Composite material GZHMU-2 (at) Ag and application of composite material GZHMU-2 (at) Ag in preparation of self-cleaning biosensor

Abstract

The invention belongs to the technical field of organic materials, and particularly relates to a composite material GZHMU-2 (at) Ag and application of the composite material GZHMU-2 (at) Ag in preparation of a self-cleaning biosensor. The composite material GZHMU-2 (at) Ag is synthesized by loading AgNPs on GZHMU-2 through a method of reducing silver nitrate through ultraviolet irradiation, and the GZHMU-2 (at) Ag can generate active oxygen through photocatalysis, so that an antibacterial effect is achieved, and the composite material has a killing effect on escherichia coli and staphylococcus aureus. Based on a metal covalent organic framework modified by silver nanoparticles, a carboxyl functionalized SA31 aptamer is fixed on GZHMU-2 (at) Ag through an amidation reaction, a self-cleaning electrochemical biosensor for sensitive detection and real-time inactivation of bacteria is developed, the photocatalytic and electrochemical properties of the sensor are remarkably enhanced, and the sensor has a wide application prospect. The method has important potential for field monitoring and photocatalytic disinfection of pathogens in practical application.

Description

Technical Field

[0001] This invention belongs to the technical field of organic materials, specifically relating to a composite material GZHMU-2@Ag and its application in the preparation of self-cleaning biosensors. Background Technology

[0002] Early detection and effective inactivation of bacteria are crucial for preventing the spread of disease. Many analytical techniques, such as enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay, and colorimetric methods, have been used for bacterial detection. However, most of these methods require complex equipment and involve time-consuming procedures.

[0003] Electrochemical biosensors, with their advantages of high speed, high sensitivity, and low cost, have shown great potential for the sensitive detection of biomolecules in complex samples. Several electrochemical immunobiosensors have also been used to detect bacteria. However, these sandwich-based immunoassay strategies involve multiple elements and steps, making reproducibility challenging, while unlabeled detection strategies are often less sensitive. Therefore, there is a need to develop a rapid, sensitive, simple, and reproducible "one-step" strategy that can both identify the target and detect the signal. Nanozymes, a specific class of artificial enzyme mimics based on nanomaterials, are gaining increasing attention as alternatives to natural enzymes due to their unique environmental adaptability, reusability, and extended durability. Furthermore, the development of nanomaterials has brought new opportunities for the development of electrochemical methods.

[0004] MOFs (Metal-Organic Facility-Based Materials) are a class of porous crystalline materials assembled from metal ions / clusters and organic ligands through coordination bonds. When used as nanozymes, their core characteristic is the combination of porous structure and diverse enzymatic activities, allowing them to mimic the catalytic functions of various natural enzymes, rather than a single type of nanozyme. They possess a wide range of porosities and strong design flexibility, offering numerous advantages in photocatalytic performance. In recent years, many nanomaterials have been used as electrodes for detection, but the detection sensitivity of these materials needs improvement, as simultaneous inactivation cannot be achieved.

[0005] Besides detection, in-situ elimination of bacteria has become increasingly attractive. Several types of nanomaterials have been used to kill bacteria. For example, Ag... + It can interact with proteins on the cell membrane of microorganisms, leading to protein inactivation and bacterial cell death. Zhong et al. found that the Au / Ir@Cu / Zn-MOF lateral flow immunosensor can kill bacteria while detecting them. Wang et al. invented an enzyme biofuel cell that can achieve the detection and in-situ elimination of bacteria.

[0006] MOFs (Metal-Organic Facility Materials) possess excellent electrochemical and photocatalytic properties, enabling simultaneous electrochemical detection and inactivation, and hold promise for developing new detection methods. Many MOF materials have been developed; however, many suffer from poor visible light responsiveness and rapid electron-electron-hole recombination caused by light, which reduces their photocatalytic efficiency. To address these shortcomings, metal nanoparticles exhibiting surface plasmon resonance (SPR), such as silver and gold, are incorporated into MOFs to enhance charge separation, allowing photoinduced charge carriers to participate in the reaction and improving photocatalytic efficiency. Although the porous structure of MOFs facilitates rapid diffusion of reactive oxygen species (ROS), their extremely short lifetime (<40 ns) and short diffusion distance (approximately 10 nm) limit their effectiveness. Therefore, further design and improvement are needed to develop MOFs with high photodynamic antibacterial activity. Summary of the Invention

[0007] To address the above-mentioned problems, the present invention aims to provide a composite material GZHMU-2@Ag and its application in the preparation of self-cleaning biosensors.

