crystal plane respectively, and the rest of the diffraction peaks match the standard card.

[0010] Further, in the XRD pattern of Nb 2 CT x , the low-angle (002) peak is the characteristic peak of MXene; TpPa-1 shows a sharp peak at 5.1° and a broad peak at 26.6°, corresponding to (100) and (001) respectively; in the XRD pattern of m-WO 3-x , it is consistent with the standard XRD pattern.

[0011] Furthermore, for the application of the self-powered triple-mode sensor based on dual photoanodes, the dual photoanode self-powered sensor is used for triple-mode bacteria detection.

[0012] Using the photoanode as the working electrode, the photocathode as the counter electrode, and the saturated calomel electrode as the reference electrode, simulating sunlight with a xenon lamp, and connecting an electrochemical workstation to perform photoelectrochemical and electrochemical detections in PBS buffer solution (pH 7.4); using ExoⅠⅠⅠ as the cleavage enzyme and SYBR Gold as the fluorescent dye for fluorescence spectroscopy detection.

[0013] In the range of 3.57×10 2 CFU / mL to 3.57×10 8 CFU / mL, a linear relationship of ΔI = 10.51LogC - 13.71 with a correlation coefficient of 0.994 and a detection limit of 6.3 CFU / mL is adopted. According to the linear relationship between the change in photocurrent and the logarithm of the Staphylococcus aureus concentration, the concentration of Staphylococcus aureus is detected.

[0014] Furthermore, a linear relationship of ΔI = 55.10LogC - 126.73 with a correlation coefficient of 0.996 and a detection limit of 3.8 CFU / mL is adopted. According to the linear relationship between the change in current and the logarithm of the Staphylococcus aureus concentration, the concentration of Staphylococcus aureus is detected.

[0015] Furthermore, in the range of 3.57×10 2 CFU / mL to 3.57×10 8 CFU / mL, a linear relationship of FL = 22.57LogC - 1.32 with a correlation coefficient of 0.997 and a detection limit of 10.7 CFU / mL is adopted. According to the good linear relationship between the relative fluorescence intensity and the logarithm of the Staphylococcus aureus concentration, the concentration of Staphylococcus aureus is detected.

[0016] Furthermore, the dual photoanode self-powered sensor is used for photocatalytic sterilization.

[0017] Furthermore, the preparation method of the self-powered triple-mode sensor based on dual photoanodes includes the following steps:

[0018] (1) After using the photocathode materials CuI and CuSCN, a CuI / CuSCN / FTO photocathode is prepared.

[0019] (2) Using the photoanode materials NTW and Au NPs to prepare a photoanode.

[0020] (3) Using the photocathode and the photoanode to prepare a self-powered triple-mode sensor.

[0021] Furthermore, during the preparation of the photoanode material NTW, Au NPs were added to the surface of the NTW / FTO electrode. To avoid the formation of disulfide bonds, after the aptamer (Ap) was incubated in tris(2-carboxyethyl)phosphine (TCEP), the activated Ap was dropped onto the surface of the Au / NTW / FTO electrode and incubated, and then the unconnected Ap was rinsed with Tris-HCL to obtain Ap / Au / NTW / FTO.

[0022] Furthermore, to block non-specific active sites, bovine serum albumin (BSA) was drop-coated onto the surface of the Ap / Au / NTW / FTO electrode and sealed for a certain period of time, and then rinsed with PBS buffer to obtain the BSA / Ap / Au / NTW / FTO electrode.

[0023] Furthermore, the specific preparation steps of the self-powered triple-mode sensor based on a dual photoanode are as follows:

[0024] (1) Preparation of the photocathode material CuI: A certain volume of a 0.2 M KI solution was added dropwise to a certain volume of a 0.08 M CuCl 2 solution, and then a certain mass of PVP dissolved in a certain volume of deionized water was added. The mixed solution was stirred evenly using a magnetic stirrer, placed in a high-pressure reaction kettle, and the reaction kettle was placed in an oven at 200 °C for 6 h. After the reaction kettle cooled to room temperature, it was washed several times with absolute ethanol and deionized water and then centrifuged, and dried in an oven at 60 °C for 12 h to obtain CuI.

[0025] Furthermore, in the step (1), the volume of the KI solution is 4 - 12 mL, and the volume of the CuCl 2 solution is 5 - 15 mL, the volume of deionized water is 50 - 150 mL, and the mass of PVP is 0.05 - 0.15 g.

[0026] (2) Preparation of the photocathode material CuSCN: A certain volume of a 0.2 M NaSCN solution was slowly added dropwise to a certain volume of a 0.2 M CuSO 4 ·5H 2 O solution. During the continuous stirring using a magnetic stirrer, the black precipitate gradually changed to a brown precipitate. Then a certain volume of the surfactant polyethylene glycol 400 was added, and stirring was continued until the mixture was homogeneous. The above mixed solution was transferred to a high-pressure reaction kettle and reacted in an oven at 80 °C for 24 h. After the reaction kettle cooled to room temperature, the obtained product was washed several times with absolute ethanol and then centrifuged, and dried in an oven at 60 °C for 12 h to obtain CuSCN.

