A MOF nanomembrane-based faraday cage type aptamer sensor and a method for detecting staphylococcus aureus therefrom

Abstract

The application relates to a Faraday cage type aptamer sensor based on a MOF nanofilm and a method for detecting Staphylococcus aureus. A 1,1'-ferrocene carboxylic acid is used as a ligand, zirconium / hafnium is used as a central atom, and a Zr-based MOF nanofilm with different hafnium doping ratios is synthesized by using a bottom-up method. A Zr / Hf-MOF nanofilm with large size and excellent conductivity is selected as a two-dimensional signal probe, and is used for constructing an aptamer sensor of a Faraday cage. When Staphylococcus aureus exists, the Zr / Hf-MOF nanofilm is attached to the surface of an electrode to form a Faraday cage sensing interface, the interface expands an outer Helmholtz plane layer, leads to direct electron transmission between the electrode and the signal probe, and thus Staphylococcus aureus can be detected through an electrochemical workstation. The detection method of the application can realize detection of a large target by requiring the size and conductivity of the MOF nanofilm, and effectively amplifies the electrical signal through the Faraday cage structure, and has excellent detection specificity and sensitivity.

Description

Technical Field

[0001] This invention relates to the fields of functional materials and biosensing technology, and in particular to a Faraday cage aptamer sensor based on MOF nanofilm and a method for detecting Staphylococcus aureus. Background Technology

[0002] Staphylococcus aureus (S. aureus) is a life-threatening pathogen that poses a significant risk to food safety and public health. Infections caused by Staphylococcus aureus can range from mild gastroenteritis to severe organ dysfunction and sepsis, making its detection crucial for food safety and medical diagnosis. Traditional methods for detecting Staphylococcus aureus are widely used, including microbial culture, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR). However, these methods are often time-consuming, have low sensitivity, are cumbersome, and require expensive equipment, making them unsuitable for rapid detection needs. These limitations have spurred the development of alternative technologies aimed at addressing the shortcomings of traditional methods while maintaining or improving accuracy and reliability.

[0003] Traditional sandwich-type electrochemical aptamer sensors consist of a three-layer structure with a pair of aptamers as biorecognition elements and a signal probe as the signal output. However, this model encounters significant steric hindrance after capturing large targets, causing most of the captured signal probes to be distributed outside the outer Helmholtz plane (OHP). This means that the electron transport required for detection is difficult to occur, affecting signal generation and making the detection of large targets challenging. Faraday cage-based sensors have been built to address this challenge, utilizing conductive two-dimensional (2D) nanomaterials as signal probes and immobilized recognition elements to construct signal units. After specific target capture on the electrode surface, a sandwich structure of recognition unit-target unit-signal unit is formed. Due to the large area of ​​2D nanomaterials, they can become part of the electrode, equivalent to stretching the OHP layer, forming an electron transport channel between the electrode and the signal unit. Furthermore, due to their large specific surface area, 2D materials themselves carry more signal molecules than traditional sandwich sensors. However, most Faraday cage sensing platforms are based on carbon-based 2D materials. The dual loading of recognition and signal probes on their surfaces often competes with each other, limiting further signal enhancement.

[0004] Metal-organic frameworks (MOFs), composed of organic ligands and metal ion subunits arranged according to topological rules, have been successfully applied in gas storage and separation, sensing, and catalysis. Compared to three-dimensional MOFs, two-dimensional MOF nanomaterials possess a larger specific surface area, more active sites, and faster electron transfer. Therefore, the application of conductive two-dimensional MOF nanomaterials as signal carriers not only expands the OHP layer and improves the electron transport rate, but also facilitates the loading of metal ions due to their high vacancy rate. These factors simultaneously enable in-situ modification of recognition and signal probes on the same carrier wave. Zr-MOFs have been reported to be highly conductive two-dimensional materials, with carboxyl groups contributing to improved conductivity. Furthermore, it has been noted that doping with metal atoms can further enhance the conductivity of the materials.