[0008] The technical content of this invention is as follows: This invention provides a high-performance MOF-based composite material GZHMU-2@Ag, which is a composite material obtained by encapsulating silver nanoparticles in the metal-organic framework material GZHMU-2. The structure of the metal-organic framework material GZHMU-2 is as follows: ; The structure of the composite material GZHMU-2@Ag is as follows:

[0009] This invention also provides a method for preparing a high-performance MOF-based composite material GZHMU-2@Ag, comprising the following steps: GZHMU-2 was added to an organic solvent and ultrasonically mixed until homogeneous. Then, silver salt was added and stirred thoroughly in the dark. After stirring, the mixture was irradiated under a UV lamp. The product was washed, stored in the dark, and finally vacuum dried to obtain the composite material GZHMU-2@Ag. The solid-liquid ratio of GZHMU-2 and organic solvent is (1~10):1 mg / mL; The mass ratio of GZHMU-2 to silver salt is (1~5):1; The organic solvent includes one of anhydrous ethanol, ethylene glycol, N,N-dimethylformamide, and N-methylpyrrolidone; The silver salt includes one of silver nitrate, silver sulfate, and silver chloride.

[0010] Modifying MOFs with silver nanoparticles not only enhances the electrochemical activity of the materials but also strengthens the photocatalytic generation of ROS under visible light, promoting the rapid transfer of ROS to bacteria. Furthermore, the GZHMU-2@Ag composite material exhibits enzyme-mimicking activity, promoting the inactivation of Staphylococcus aureus under simulated sunlight irradiation conditions. These inactivated bacteria can desorb from the photoelectrode surface, exposing more active sites and restoring the electrochemical signal.

[0011] This invention also provides an application of the high-performance MOF-based composite material GZHMU-2@Ag in the preparation of self-cleaning biosensors; The self-cleaning biosensor is prepared by dissolving the composite material GZHMU-2@Ag and dropping it onto the working electrode to obtain the GZHMU-2@Ag / SPE photoelectrode. The SA31 aptamer is then fixed onto the photoelectrode to obtain the self-cleaning biosensor. The resulting self-cleaning biosensor enables the detection and in-situ elimination of bacteria (Staphylococcus aureus).

[0012] The beneficial effects of this invention are as follows: The high-performance MOF-based composite material GZHMU-2@Ag of this invention is synthesized by loading AgNPs onto GZHMU-2 through a method of reducing silver nitrate under ultraviolet light. GZHMU-2@Ag can generate reactive oxygen species through photocatalysis, thereby exerting an antibacterial effect, and it has a killing effect on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Based on a silver nanoparticle-modified metal covalent organic framework (MCOF), a carboxyl-functionalized SA31 aptamer is immobilized on GZHMU-2@Ag through an amide reaction to develop a self-cleaning electrochemical biosensor for sensitive detection and real-time inactivation of bacteria. GZHMU-2@Ag is prepared by in-situ deposition of silver on GZHMU-2, and its photocatalytic and electrochemical performance is significantly enhanced. The SA31 aptamer, which is specific to Staphylococcus aureus, is immobilized on the surface of the GZHMU-2@Ag electrode. In the presence of Staphylococcus aureus, target binding causes a measurable decrease in the electrochemical signal, thus enabling highly sensitive detection of the bacteria. This sensor can detect concentrations ranging from 1 to 10 within 10 minutes. 5Logarithmic quantification of Staphylococcus aureus at CFU / mL was performed, with a detection limit as low as 1 CFU / mL. Notably, due to the excellent photocatalytic activity of GZHMU-2@Ag, the functionalized electrode generated reactive oxygen species (ROS) within 20 minutes of illumination, achieving a 99.4% inactivation rate of captured bacteria. Subsequently, the desorption of inactivated bacteria restored the electrode's detection activity. This work provides a novel strategy for constructing MCOF-based biosensors that integrate bacterial detection and efficient inactivation, showing significant potential for on-site pathogen monitoring and photocatalytic disinfection in practical applications. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the synthesis process of the GZHMU-2@Ag biosensor of the present invention; Figure 2 This is a graph showing the results of photocatalytic bactericidal activity of the GZHMU-2@Ag powder of this invention; Figure 3 The XRD patterns of GZHMU-2 powder and GZHMU-2@Ag of this invention are shown below. Figure 4 This is the SEM image of GZHMU-2@Ag from the present invention; Figure 5 The image shows the morphological differences of bacteria with and without GZHMU-2@Ag photocatalytic sterilization observed by low-pressure transmission electron microscopy. Figure 6 Fluorescent staining images of bacteria with and without GZHMU-2@Ag photocatalytic bactericidal activity observed under a fluorescence microscope; Figure 7 The spectrum of reactive oxygen species in GZHMU-2 and other MOF materials of the present invention was determined by EPR. Figure 8 This is an LB plate bacterial culture diagram of GZHMU-2@Ag photocatalytic sterilization according to the present invention; Figure 9 The standard curve and it curve for detecting Staphylococcus aureus using GZHMU-2@Ag / SPE / SA31 in this invention are provided. Detailed Implementation