[0027] Furthermore, in the step (2), the volume of the NaSCN solution is 20 - 60 mL, and the CuSO 4 ·5H2 The volume of the O solution is 10 - 30 mL, and the volume of polyethylene glycol 400 is 1 - 3 mL.

[0028] (3) Preparation of the photocathode: Weigh a certain amount of CuI powder and add it to a certain volume of ethyl sulfide, and use a magnetic stirrer to stir it evenly. The preparation method of the CuSCN solution is the same as that of CuI. After mixing the two in a 1:1 ratio, perform ultrasonic bath for 24 h. The obtained product is washed multiple times with absolute ethanol and deionized water and then centrifuged, and dried in an oven at 60 °C for 12 h to obtain CuI / CuSCN. Prepare a 10 mg / mL CuI / CuSCN suspension, then grind the suspension thoroughly for about 30 min, and drop a certain volume of the CuI / CuSCN suspension on a fluorine-doped tin oxide (FTO) electrode, and dry it at room temperature to obtain a CuI / CuSCN / FTO photocathode;

[0029] Further, in the step (3), the mass of CuI is 0.2 - 0.6 g, the volume of CuI is 10 - 30 mL, the mass of CuSCN is 0.2 - 0.6 g, the volume of CuSCN is 10 - 30 mL, and the volume of the CuI / CuSCN suspension is 30 μL.

[0030] (4) Preparation of the photoanode material m-WO 3-x : First, add a certain mass of tungsten hexachloride (WCl 6 ) to a certain volume of absolute ethanol under ultrasonic conditions to form a yellow clear solution. Subsequently, add a certain volume of acetylacetone, and the yellow solution immediately turns dark blue. Slowly add a certain mass of mesoporous silica (KIT-6), and after 2 h of continuous ultrasonic treatment, transfer the above solution to a high-pressure reaction kettle. React in an oven at 150 °C for 24 h, cool to room temperature, wash with deionized water and ethanol in sequence, and then dry in an oven at 60 °C. Immerse the obtained solid product in a 10% mass fraction hydrofluoric acid solution for 6 h to remove KIT-6, then separate and centrifuge, and wash with deionized water until the pH value is neutral. Wash the sample with absolute methanol 3 - 5 times, centrifuge, and dry in an oven at 70 °C for 18 h to obtain m-WO 3-x ;

[0031] Further, in the step (4), the mass of WCl 6 is 0.4 - 1.2 g, the volume of absolute ethanol is 50 - 150 mL, the volume of acetylacetone is 0.6 - 1.8 mL, and the mass of KIT-6 is 0.2 - 0.6 g.

[0032] (5) Preparation of the photoanode material Nb 2 CT x : Weigh a certain mass of Nb 2AlC was added to a certain volume of hydrofluoric acid solution with a mass fraction of 40%, and stirred at 55 °C. After the reaction ended, the prepared solid was washed and centrifuged with deionized water multiple times until the pH value of the solution was slightly neutral. The centrifuged precipitate was dried to collect the product. Then, it was dispersed in a certain volume of tetramethylammonium hydroxide solution with a mass fraction of 26% at room temperature for a period of time to form a suspension. The suspension was centrifuged and washed with deionized water to remove the residual tetramethylammonium hydroxide solution. Then, the obtained solid was redispersed in deionized water and ultrasonicated for 1 h. After ultrasonic treatment, it was centrifuged, washed, and dried at a low speed to obtain monolayer or few-layer Nb 2 CT x . A certain mass of Nb 2 CT x was dissolved in deionized water, stirred for 10 min, and then a certain volume of ethanol was added and continuously stirred. Then, a certain volume of triethoxysilane was added, and the mixture was stirred under a nitrogen atmosphere for 24 h. The obtained product was washed three times with ethanol to remove the unreacted triethoxysilane, and washed three times with deionized water to remove ethanol. The precipitate was freeze-dried to obtain amino-functionalized Nb 2 CT x (N-Nb 2 CT x );

[0033] Further, in the step (5), the mass of Nb 2 AlC is 1 - 3 g, the volume of the hydrofluoric acid solution is 60 - 180 mL, the volume of the tetramethylammonium hydroxide solution is 5 - 15 mL, the mass of Nb 2 CT x is 1 - 3 g, the volume of ethanol is 3 - 9 g, and the volume of triethoxysilane is 200 - 600 μL.

[0034] (6) Preparation of the photoanode material NTW: A certain mass of N-Nb 2 CT x and m-WO 3-x were added to a reaction kettle containing a certain volume of N,N-dimethylformamide (DMF). Trihydroxybenzene and p-phenylenediamine with a certain mass, mesitylene and 1,4-dioxane with a certain volume were added in sequence, and then a certain volume of 3M acetic acid aqueous solution was added as a catalyst. The mixture was ultrasonicated for 30 min to make it disperse evenly, and then transferred to a high-pressure reaction kettle. Nitrogen was bubbled through it for 10 min, and the reaction kettle was placed in an oven at 120 °C for reaction for 72 h. After the reaction kettle cooled to room temperature, it was washed three times with 1,4-dioxane and ethanol and then centrifuged and separated. Finally, it was freeze-dried for 12 h to obtain NTW.