[0005] Therefore, there is an urgent need to provide a method for constructing an electrochemical Faraday cage immunosensor for detecting Staphylococcus aureus that is specific, sensitive, fast, accurate, reliable, and low-cost, and to achieve the detection of Staphylococcus aureus by electrochemical catalysis of 1,1′-ferrocene carboxylic acid and the construction of a Faraday cage sensor using a nanofilm. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a Faraday cage-based aptamer sensor based on MOF nanofilms and a method for detecting Staphylococcus aureus. This invention constructs a Faraday cage-based aptamer sensor based on conductive two-dimensional Zr / Hf-MOF nanofilms for whole-cell detection of Staphylococcus aureus. The detection method of this invention synthesizes a series of Zr-based MOF nanofilms with different hafnium doping ratios using 1,1′-ferrocene carboxylic acid as a ligand and zirconium / hafnium as the central atom via a bottom-up approach. AuNPs and Apt are modified onto the nanofilms. The in-situ grown AuNPs improve the conductivity of the two-dimensional MOF nanofilms and facilitate the modification of Apt onto the material via Au-S bonds, forming signal units (Zr / Hf-MOF / AuNPs / Apt). When Staphylococcus aureus is present, the large surface area and highly conductive Zr / Hf-MOF adhere to the electrode surface, forming multiple Faraday cage structures. In this way, electron transfer in the aptamer sensor for whole-cell detection of Staphylococcus aureus is no longer hindered by the large target volume. Electrons can be directly transferred between the ferrocene (Fc) signal probe inside the cage and the electrode surface, greatly improving the detection signal. The detection method of the Faraday cage sensor of this invention can detect large targets, and this invention achieves effective amplification of the electrical signal through the Faraday cage structure system, exhibiting excellent detection specificity and sensitivity.

[0007] This invention is achieved through the following technical solution:

[0008] The purpose of this invention is to provide a method for detecting Staphylococcus aureus using a Faraday cage aptamer sensor based on MOF nanofilms, comprising the following steps:

[0009] S1. Add HAuCl4 and a reducing agent to an aqueous solution of Zr / Hf-MOF nanofilm to obtain a Zr / Hf-MOF nanofilm loaded with AuNPs; add an activated aptamer and an electron blocking agent to an aqueous solution of the obtained Zr / Hf-MOF nanofilm loaded with AuNPs to obtain the signal unit Zr / Hf-MOF / AuNPs / Apt.

[0010] S2. The activated aptamer is drop-coated onto the surface of a gold electrode and incubated to obtain an AuR / Apt electrode; the obtained AuR / Apt electrode is inserted into an electron blocking agent solution and incubated to obtain an AuR / Apt electrode that blocks non-specific binding sites.

[0011] S3. Add the solution containing Staphylococcus aureus to the surface of the AuR / Apt electrode with blocked nonspecific binding sites obtained in step S2 and incubate. Drop-coat the signal unit Zr / Hf-MOF / AuNPs / Apt obtained in step S1. After incubation at room temperature, detect the electrochemical differential pulse voltammetry signal of the obtained solution to perform qualitative or quantitative analysis of Staphylococcus aureus.

[0012] In one embodiment of the present invention, in step S1, the doping amount of hafnium in the Zr / Hf-MOF nanofilm is 1%-5%.

[0013] In one embodiment of the present invention, in step S1, the Zr / Hf-MOF nanofilm is prepared by the following method:

[0014] Zirconium salt, hafnium salt, and redox mediator were dissolved in a mixed solution of organic solvent and HAC, and then heated to obtain Zr / Hf-MOF nanofilms.

[0015] In one embodiment of the present invention, the zirconium salt is selected from ZrCl4;

[0016] And / or, the hafnium salt is selected from HfCl4;

[0017] And / or, the molar ratio of the zirconium salt to the hafnium salt is 9.0-9.9:0.1-0.5; preferably 9.5:0.5.

[0018] In one embodiment of the present invention, the redox mediator is selected from 1,1′-ferrocene carboxylic acid;

[0019] And / or, the organic solvent is selected from DMF.

[0020] In one embodiment of the present invention, in steps S1 and S2, the nucleotide sequence of the activated aptamer is shown in SEQ ID NO. 1.

[0021] In one embodiment of the present invention, in steps S1 and S2, the electron blocking agent comprises 6-mercaptohexanol.

[0022] In one embodiment of the present invention, in step S2, the activated aptamer is obtained by dissolving the aptamer in a buffer solution and reacting it with TCEP solution at room temperature.