[0014] The present invention will be further described in detail below through specific implementation examples and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope of the appended claims.

[0015] Unless otherwise specified, all raw materials and reagents used in this invention are from the conventional market.

[0016] Example 1 Preparation of a high-performance photocatalytic composite material GZHMU-2@Ag 1) Synthesis of Cu3L: 2.0 g of 1H-pyrazole-4-carboxaldehyde and 4.0 g of copper nitrate were dissolved in 51 mL of deionized water, 67 mL of DMF and anhydrous ethanol were added, and the mixture was heated in an oil bath at 110 °C. The blue color faded, and the product was obtained by filtration and washing. The reaction formula is as follows: ; 2) Synthesis of GZHMU-2: 0.44 g p-toluenesulfonic acid, 80 mg tris(4-aminophenyl)amine, and 0.14 g Cu3L were added to 20 mL of polyethylene glycol (PEG: 400: 1000). After stirring and mixing, the mixture was reacted at 170 °C for 30 min. The precipitate was collected by centrifugation, washed with tetrahydrofuran, deionized water, and DMF, and dried to obtain GZHMU-2 powder. The reaction formula is as follows: ; 3) Synthesis of GZHMU-2@Ag: 40 mg of GZHMU-2 was added to 20 mL of anhydrous ethanol and sonicated for 10 min to ensure thorough mixing. 30 mg of silver nitrate was added to the mixed solution, and the mixture was stirred in the dark for 1 h. After stirring, the mixture was irradiated under a UV lamp for 1 h. The resulting material was washed twice with deionized water, stored in the dark, and finally vacuum dried to obtain GZHMU-2@Ag. The reaction formula is as follows: ; The photocatalytic bactericidal activity of the GZHMU-2@Ag powder prepared in Example 1 was investigated. Staphylococcus aureus was used as a model bacterium, and the bactericidal efficiency was statistically analyzed using the plate count method under both light and dark conditions. The results are as follows: Figure 1 , 2 As shown, under light conditions, GZHMU-2 powder material can effectively kill Staphylococcus aureus, and the antibacterial rate can reach 99.4% when the material concentration is 300 μg / mL.

[0017] Example 2 Preparation of a high-performance photocatalytic composite material GZHMU-2@Ag 1) Synthesis of Cu3L: 2 Same as in Example 1; 2) Synthesis of GZHMU-2: Same as in Example 1; 3) Synthesis of GZHMU-2@Ag: 60 mg of GZHMU-2 was added to 20 mL of ethylene glycol and sonicated for 12 min to mix thoroughly. 40 mg of silver sulfate was added to the mixed solution, and the mixture was stirred in the dark for 1 h. After stirring, the mixture was irradiated under a UV lamp for 1 h. The material obtained from the reaction was washed twice with deionized water, stored in the dark, and finally vacuum dried to obtain GZHMU-2@Ag.

[0018] Example 3 Preparation of a high-performance photocatalytic composite material GZHMU-2@Ag 1) Synthesis of Cu3L: 2 Same as in Example 1; 2) Synthesis of GZHMU-2: Same as in Example 1; 3) Synthesis of GZHMU-2@Ag: 80 mg of GZHMU-2 was added to 20 mL of N-methylpyrrolidone and sonicated for 15 min to mix thoroughly. 30 mg of silver chloride was added to the mixed solution, and the mixture was stirred in the dark for 1 h. After stirring, the mixture was irradiated under a UV lamp for 1 h. The material obtained from the reaction was washed twice with deionized water, stored in the dark, and finally vacuum dried to obtain GZHMU-2@Ag.