[0035] Further, in the step (6), N-Nb 2 CTx with a mass of 27 - 81 g, m-WO 3-x with a mass of 90 - 270 g, a DMF volume of 3 - 9 mL, a phloroglucinol tricarboxaldehyde mass of 63 - 189 mg, a p-phenylenediamine mass of 48 - 144 mg, both a mesitylene and a 1,4-dioxane volume of 1.5 - 4.5 mL, and an acetic acid aqueous solution volume of 0.5 - 1.5 mL.

[0036] (7) Preparation of the photoanode material Au NPs: Inject a certain volume of 1 mmol / L HAuCl 4 solution into a two-necked flask, then place it in an oil bath and heat it to 120 °C for reflux, and continuously stir. When the reflux starts, quickly add a certain volume of sodium citrate solution (38.8 mmol / L). Stop heating when the solution color turns into a stable wine red color, continuously stir until it cools to room temperature, and then store it in a 4 °C refrigerator.

[0037] Further, in the step (7), the volume of the HAuCl 4 solution is 100 - 300 mL, and the volume of the sodium citrate solution is 10 - 30 mL.

[0038] (8) Preparation of the photoanode: First, prepare a 10 mg / mL NTW suspension and grind it thoroughly for about 30 min. Then, drop a certain volume of the NTW suspension onto the FTO electrode. Place it at room temperature to dry naturally to obtain the NTW / FTO electrode. Drop a certain volume of Au NPs onto the electrode surface and name it the Au / NTW / FTO electrode. To avoid the formation of disulfide bonds, incubate 10 μM aptamer (Ap) in 1.2 mM tris(2-carboxyethyl)phosphine (TCEP) at 4 °C for 1 h. Drop a certain volume of the activated Ap onto the surface of the Au / NTW / FTO electrode and incubate it at 4 °C for 12 h. Rinse off the unconnected Ap with Tris-HCL to obtain Ap / Au / NTW / FTO. To block non-specific active sites, drop a certain volume of 1% bovine serum albumin (BSA) by mass percentage onto the surface of the Ap / Au / NTW / FTO electrode and block it at 4 °C for 1 h. After rinsing with PBS buffer, obtain the BSA / Ap / Au / NTW / FTO electrode. Drop a certain volume of 10 μM complementary strand of the aptamer (cDNA) onto the electrode and incubate it at 4 °C for 1 h, and name it the cDNA / BSA / Ap / Au / NTW / FTO electrode. Prepare a 10 mM methylene blue (MB) solution, pipette a certain volume of the MB solution onto the electrode and incubate it for 30 min to prepare the MB / cDNA / BSA / Ap / Au / NTW / FTO electrode. Then, add a certain volume of different concentrations of Staphylococcus aureus suspension and incubate it for 1 h, and name it the SA / MB / cDNA / BSA / Ap / Au / NTW / FTO electrode. Drop a certain volume of Exo ⅠⅠⅠ containing 30 U onto the electrode and incubate it for 2 h, and name it Exo ⅠⅠⅠ / SA / MB / cDNA / BSA / Ap / Au / NTW / FTO.

[0039] Further, in the step (8), the volume of the NTW suspension is 30 μL, the volume of the Au NPs is 30 μL, the volume of the Ap is 30 μL, the volume of the BSA is 30 μL, the volume of the cDNA is 30 μL, the volume of the MB is 30 μL, the volume of the Staphylococcus aureus is 30 μL, the concentration of the Staphylococcus aureus is 3.57×10 2 CFU / mL to 3.57×10 8 CFU / mL, and the volume of the Exo ⅠⅠⅠ is 30 μL.

[0040] Prepare the photocathode and photoanode of the dual-photoelectrode according to the above preparation method.

[0041] The preparation method and multifunctional application of the self-powered triple-mode sensor based on the dual-photoelectrode, wherein the multifunctional application is bacteria detection and sterilization, and the bacteria is Staphylococcus aureus.

[0042] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0043] (1) The present invention constructs a dual-photoelectrode self-powered sensor. Utilizing the Fermi level difference between two electrodes, without the need for an external bias voltage and redox mediator, it has high light energy utilization efficiency, portability, and anti-interference ability, providing a new idea for the development of a novel and efficient sensing platform.

[0044] (2) The present invention uses aptamers as recognition elements to construct a triple-mode detection of photoelectrochemical, electrochemical, and fluorescence sensors, improving the detection accuracy and sensitivity, and providing strong support for the sensitive detection of foodborne pathogenic bacteria.