[0023] In one embodiment of the present invention, the buffer solution is selected from PBS buffer solution.

[0024] In one embodiment of the present invention, in step S3, the concentration of the solution containing Staphylococcus aureus is 10. 1 CFU·mL −1 ~10 7 CFU·mL −1 .

[0025] The second objective of this invention is to provide a Faraday cage aptamer sensor based on MOF nanofilm, comprising a signal unit Zr / Hf-MOF / AuNPs / Apt, an aptamer, and electrodes;

[0026] The Zr / Hf-MOF nanofilm in the signal unit is modified with redox mediators and electron blocking agents.

[0027] The technical solution of the present invention has the following advantages compared with the prior art:

[0028] (1) This invention provides a Faraday cage-type aptamer sensor based on MOF nanofilms and a method for detecting Staphylococcus aureus. Using 1,1′-ferrocene carboxylic acid as a ligand and zirconium / hafnium as the central atom, a series of Zr-based MOF nanofilms with different hafnium doping ratios were synthesized using a bottom-up method. 1,1′-ferrocene carboxylic acid not only serves as an electrochemical signal molecule but can also be used for in-situ reduction of gold nanoparticles (AuNPs), providing a fixed site for the aptamer (Apt). By doping with hafnium and in-situ growing AuNPs, the conductivity of the two-dimensional MOF nanofilm is improved. When Staphylococcus aureus is present, the two-dimensional Zr / Hf-MOF / AuNPs / Apt nanofilm, acting as the signal unit, adheres to the electrode surface due to its large surface area and high conductivity, forming a Faraday cage structure. Thus, electron transfer in the aptamer sensor for whole-cell detection of Staphylococcus aureus is no longer hindered by its large size. Electrons can be directly transferred between the signal probe within the cage and the electrode surface, greatly improving the detection signal.

[0029] (2) This invention designs and prepares a Staphylococcus aureus response unit Zr / 0.05Hf-MOF / AuNPs / Apt, which generates an electrochemical signal change based on its specificity with the Staphylococcus aureus site. Furthermore, by connecting AuR with Zr / 0.05Hf-MOF / AuNPs / Apt to form a Faraday cage structure, the electrochemical signal is greatly increased, thus detecting Staphylococcus aureus. Compared with traditional detection methods, this invention has the advantages of high specificity and high sensitivity. Attached Figure Description

[0030] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0031] Figure 1 This is a schematic diagram of the electrochemical method for detecting whole cells of Staphylococcus aureus based on a Faraday cage-type aptamer sensor with two-dimensional high conductivity Zr / 0.05Hf-MOF nanofilm.

[0032] Figure 2 This invention presents the signal response and detection standard curves of the Faraday cage biosensor for different concentrations of Staphylococcus aureus, where af represents the following concentrations: 0 CFU·mL −1 10 1 CFU·mL −1 10 2 CFU·mL −1 10 3 CFU·mL −1 10 4 CFU·mL −1 10 5 CFU·mL −1 10 6 CFU·mL −1 10 7 CFU·mL −1 .

[0033] Figure 3 This invention describes the measurement of the dimensions of different MOFs under an atomic force microscope; where A is Zr-MOF, B is Zr / 0.01Hf-MOF, C is Zr / 0.05Hf-MOF, D is Zr / 0.10Hf-MOF, and E is Zr / 0.20Hf-MOF; F is a height analysis profile of different MOFs: Zr-MOF (a), Zr / 0.01Hf-MOF (b), Zr / 0.05Hf-MOF (c), Zr / 0.10Hf-MOF (d), and Zr / 0.20Hf-MOF (e).