[0019] Example 4 Preparation of a high-performance photocatalytic composite material GZHMU-2@Ag 1) Synthesis of Cu3L: 2 Same as in Example 1; 2) Synthesis of GZHMU-2: Same as in Example 1; 3) Synthesis of GZHMU-2@Ag: 100 mg of GZHMU-2 was added to 20 mL of N-methylpyrrolidone and sonicated for 10 min to mix thoroughly. 20 mg of silver chloride was added to the mixed solution, and the mixture was stirred in the dark for 1 h. After stirring, the mixture was irradiated under a UV lamp for 1 h. The material obtained from the reaction was washed twice with deionized water, stored in the dark, and finally vacuum dried to obtain GZHMU-2@Ag.

[0020] Test case 3 mg of GZHMU-2@Ag was weighed and dissolved in 1 mL of H2O:C2H5OH = 1:1 solution. The solution was sonicated for 20 minutes to obtain a well dispersed solution. 10 μL of the dispersion solution was added dropwise to the working electrode of a screen-printed electrode (SPE). The electrode was allowed to dry at room temperature to obtain a GZHMU-2@Ag / SPE photoelectrode. Electrochemical tests were performed using 0.5 mol / L (pH = 6.22) sodium sulfate as electrolyte, silver chloride as reference electrode, carbon black as counter electrode, and a carbon black electrode coated with the material as working electrode.

[0021] SA31 aptamers were immobilized on the GZHMU-2@Ag / SPE photoelectrode via intermolecular forces between the -COOH group of the SA31 aptamer and the -NH2 group on GZHMU-2@Ag. 10 μL of 10 mg / mL EDC and 10 μL of 1000 mM aptamer were added to the photoelectrode surface and incubated at 37°C for 35 min. Afterward, the surface was washed with PBS to remove any unbound SA31 aptamers.

[0022] The GZHMU-2 powder and GZHMU-2@Ag powder from Example 1 were characterized by X-ray powder diffraction (XRD) using a Bruker D8 Advance (Germany). The test conditions were as follows: voltage 40 kV, current 40 mA. The results are as follows: Figure 3 As shown, the diffraction peaks of GZHMU-2@Ag correspond completely to the diffraction peaks of GZHMU-2, and at the same time, the characteristic peaks of Ag are newly added. The XRD results confirm the successful synthesis of the GZHMU-2@Ag composite material.

[0023] The microstructure of GZHMU-2 and GZHMU-2@Ag from Example 1 was observed using a MARA3 scanning electron microscope manufactured by a Czech company. The test conditions were as follows: accelerating voltage 40 kV, current 5 mA. The results are as follows: Figure 4 As shown in the electron microscopy results, AgNPs nanoparticles are uniformly grown in situ on GZHMU-2.

[0024] The morphological differences of bacteria with and without GZHMU-2@Ag photocatalytic bactericidal activity were observed using low-pressure transmission electron microscopy. The results are as follows: Figure 5 As shown, after being treated with GZHMU-2@Ag material by light, the bacterial outer mold became severely wrinkled, damaged, and even broken.

[0025] The inactivation of bacteria with and without GZHMU-2 photocatalytic sterilization was observed using fluorescence microscopy. The results are as follows: Figure 6 As shown, the red fluorescence was significantly enhanced after being treated with GZHMU-2 material, indicating that the cell membrane was damaged.

[0026] Electron paramagnetic resonance (EPR) spectroscopy was used to identify the types of ROS generated during photocatalysis, such as... Figure 7 As shown, no characteristic signals were detected in the spectra of either material under dark conditions. However, DMPO-·O2 was observed under visible light illumination. - DMPO-·OH and TEMP- 1The characteristic peak of O2 confirms that visible light radiation is essential for ROS generation, and all three reactive oxygen species participate in the photocatalytic process. In summary, AgNP incorporation into GZHMU-2 induces a redshift in light absorption and a narrowing of the band gap, which together enhances the separation of photogenerated charges. Therefore, under simulated sunlight irradiation, GZHMU-2@Ag efficiently generates ROS, including O2. - ·OH and 1 O2 exhibits excellent photocatalytic activity. It can be seen that the peak positions in the EPR spectrum are related to ·OH and ·O2. - and 1 The characteristic peaks of O2 are consistent, and the peaks of the EPR spectrum of GZHMU-2@Ag are the most obvious.