[0045] (3) The present invention uses aptamers as capture units, gold nanoparticles as linking units, and a photoanode material as a killing unit to further construct a multifunctional application platform integrating bacteria detection and killing, providing a feasible solution for simultaneously detecting and killing foodborne pathogenic bacteria. Description of the Drawings

[0046] Figure 1 (a) in [the figure] is the XRD pattern of the photocathode material CuI / CuSCN, Figure 1 (b) in [the figure] is the XRD pattern of the photoanode material NTW.

[0047] Figure 2 ([The figure]) is a schematic diagram of the dual-photoelectrode self-powered sensor.

[0048] Figure 3 ([The figure]) is the photocurrent response curve of different electrodes.

[0049] Figure 4 ([The figure]) is the photocurrent of the photoanode. In the figure:

[0050] a: NTW / FTO,

[0051] b: Au / NTW / FTO,

[0052] c: Ap / Au / NTW / FTO,

[0053] d: BSA / Ap / Au / NTW / FTO,

[0054] e: cDNA / BSA / Ap / Au / NTW / FTO,

[0055] f: MB / cDNA / BSA / Ap / Au / NTW / FTO,

[0056] g: SA / MB / cDNA / BSA / Ap / Au / NTW / FTO.

[0057] Figure 5(a) among them is the photocurrent curve of Staphylococcus aureus at different concentrations, Figure 5 (b) among them is the detection calibration curve. From a to g in the figure are respectively: 3.57×10 2 CFU / mL → 3.57×10 8 CFU / mL.

[0058] Figure 6 (a) among them is the current curve of Staphylococcus aureus at different concentrations, Figure 6 (b) among them is the detection calibration curve. From a to g in the figure are respectively: 3.57×10 2 CFU / mL → 3.57×10 8 CFU / mL.

[0059] Figure 7 (a) among them is the fluorescence spectrogram of Staphylococcus aureus at different concentrations, Figure 7 (b) among them is the detection calibration curve. From a to g in the figure are respectively: 3.57×10 2 CFU / mL → 3.57×10 8 CFU / mL.

[0060] Figure 8 (a) among them is the photocatalytic sterilization efficiency diagram of the photoanode, Figure 8 (b) among them is the colony photo. Specific implementation manners

[0061] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only for illustration and are not used to limit the present application.

[0062] The present invention will be described in detail below in conjunction with the embodiments and the accompanying drawings. However, it should be understood that the embodiments and the accompanying drawings are only used for exemplary description of the present invention and cannot constitute any limitation to the protection scope of the present invention. All reasonable transformations and combinations within the scope of the inventive concept of the present invention fall within the protection scope of the present invention.

[0063] 1. Preparation method of the photocathode, the steps are as follows:

[0064] (1) Gradually add 4 mL of 0.2 M KI solution dropwise to 5 mL of 0.08 M CuCl 2 solution, then add 0.05 g of PVP dissolved in 50 mL of deionized water, stir the mixed solution evenly with a magnetic stirrer, place the mixed solution in a high-pressure reaction kettle, and put the reaction kettle into an oven at 200°C for reaction for 6 h. After the reaction kettle cools to room temperature, wash it with anhydrous ethanol and deionized water multiple times and then centrifuge it, and dry it in an oven at 60°C for 12 h to obtain CuI.

[0065] (2) Slowly add 20 mL of 0.2 M NaSCN solution dropwise to 10 mL of 0.2 M CuSO 4 ·5H 2 O solution. During the process of continuously stirring with a magnetic stirrer, the black precipitate gradually turns into a brown precipitate. Then add 1 mL of the surfactant polyethylene glycol 400 and continue stirring until evenly mixed. Transfer the above mixed solution to a high-pressure reactor and react in an oven at 80 °C for 24 h. After the reactor cools to room temperature, the obtained product is washed repeatedly with anhydrous ethanol and then centrifuged, and dried in an oven at 60 °C for 12 h to obtain CuSCN.

[0066] (3) Weigh 0.2 g of CuI powder into 10 mL of ethyl sulfide and stir it evenly with a magnetic stirrer. Similarly, weigh 0.2 g of CuSCN powder into 10 mL of ethyl sulfide and stir it evenly. Mix the two in a 1:1 ratio and perform ultrasonic bath for 24

[0067] h. The obtained product is washed repeatedly with anhydrous ethanol and deionized water and then centrifuged, and dried in an oven at 60 °C for 12 h to obtain CuI / CuSCN. Prepare a 10 mg / mL CuI / CuSCN suspension, then grind the suspension sufficiently for about 30 min, and drop 30 μL of the CuI / CuSCN suspension on the FTO electrode and dry it at room temperature to obtain the CuI / CuSCN / FTO photoanode.