[0034] Figure 4 This invention analyzes the conductivity and signal quantity of different MOFs; where A represents the differential pulse voltammetry curves of different Zr / Hf-MOFs; B represents the peak current of different Zr / Hf-MOFs; C represents the electrochemical impedance spectroscopy of different Zr / Hf-MOFs; and D represents the electron transfer resistance of different Zr / Hf-MOFs; in A and C: Zr / 0.01Hf-MOF (a), Zr / 0.05Hf-MOF (c), Zr / 0.10Hf-MOF (d), and Zr / 0.20Hf-MOF (e);

[0035] Figure 5 This invention relates to the specificity and stability testing of the Faraday cage sensor; wherein A is a graph showing the DPV current values ​​of the Faraday cage ligand sensor for different bacteria; and B is a graph showing the stability results of the Faraday cage aptamer sensor. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0037] This invention provides an electrochemical method for the detection of whole cells of Staphylococcus aureus using a Faraday cage-type aptamer sensor based on a Zr / 0.05Hf-MOF nanofilm, comprising the following steps:

[0038] S1. ZrCl4, HfCl4 in different proportions, and 1,1′-ferrocene carboxylic acid were added to DMF and HAC solutions. The mixed suspension was heated in a stainless steel autoclave to obtain Zr / Hf-MOF nanofilms. Highly conductive, large-area Hf-doped Zr / Hf-MOF nanofilms (Zr / Hf-MOF) were selected by morphological and electrochemical characterization. HAuCl4 was added to the nanofilm solution to react and obtain Zr / Hf-MOF nanofilms loaded with AuNPs. The nanofilms were purified by centrifugation for later use. An aptamer and MCH were added to the nanofilm solution and reacted at room temperature to obtain a Zr / Hf-MOF / AuNPs / Apt complex as a signaling unit.

[0039] S2. Preparation of AuR / Apt: Dissolve the aptamer in PBS buffer solution, add TCEP solution and react at room temperature. Drop the reaction solution onto the surface of a gold electrode and incubate. Then drop MCH solution onto AuR / Apt and incubate. MCH fills the vacancies to avoid non-specific adsorption.

[0040] S3. Preparation of electrochemical Faraday cage sensor: Staphylococcus aureus was placed on the gold electrode surface of S2 and incubated. Then, the signal unit of S1 was drop-coated onto the electrode surface and incubated at room temperature. Subsequently, the sensor was placed in PBS buffer solution for electrochemical differential pulse voltammetry (DPV) detection.

[0041] Furthermore, the method for selecting the Zr / Hf-MOF nanofilm is as follows:

[0042] AFM was used to confirm that Zr-MOFs with Hf doping of ≤5% were dimensionally complete and thin. Then, Nafion reagent diluted to 0.5% was added to Zr / Hf-MOFs with different Hf ratios to form concentrations of 1 mg·mL⁻¹. −1 The suspension was dropped 8 µL onto a 3 mm gold electrode and dried at room temperature to fix Zr / Hf-MOF. Electrochemical DPV and EIS detection determined that the 0.05 Hf Zr-MOF nanofilm had the best electrochemical signal.

[0043] Furthermore, the aptamer sequence aptamers need to be activated by the thiol activator TCEP to avoid the formation of disulfide bonds between aptamers and to facilitate the formation of Au-S bonds.

[0044] Furthermore, the 5' end of the aptamer sequence is modified with a thiol group. This modification is achieved via Au-S bonds on the electrode and the Zr / Hf-MOF / AuNPs nanofilm.

[0045] Furthermore, the aptamer sequence is as follows:

[0046] Apt:5′-SH-GCAATGGGTACGGTACTTCCTCGGCACGTTCTCAGTAGCGCTCGCTGGTCATCCCACAGCTACGTCAAAAGTGCACGCTACTTTGCTAA-3′

[0047] Furthermore, the aptamer specifically binds to the cell wall of Staphylococcus aureus, which is beneficial to the specificity and sensitivity of the detection.

[0048] Furthermore, the detection system also includes redox mediators and electron blocking agents modified on the Zr / Hf-MOF signaling units.

[0049] Furthermore, the redox mediator comprises 1,1′-ferrocene carboxylic acid, and the electron blocking agent comprises 6-mercaptohexanol. The electron blocking agent can block electron transfer between the electrode surface and ions in the solution, ensuring that electron transfer can only occur through the ferrocene on the nanofilm, thereby amplifying the electrical signal.

[0050] Furthermore, the detection method described herein is applied in the preparation of Staphylococcus aureus detection products.

[0051] Furthermore, when detecting Staphylococcus aureus, the aptamers on the nanofilm form a Faraday cage structure on the gold electrode by specifically recognizing the target.