[0027] The Gram-positive bacterium Staphylococcus aureus was used as the experimental bacteria. Cells from P4 to P6 were prepared to a concentration of 1×10⁻⁶. 5 A bacterial suspension of CFU / mL was prepared, and 100 μL of the suspension was added dropwise to the surface of the GZHMU-2@Ag / SPE / SA31 photoelectrode. The electrode was then heated with 110 mW / cm². 2 Irradiate the bacterial suspension with visible light for 20 min (using a 300 W xenon lamp equipped with an AM 1.5 filter and attenuator). After the reaction, serially dilute the bacterial suspension twice with sterile physiological saline, each time by a factor of 10. Take the diluted bacterial suspension (10... 3 100 μL of (CFU / mL) was evenly spread onto LB agar medium and incubated at 37°C for 18–24 h. Colony counts were then performed. A series of experiments were conducted in the dark under the same conditions as described above. Light and dark control groups were performed without the addition of a photocatalyst. Experimental results are as follows: Figure 8 As shown, the bacterial mortality rate on the electrode surface after light irradiation was 99.4%. Compared with previously reported inactivation methods, no addition of H2O2 is required. Furthermore, the use of visible light for inactivation prevents potential damage to the aptamers that might be caused by laser or ultraviolet irradiation.

[0028] Different concentrations (from 1, 10, 10) were used. 2 10 3 10 4 10 5 A bacterial suspension (CFU / mL) was added to the surface of GZHMU-2@Ag / SPE / SA31 and incubated at 37°C for 40 min. In the blank solution, the same volume of physiological saline was used instead of Staphylococcus aureus. 0.5 M Na₂SO₄ (pH=6.22) was used as the electrolyte to detect the electrochemical signal. Results are as follows: Figure 9As shown, due to the specific binding between Staphylococcus aureus and the SA31 aptamer on the electrode, Staphylococcus aureus can be quantitatively detected by monitoring changes in electrical signal intensity. To quantitatively detect Staphylococcus aureus, different doses of Staphylococcus aureus (range: 1-10) were added to the GZHMU-2@Ag / SPE / SA31 electrode. 5 The electrical signals of different samples were recorded using a method involving the concentration of *Staphylococcus aureus* (CFU / mL). The results showed that the electrical signal intensity consistently decreased with increasing *Staphylococcus aureus* concentration. Furthermore, the linear regression equation between the current decrease (ΔI) and the *Staphylococcus aureus* concentration was I = 6.031 - 6.931 log C (CFU / mL). The calculated limit of detection (LOD) was 1 CFU / mL (S / N = 3). Therefore, due to the excellent electrochemical properties of GZHMU-2@Ag, a one-step quantitative detection of *Staphylococcus aureus* can be achieved, which is more sensitive and simpler than previously reported electrochemical methods.

Claims

1. A high-performance MOF-based composite material GZHMU-2@Ag, characterized in that, It is a composite material obtained by encapsulating silver nanoparticles in the metal-organic framework material GZHMU-2; The structure of the metal-organic framework material GZHMU-2 is as follows: ； The structure of the composite material GZHMU-2@Ag is as follows: .

2. A method for preparing the high-performance MOF-based composite material GZHMU-2@Ag as described in claim 1, characterized in that, Includes the following steps: GZHMU-2 was added to an organic solvent and ultrasonically mixed until homogeneous. Then, silver salt was added and stirred thoroughly in the dark. After stirring, the mixture was irradiated under a UV lamp. The product was washed, stored in the dark, and finally vacuum dried to obtain the composite material GZHMU-2@Ag.

3. The method for preparing the composite material GZHMU-2@Ag according to claim 2, characterized in that, The solid-liquid ratio of GZHMU-2 and organic solvent is (1~10):1 mg / mL.

4. The preparation method of the composite material GZHMU-2@Ag according to claim 2, characterized in that, The mass ratio of GZHMU-2 to silver salt is (1~5):

1.

5. The method for preparing the composite material GZHMU-2@Ag according to claim 2, characterized in that, The organic solvent includes one of anhydrous ethanol, ethylene glycol, N,N-dimethylformamide, and N-methylpyrrolidone.

6. The method for preparing the composite material GZHMU-2@Ag according to claim 2, characterized in that, The silver salt includes one of silver nitrate, silver sulfate, and silver chloride.

7. The application of the high-performance MOF-based composite material GZHMU-2@Ag as described in claim 1 in the preparation of a self-cleaning biosensor.

8. The application according to claim 7, characterized in that, The self-cleaning biosensor is prepared by dissolving the composite material GZHMU-2@Ag and dropping it onto the working electrode to obtain the GZHMU-2@Ag / SPE photoelectrode. The SA31 aptamer is then fixed onto the photoelectrode to obtain the self-cleaning biosensor.

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