[0068] Figure 1 (a) XRD pattern of the photoanode material CuI / CuSCN. In the XRD pattern of CuI, the main diffraction peaks appear at 2θ values of 25.5°, 29.5°, 42.2°, 50.0°, 52.4°, 61.3°, 67.5° and 69.5°, corresponding to (111), (200), (220), (311), (222), (400), (331) and (420) respectively. CuSCN has obvious diffraction peaks at 16.1° and 28.8°, corresponding to the 003 crystal plane and the 012 crystal plane respectively, and the rest of the diffraction peaks are in good agreement with the standard card (JCPDS29 - 0581). For the XRD pattern of CuI / CuSCN, both the characteristic diffraction peaks of CuI and the diffraction peaks of CuSCN can be observed, indicating that the CuI / CuSCN nanocomposite material has been successfully synthesized.

[0069] 2. Preparation method of the photoanode, the steps are as follows:

[0070] (1) First, a certain mass of WCl 6It was added to 50 mL of absolute ethanol under ultrasonic conditions to form a yellow clear solution. Subsequently, 0.6 mL of acetylacetone was added, and the yellow solution immediately turned dark blue. 0.2 g of KIT-6 was slowly added. After 2 h of continuous ultrasonic treatment, the above solution was transferred to a high-pressure reaction kettle. It was reacted in an oven at 150 °C for 24 h. After cooling to room temperature, it was washed successively with deionized water and ethanol, and then dried in an oven at 60 °C. The obtained solid product was soaked in a 10% mass fraction hydrofluoric acid solution for 6 h to remove KIT-6, then separated and centrifuged, and then washed with deionized water until the pH value was neutral. The sample was washed with absolute methanol 3 - 5 times, centrifuged, and dried in an oven at 70 °C for 18 h to obtain m-WO 3-x .

[0071] (2) 1 g of Nb 2 AlC powder was added to 120 mL of a 40% mass fraction hydrofluoric acid solution and stirred at 55 °C. After the reaction ended, the prepared solid was washed and centrifuged with deionized water multiple times until the pH value of the solution was slightly neutral. The centrifuged precipitate was dried to collect the product. Then it was dispersed in 5 mL of a 26% mass fraction tetramethylammonium hydroxide solution at room temperature for a period of time to form a suspension. The suspension was centrifuged and washed with deionized water to remove the residual tetramethylammonium hydroxide solution. Then the obtained solid was redispersed in deionized water and ultrasonicated for 1 h. After ultrasonic treatment, it was centrifuged at low speed, washed, and dried to obtain monolayer or few-layer Nb 2 CT x . The obtained monolayer or few-layer Nb 2 CT x was dissolved in 12 g of deionized water, stirred for 10 min, 3 g of ethanol was added and continuously stirred. Then 200 μL of triethoxysilane was added, and the mixture was stirred under a nitrogen atmosphere for 24 h. The obtained product was washed three times with ethanol to remove the unreacted triethoxysilane, then washed three times with deionized water to remove ethanol, and the precipitate was freeze-dried to obtain N-Nb 2 CT x。

[0072] (3) 27 mg of N-Nb 2 CT x and 90 mg of m-WO 3-xAdd 63 mg of phloroglucinol trialdehyde and 48 mg of p-phenylenediamine, 1.5 mL of mesitylene and 1.5 mL of 1,4-dioxane into a reaction kettle containing 3 mL of DMF in sequence. Then add 0.5 mL of acetic acid aqueous solution with a concentration of 3 M as a catalyst into it. Ultrasonicate the mixture for 30 min to disperse it evenly, then transfer it into a high-pressure reaction kettle, bubble nitrogen for 10 min, and place the reaction kettle in an oven at 120 °C for reaction for 72 h. Wait for the reaction kettle to cool to room temperature, wash it three times with 1,4-dioxane and ethanol and then centrifuge and separate, and finally freeze-dry for 12 h to obtain NTW.

[0073] (4) Inject 100 mL of 1 mmol / L HAuCl 4 solution into a two-necked flask, then place it in an oil bath and heat it up to 120 °C for reflux, and continuously stir. When the reflux starts, quickly add sodium citrate solution (10 mL of 38.8 mmol / L). Stop heating when the solution color turns into a stable wine red color, continuously stir until it cools to room temperature and then store it in a refrigerator at 4 °C.

[0074] (5) First, prepare a 10 mg / mL NTW suspension, and grind the suspension sufficiently for about 30 min. Drop 30 μL of the NTW suspension onto the FTO electrode. Place it at room temperature and let it dry naturally to obtain the NTW / FTO electrode. Drop 30 μL of Au NPs onto the electrode surface and name it the Au / NTW / FTO electrode. To avoid the formation of disulfide bonds, incubate 10 μM of aptamer (Ap) in 1.2 mM of TCEP at 4 °C for 1 h. Drop 30 μL of Ap onto the surface of the Au / NTW / FTO electrode and incubate it at 4 °C for 12 h, and rinse the unconnected Ap with Tris-HCL to obtain Ap / Au / NTW / FTO. To block non-specific active sites, drop 30 μL of 1% (mass percentage) BSA onto the surface of the Ap / Au / NTW / FTO electrode and block it at 4 °C for 1 h, and rinse it with PBS buffer solution to obtain the BSA / Ap / Au / NTW / FTO electrode. Drop 30 μL of 10 μM cDNA onto the electrode and incubate it at 4 °C for 1 h, and name it the cDNA / BSA / Ap / Au / NTW / FTO electrode. Prepare a 10 mM MB solution, pipette 30 μL of the MB solution and drop it onto the electrode and incubate it for 30 min to prepare the MB / cDNA / BSA / Ap / Au / NTW / FTO electrode. Then add 30 μL of Staphylococcus aureus suspension with different concentrations and incubate it for 1 h, and name it the SA / MB / cDNA / BSA / Ap / Au / NTW / FTO electrode. Drop 30 μL of the solution containing 30 U of Exo ⅠⅠⅠ onto the electrode and incubate it for 2 h, and name it Exo ⅠⅠⅠ / SA / MB / cDNA / BSA / Ap / Au / NTW / FTO.