[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0053] Example 1

[0054] This embodiment provides a Faraday cage aptamer sensor based on MOF nanofilm and a method for detecting Staphylococcus aureus (the schematic diagram is shown below). Figure 1 As shown), the details are as follows:

[0055] (1) Preparation of Zr / 0.05Hf-MOF nanofilm: 393.8 mg of ZrCl4, 28.3 mg of HfCl4 and 493.8 mg of 1,1′-ferrocene carboxylic acid were added to 60 mL of DMF and 3.4 mL of HAC, and sonicated at 25 °C for 30 min until completely dissolved. The mixed suspension was added to a 100 mL stainless steel autoclave and heated at 120 °C for 12 h. After cooling to room temperature, it was centrifuged at 7000 rpm for 15 min to remove unreacted substances. It was washed three times with fresh DMF and deionized water to obtain a black precipitate Zr / 0.05Hf-MOF nanofilm, i.e., 5% Hf-doped Zr / Hf-MOF nanofilm. After freezing at -80 °C for 12 h, the material was dried in a freeze dryer for later use.

[0056] (2) Preparation of signal unit: 100 mg Zr / 0.05Hf-MOF was dispersed in 100 mL of deionized water by ultrasonication for 30 min. 1 mL of 10 mg·mL⁻¹ was then added dropwise to the resulting dispersion. −1The Zr / Hf-MOF / AuNPs nanofilm precipitate was obtained by stirring the HAuCl4 solution at 30°C for 30 min, followed by continuous stirring and the addition of fresh NaBH4 (1 mL, 0.1 M) solution, and reacting for 10 min. The precipitate was dispersed in 5 mL of deionized water. The Zr / Hf-MOF / AuNPs nanofilm was added to PBS buffer containing 12 μM aptamers and incubated in the dark at 25°C. 5 μL of 11 mM MCH solution was added to the mixture for 1 h to block nonspecific sites. After centrifugation at 7000 rpm for 15 min to remove free aptamers, the nanofilm was washed and resuspended in 100 μL of DNA hybridization buffer (10 mM PBS, 1 M NaCl, pH 7.4) to obtain the signal unit Zr / 0.05Hf-MOF / AuNPs / Apt.

[0057] The sequence of the aptamer Apt is as follows:

[0058] 5′-SH-GCAATGGGTACGGTACTTCCTCGGCACGTTCTCAGTAGCGCTCGCTGGTCATCCCACAGCTACGTCAAAAGTGCACGCTACTTTGCTAA-3′

[0059] (3) Electrode cleaning and activation: The gold electrode (Au rod electrode, AuR) was polished with alumina polishing powder, followed by ultrasonic cleaning in n-propanol, anhydrous ethanol, and deionized water for 5 min each. A chemical cleaning process was performed on the AuR using platinum as the auxiliary electrode and an Ag / AgCl electrode as the reference electrode. First, in 0.5 mol·L⁻¹… −1 Alkali washing was performed in NaOH solution using a CV program (voltage scan from −1.5 V to −0.35 V, 500 scans at a scan rate of 4 V / s). Finally, alkaline washing was performed in 0.5 mol·L⁻¹ solution. −1 The electrode was cleaned by acid washing in H2SO4 solution using a CV program (scanning voltage from −0.35 V to 1.5 V, 100 scans at a scan rate of 1 V / s).

[0060] (4) Modification of the electrochemical sensing interface: 2 µL of 10 µmol L −1 The modified Apt was added to 16 µL of PBS buffer and heated at 95 °C for 5 min, then slowly cooled to room temperature to form the correct DNA structure. Subsequently, 2 µL of 10 mmol·L⁻¹ PBS was added. −1TCEP was applied and incubated at 25°C for 1 h to reduce disulfide bonds and form thiol groups. 200 µL of incubation buffer was added, followed by overnight incubation with AuR at 25°C to obtain the AuR / Apt electrode. The AuR / Apt electrode was then washed with 3 mL of PBS buffer and 3 mL of deionized water. The AuR / Apt electrode was then inserted into a container containing 200 µL of 0.5 mmol·L⁻¹ PBS buffer. −1 The electrodes were incubated in MCH solution at 25°C for 1 hour. The electrodes were then washed with 6 mL of PBS buffer and 6 mL of deionized water, respectively, to block non-specific binding sites with MCH.