[0075] Figure 1 (b) is the XRD pattern of the photoanode material NTW. Nb 2 CT x The low-angle (002) peak in the XRD pattern is the characteristic peak of MXene, which confirms the successful synthesis of Nb 2 CT x TpPa-1 shows a sharp peak at 5.1° and a broad peak at 26.6°, corresponding to (100) and (001) respectively. In the XRD pattern of m-WO 3-x is consistent with the standard XRD pattern (JCPDS 89-1287). No characteristic peaks of Nb are observed in NT and NTW, which may be due to the low content of Nb 2 CT x The characteristic peaks of Nb are not observed, which may be due to the low content of Nb 2 CT x At the same time, no characteristic peaks of TpPa-1 are observed in NTW, which may be due to the overlap with the XRD diffraction peaks of m-WO 3-x

[0076] Figure 2 is the schematic diagram of the dual-photoelectrode self-powered sensor. Using NTW as the photoanode material and CuI / CuSCN as the photocathode material, a dual-photoelectrode self-powered multifunctional platform for the three-mode detection and killing of Staphylococcus aureus is constructed. By introducing aptamers and photocatalysis into the dual-photoelectrode, sensitive three-mode detection and excellent sterilization performance are achieved.

[0077] Application Example 1:

[0078] The above-obtained dual-photoelectrode self-powered sensor is applied to three-mode bacteria detection.

[0079] The Staphylococcus aureus frozen at -80 °C is activated on the LB medium by the streaking method. A single colony is picked and inoculated into the LB broth at 37 °C and 120 rpm for 15 h, and then serially diluted with 0.85% saline solution.

[0080] To explore the detection ability for Staphylococcus aureus, the photoanode is incubated with a series of suspensions of Staphylococcus aureus with concentrations ranging from 3.57×10 2 ~ 3.57×10 8 CFU / mL. Then, using the photoanode as the working electrode, the photocathode as the counter electrode, and the saturated calomel electrode as the reference electrode, the photoelectrochemical and electrochemical detections are respectively carried out in PBS buffer solution (pH 7.4) by simulating sunlight with a xenon lamp. In addition, fluorescence spectroscopy detection is carried out using ExoⅠⅠⅠ as the cleavage enzyme and SYBR Gold as the fluorescent dye.

[0081] Figure 3 The photocurrent response curves of different electrodes. When the Pt wire is the counter electrode, the photocurrents of CuI / / Pt, CuSCN / / Pt, CuI / CuSCN / / Pt, and NTW / / Pt are -90.6 μA, -9.2 μA, -179.3 μA, and 93.2 μA, respectively. When CuI / CuSCN is the counter electrode, the photocurrent of NTW / / CuI / CuSCN is 146.7 μA, and the photocurrent intensity is 1.57 times that of NTW / / Pt. The results show that the dual photoanode can enhance the anodic photocurrent.

[0082] Figure 4 The photocurrent of the photoanode. When the FTO surface is modified with NTW nanomaterials, a certain photocurrent is generated (curve a). After Au NPs are modified onto the electrode surface, the photocurrent increases significantly (curve b). On the one hand, the surface plasmon resonance effect of Au NPs injects a large number of hot electrons. On the other hand, Au NPs can promote the separation of photo-generated electron-hole pairs. The synergistic effect of the above two aspects improves the photoelectrochemical performance of Au / NTW. When the aptamer is modified onto the electrode surface, the photocurrent shows a downward trend (curve c). This may be because the presence of the aptamer increases the steric hindrance and reduces the electron transfer. After BSA and cDNA are successively modified onto the electrode, due to the poor charge transfer of organic small molecules and the DNA backbone, the photocurrents of curves d and e decrease. After MB is modified, MB is embedded into the DNA double strand, and the photocurrent increases significantly (curve f). When the target Staphylococcus aureus is modified, the photocurrent decreases (curve g). This is because the binding of Staphylococcus aureus to Ap breaks the double strand, and MB detaches from the electrode surface, weakening the dye-sensitization effect.

[0083] Figure 5 The photocurrent curves (a) and detection calibration curves (b) of different concentrations of Staphylococcus aureus. The photocurrent gradually decreases within a certain range as the concentration of Staphylococcus aureus increases. This is because the aptamer binds to its complementary DNA or Staphylococcus aureus, and after MB modification, it is embedded into the double strand. The binding affinity of Staphylococcus aureus to the aptamer is greater than that of the aptamer and cDNA. Therefore, Staphylococcus aureus can competitively bind to the aptamer and release cDNA and MB, resulting in a gradual decrease in the photocurrent. Figure 5 (a) shows that in the range of 3.57×10 2 CFU / mL to 3.57×10 8 CFU / mL, there is a good linear relationship between the change in photocurrent and the logarithm of the Staphylococcus aureus concentration. The linear equation is ΔI = 10.51LogC - 13.71, the correlation coefficient is 0.994, and the detection limit is 6.3 CFU / mL.

[0084] Figure 6 Current curves (a) and detection calibration curves (b) for Staphylococcus aureus at different concentrations. As Figure 6 (a) shows, the change and mechanism of the current are consistent with the photocurrent, and the current gradually decreases as the concentration of Staphylococcus aureus increases. Figure 6 (b) shows that there is also a good linear relationship between the change in current and the logarithm of the Staphylococcus aureus concentration. The linear equation is ΔI = 55.10LogC - 126.73, the correlation coefficient is 0.996, and the detection limit is 3.8 CFU / mL.

[0085] Figure 7 Fluorescence spectra (a) and detection calibration curves (b) for Staphylococcus aureus at different concentrations. The aptamer first hybridizes and binds with the complementary strand to form a DNA double-stranded structure. In the presence of Staphylococcus aureus, since the aptamer has a stronger binding ability to Staphylococcus aureus compared to the complementary strand, the double-stranded DNA is dissociated, and Staphylococcus aureus binds to the aptamer to form a G-quadruplex structure. At the same time, the complementary strand exists in a single-stranded free state, thus preventing the digestion by Exo III. When SYBR Gold dye is added, SYBR Gold binds to the intact single strand and emits strong fluorescence. On the contrary, in the absence of Staphylococcus aureus, the double-stranded DNA will not be unwound, and the intact double-stranded DNA is hydrolyzed into single nucleotides by Exo III. When SYBR Gold is added, SYBR Gold cannot bind to the single nucleotides produced by DNA hydrolysis, and thus only a weak fluorescence signal can be detected. Therefore, the quantitative detection of Staphylococcus aureus can be achieved according to the intensity of the detected fluorescence signal. As Figure 7 (a) shows, as the concentration of Staphylococcus aureus increases, the fluorescence intensity also shows an increasing trend. Figure 7 (b) shows the standard curve for the quantitative analysis of the target molecule Staphylococcus aureus. In the range of 3.57×10 2 CFU / mL to 3.57×10 8 CFU / mL, the relative fluorescence intensity has a good linear relationship with the logarithm of the Staphylococcus aureus concentration. The linear equation is FL = 22.57LogC - 1.32, the correlation coefficient is 0.997, and the detection limit is 10.7 CFU / mL.

[0086] Application Example 2:

[0087] The obtained photoanode above is applied to photocatalytic sterilization.

[0088] The photoanode was placed in a PBS buffer solution containing Staphylococcus aureus, and then irradiated with a 300 W xenon lamp equipped with an ultraviolet cut-off filter (λ>420 nm) for 30 min, and samples were taken every 5 min. 100 μL of the Staphylococcus aureus suspension was taken from different illumination times and spread on a newly prepared LB agar plate, and cultured at 37 °C for 24 h. Each group was tested in parallel three times, and the bacterial solution was diluted and then plate counted. At the same time, a light control experiment (without a photocatalyst under light illumination) and a dark control experiment (with a photocatalyst in the dark) were also carried out. The sterilization efficiency of the photoanode was calculated using Equation 1.

[0089] Sterilization rate (%) = (N 0 -N t ) / N 0 *100% (1)

[0090] where N 0 and N t correspond to the number of colonies counted on the control and sample culture medium plates, respectively.

[0091] Figure 8 Figures (a) and (b) are the photocatalytic sterilization efficiency diagram and colony photos of the photoanode. As Figure 8 (a) shows, there is almost no sterilization effect in the control group under light illumination, indicating that simple light illumination does not sterilize. And the sterilization efficiency only increased slightly under the condition of only the catalyst without light illumination, which may be due to the certain sterilization performance of Au NPs. Under light illumination, the sterilization efficiency of NTW / FTO is 65.3%, while that of Au / NTW / FTO is 82.8%. This is because Au NPs not only have certain sterilization performance by themselves, but also can promote the separation of photo-generated electron-hole pairs. In addition, the highest sterilization efficiency of Ap / Au / NTW / FTO is 89.1%, which may be because the aptamer can specifically capture Staphylococcus aureus, causing the aggregation of Staphylococcus aureus, thereby improving the sterilization efficiency. To make the experimental results more intuitive, Figure 8 (b) shows the number of bacterial colonies of Staphylococcus aureus growing on the agar plate under photocatalytic conditions for different photoanodes.