[0061] (5) Electrochemical detection of Staphylococcus aureus: Sensitivity analysis of the constructed electrochemical biosensor was performed under optimal electrochemical analysis conditions. Staphylococcus aureus (10⁻¹⁰) was introduced at equal concentration gradients. 1 CFU·mL −1 10 2 CFU·mL −1 10 3 CFU·mL −1 10 4 CFU·mL −1 10 5 CFU·mL −1 10 6 CFU·mL −1 10 7 CFU·mL −1 The samples were placed on the gold electrode surface from step (4) and incubated. Then, the signal unit from step (2) was drop-coated onto the electrode surface and incubated at room temperature. Subsequently, the prepared sensor was placed in PBS (0.1 M, pH 7.0) for electrochemical differential pulse voltammetry (DPV) detection. Figure 2 Analysis revealed a clear linear relationship between the logarithmic value of Staphylococcus aureus concentration and the change in current (ΔI) of its output current response. The corresponding log-linear regression equation is y = 172.30 lg C S. aureus + 55.98,(R 2 = 0.992)(where the letter y represents the change in current ΔI, C S. aureus The limit of detection (LOD), representing the concentration of Staphylococcus aureus, was calculated to be 1.8 CFU / mL using the 3σ / slope rule. −1 (where σ is the standard deviation).

[0062] Example 2

[0063] This embodiment provides a Faraday cage aptamer sensor based on MOF nanofilm and a method for detecting Staphylococcus aureus, similar to Embodiment 1, except that the molar ratio of ZrCl4 to HfCl4 is 9.9:0.1, resulting in a 1% Hf-doped Zr / Hf-MOF nanofilm (Zr / 0.01Hf-MOF).

[0064] Comparative Example 1

[0065] This comparative example provides a Faraday cage aptamer sensor based on MOF nanofilm and a method for detecting Staphylococcus aureus, similar to Example 1, except that HfCl4 is not added to obtain a Zr / MOF nanofilm.

[0066] Comparative Example 2

[0067] This comparative example provides a Faraday cage aptamer sensor based on MOF nanofilm and a method for detecting Staphylococcus aureus. It is similar to Example 1, except that the molar ratio of ZrCl4 to HfCl4 is 9:1, resulting in a 10%f-doped Zr / Hf-MOF nanofilm (Zr / 0.1Hf-MOF).

[0068] Comparative Example 3

[0069] This comparative example provides a Faraday cage aptamer sensor based on MOF nanofilm and its method for detecting Staphylococcus aureus. It is similar to Example 1, except that the molar ratio of ZrCl4 to HfCl4 is 8:2, resulting in a 20% f-doped Zr / Hf-MOF nanofilm (Zr / 0.2Hf-MOF).

[0070] Test Example 1: Selection of Zr / Hf-MOF Nanofilms

[0071] AFM analysis revealed that Zr-MOFs with Hf doping of ≤5% were larger and thinner. Figure 3 Then, Nafion reagent diluted to 0.5% was added to 1 mg of Zr / Hf-MOF with different Hf ratios to form 1 mg·mL⁻¹ −1 The suspension was dropwise 8 μL onto a 3 mm gold electrode, dried at room temperature to fix Zr / Hf-MOF, and detected by electrochemical differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The results are as follows: Figure 4 As shown, the Zr-MOF nanofilm with 5% Hf exhibited the best electrochemical signal.

[0072] Test Example 2: Specificity and stability testing of Staphylococcus aureus

[0073] The selectivity of the Faraday cage-type ligand sensor was evaluated using five control experiments: blank, Bacillus subtilis, Salmonella typhi, Escherichia coli, and Staphylococcus aureus. Figure 5 As shown in Figure A, a significant DPV current was generated in the presence of Staphylococcus aureus. No significant current was observed when identifying other bacteria, equivalent to a blank sample. These results indicate that the Faraday cage-based aptamer sensor has high specificity for Staphylococcus aureus. To investigate the stability of the Faraday cage-based aptamer sensor, five sets of electrodes were prepared and stored at 4°C for subsequent use. Staphylococcus aureus was tested every four days for 16 days. Figure 5 As shown in B, the detection percentages remained at 94.1%, 97.3%, 94.1%, and 93.1% of their original values. The results indicate that the sensor exhibits good stability.