[0092] The above are only the preferred embodiments of the present invention, and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent structural changes made to the above embodiments according to the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A self-powered three-mode sensor based on dual photoelectrodes, characterized in that: The sensor includes three sensing modes: photoelectrochemical, electrochemical and fluorescent. The dual photoelectrode is a photocathode and a photoanode. The photocathode material is CuI / CuSCN, and the photoanode material is NTW.

2. A self-powered three-mode sensor based on dual photoelectrodes according to claim 1, characterized in that: The photocathode material CuI / CuSCN, in the XRD spectrum of CuI, diffraction peaks appear at 2θ values ​​of 25.5°, 29.5°, 42.2°, 50.0°, 52.4°, 61.3°, 67.5° and 69.5°, corresponding to (111), (200), (220), (311), (222), (400), (331) and (420), respectively; the CuSCN has obvious diffraction peaks at 16.1° and 28.8°, corresponding to the 003 crystal plane and the 012 crystal plane, respectively, and the remaining diffraction peaks are consistent with the standard card.

3. The photoanode material NTW according to claim 2, characterized in that: The Nb2CT x In the XRD spectrum of m-WO, the low-angle (002) peak is the characteristic peak of MXene; the TpPa-1 shows a sharp peak at 5.1° and a broad peak at 26.6°, corresponding to (100) and (001), respectively; 3-x The XRD pattern is consistent with the standard XRD pattern.

4. The method for preparing the self-powered three-mode sensor based on dual photoelectrodes according to any one of claims 1 to 3, characterized in that: The steps include: (1) The photocathode is prepared using CuI and CuSCN photocathode materials. (2) The photoanode was prepared using the photoanode materials NTW and Au NPs. (3) A self-powered three-mode sensor is prepared using the photocathode and photoanode.

5. The method for preparing the self-powered three-mode sensor based on dual photoelectrodes according to claim 4, characterized in that: During the preparation of the photoanode material NTW, Au NPs were added to the surface of the NTW / FTO electrode. To avoid the formation of disulfide bonds, the aptamer (Ap) was incubated in tri(2-carboxyethyl)phosphine (TCEP), and the activated Ap was dropped on the surface of the Au / NTW / FTO electrode and incubated. The unconnected Ap was rinsed with Tris-HCL to obtain Ap / Au / NTW / FTO.

6. The method for preparing the self-powered three-mode sensor based on dual photoelectrodes according to claim 5, characterized in that: In order to block nonspecific active sites, bovine serum albumin (BSA) was drop-coated on the surface of the Ap / Au / NTW / FTO electrode and blocked for a certain period of time, and then rinsed with PBS buffer to obtain the BSA / Ap / Au / NTW / FTO electrode.

7. Application of the self-powered three-mode sensor based on dual photoelectrodes according to any one of claims 1 to 3, characterized in that: The dual-photoelectrode self-powered sensor is used for three-mode bacteria detection, with a photoanode as the working electrode, a photocathode as the counter electrode, a saturated calomel electrode as the reference electrode, a xenon lamp to simulate sunlight, and an electrochemical workstation connected to a PBS buffer solution (pH 7.4) for photoelectrochemical and electrochemical detection, respectively; ExoⅠⅠⅠ is used as a shearing enzyme, and SYBR Gold is used as a fluorescent dye for fluorescence spectrum detection; At 3.57×10 2 CFU / mL to 3.57×10 8 In the range of CFU / mL, a linear relationship of ΔI=10.51LogC-13.71, a correlation coefficient of 0.994, and a detection limit of 6.3CFU / mL was adopted. The concentration of Staphylococcus aureus was detected based on the linear relationship between the change in the above photocurrent and the logarithmic value of the concentration of Staphylococcus aureus.

8. The application of the self-powered three-mode sensor based on dual photoelectrodes as claimed in claim 7, characterized in that: The linear relationship of ΔI=55.10LogC-126.73, correlation coefficient of 0.996 and detection limit of 3.8 CFU / mL was adopted, and the concentration of Staphylococcus aureus was detected according to the linear relationship between the change of the above current and the logarithmic value of the concentration of Staphylococcus aureus.

9. The application of the self-powered three-mode sensor based on dual photoelectrodes as claimed in claim 8, characterized in that: At 3.57×10 2 CFU / mL to 3.57×10 8 Within the range of CFU / mL, a linear relationship of FL=22.57LogC-1.32, a correlation coefficient of 0.997, and a detection limit of 10.7CFU / mL was adopted. The concentration of Staphylococcus aureus was detected based on the linear relationship between the relative fluorescence intensity and the logarithm of the concentration of Staphylococcus aureus, which showed a good logarithmic value.

10. The application of the self-powered three-mode sensor based on dual photoelectrodes according to claim 9, characterized in that: The dual-photoelectrode self-powered sensor is used for photocatalytic sterilization.

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