[0074] Test Example 3: Practical Application and Reliability Analysis of Faraday Cage Adaptor Sensors

[0075] To further verify the practical application and analytical reliability of the Faraday cage-based aptamer sensor, serum and milk were selected for analysis. The analytical capability of the aptamer sensor for real samples was evaluated using the standard additive method, with each sample tested three times. The results are shown in Table 1:

[0076] Table 1. Results of Staphylococcus aureus test

[0077]

[0078] According to the results in Table 1, the recovery rates of serum samples were 91.6%–97.7%, and those of milk samples were 101.9%–109.4%. The RSDs of serum samples were 0.3%–6.6%, and those of milk samples were 0.3%–6.8%. These results indicate that the electrochemical biosensor has great potential for clinical detection of Staphylococcus aureus.

[0079] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for detecting Staphylococcus aureus using a MOF nanomembrane based Faraday cage type aptasensor, characterized in that, Includes the following steps: S1. Add HAuCl4 and a reducing agent to an aqueous solution of Zr / Hf-MOF nanofilm to obtain a Zr / Hf-MOF nanofilm loaded with AuNPs; add an activated aptamer and an electron blocking agent to an aqueous solution of the obtained Zr / Hf-MOF nanofilm loaded with AuNPs to obtain the signal unit Zr / Hf-MOF / AuNPs / Apt. S2. The activated aptamer is drop-coated onto the surface of a gold electrode and incubated to obtain an AuR / Apt electrode; the obtained AuR / Apt electrode is inserted into an electron blocking agent solution and incubated to obtain an AuR / Apt electrode that blocks non-specific binding sites. S3. Add the solution containing Staphylococcus aureus to the surface of the AuR / Apt electrode with blocked non-specific binding sites obtained in step S2 and incubate. Drop-coat the signal unit Zr / Hf-MOF / AuNPs / Apt obtained in step S1. After incubation at room temperature, detect the electrochemical differential pulse voltammetry signal of the obtained solution to perform qualitative or quantitative analysis of Staphylococcus aureus. In step S1, the Zr / Hf-MOF nanofilm is prepared by the following method: Zirconium salt, hafnium salt, and redox mediator were dissolved in a mixed solution of organic solvent and HAC, and then heated to obtain Zr / Hf-MOF nanofilms. The redox mediator is selected from 1,1′-ferrocene carboxylic acid.

2. The method of claim 1, wherein, In step S1, the doping amount of hafnium in the Zr / Hf-MOF nanofilm is 1%-5%.

3. The method of claim 1, wherein, The zirconium salt is selected from ZrCl4; And / or, the hafnium salt is selected from HfCl4; And / or, the molar ratio of the zirconium salt to the hafnium salt is 9.0-9.9:0.1-0.

5.

4. The method of claim 1, wherein, The organic solvent is selected from DMF.

5. The method of claim 1, wherein, In steps S1 and S2, the nucleotide sequence of the activated aptamer is shown in SEQ ID NO.

1.

6. The method of claim 1, wherein, In steps S1 and S2, the electron blocking agent includes 6-mercaptohexanol.

7. The method of claim 1, wherein, In step S2, the activated aptamer is obtained by dissolving the aptamer in a buffer solution and then adding TCEP solution to react at room temperature.

8. The method of claim 1, wherein, In step S3, the concentration of the solution containing S. aureus is 10 1 CFU·mL −1 ~10 7 CFU·mL −1 .

9. The MOF nanomembrane based Faraday cage type aptasensor of claim 1, wherein, Includes signal units Zr / Hf-MOF / AuNPs / Apt, aptamers, and electrodes; The Zr / Hf-MOF nanofilm in the signal unit is modified with redox mediators and electron blocking agents; The Zr / Hf-MOF nanofilm was prepared by the following method: Zirconium salt, hafnium salt, and redox mediator were dissolved in a mixed solution of organic solvent and HAC, and then heated to obtain Zr / Hf-MOF nanofilms. The redox mediator is selected from 1,1′-ferrocene carboxylic acid.

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