Novel Fe-C-N-coated Au material and preparation method and application thereof

Abstract

The invention discloses a novel Fe-C-N-coated Au material and a preparation method and application thereof.According to the Fe-C-N-coated Au material, iron monatomic nano-enzyme Fe-C-N serves as a core, gold nanoparticles are loaded on the surface, the Fe-C-N nanoparticles and a HAuCl4 aqueous solution are stirred at low temperature, then an ice-cold NaBH4 solution is slowly added into a mixed solution, and the Fe-C-N-coated Au material is obtained after reaction and treatment. The novel Fe-C-N-coated Au material disclosed by the invention can be used for rapidly and sensitively detecting the azithromycin or macrolide drug-resistant gene ermB.

Description

Technical Field

[0001] This invention belongs to the field of novel materials and their analytical applications, and particularly relates to a novel Fe-CN@Au material and its preparation method and application. Background Technology

[0002] Since their discovery in the early 20th century, antibiotics have played a vital role in improving the treatment of diseases in humans and animals, effectively suppressing bacterial infections and saving numerous lives. However, the widespread use of antibiotics has also brought about some problems, the most prominent of which is antibiotic residue. When antibiotics are ingested by humans or animals, the unmetabolized portion enters the natural environment, such as water bodies and soil, through excretion. This can affect water sources, the food chain, and even the entire ecosystem, causing environmental pollution and potentially promoting the emergence and spread of drug-resistant bacteria, posing a threat to public health.

[0003] Traditional methods for detecting antibiotic residues mainly include high-performance liquid chromatography (HPLC), gas chromatography (GC), and mass spectrometry (MS) coupled techniques, such as liquid chromatography-tandem mass spectrometry (LC-MS / MS) and gas chromatography-mass spectrometry (GC-MS). These methods are currently the "gold standard" for antibiotic residue detection, possessing high specificity and sensitivity. However, these methods also have some limitations, such as long detection cycles, high costs, strong dependence on equipment, and relatively complex operation procedures, which limit their application in routine monitoring and rapid on-site screening. Methods for detecting antibiotic resistance genes mainly include polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), multiplex PCR, nucleic acid hybridization technology, and gene chips. While these nucleic acid detection methods are highly efficient, they also have drawbacks. For example, PCR is prone to false positives or false negatives and requires high operational skills; qPCR is costly; multiplex PCR primer design is complex; nucleic acid hybridization is time-consuming; and gene chip technology is costly and requires sophisticated equipment, limiting its widespread application.

[0004] In recent years, the development of various novel sensor technologies and nanomaterials has brought new opportunities for the detection of antibiotic residues and resistance genes. Although colorimetric sensors are convenient to operate and highly sensitive, their single-channel output signal makes the detection results susceptible to interference from the external environment. Nanomaterials have advantages such as high specific surface area, enhanced signal detection capability, and excellent biocompatibility. In particular, the high specific surface area and abundant enzyme-like activity of nanozymes provide the possibility for constructing highly sensitive and selective detection platforms. The application of nanomaterials in the field of antibiotic sensing has been reported, but challenges remain in multi-mode integrated detection and rapid quantitative analysis of antibiotic residues and resistance genes in real samples (such as water samples). How to design and synthesize a nanomaterial that possesses both high catalytic activity and can achieve integrated recognition, detection, and degradation of antibiotics and resistance genes has become a key focus and challenge of current research. Summary of the Invention

[0005] To overcome the problems in the prior art, this invention provides a novel Fe-CN@Au material, its preparation method, and its application, enabling rapid and sensitive detection of the ermB gene, which is a resistance gene to azithromycin or macrolides.

[0006] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows: This invention provides a novel Fe-CN@Au material, wherein the Fe-CN@Au material has an iron single-atom nanozyme Fe-CN as the core and gold nanoparticles loaded on the surface.

[0007] The Fe-CN@Au material in this invention uses the iron single-atom nanozyme Fe-CN as the core and gold nanoparticles loaded on the surface. Fe-CN provides strong peroxidase-like activity while the gold nanoparticles provide a large number of thiol-modified DNA linking sites, enabling the composite material to have catalytic activity and DNA linking ability.

[0008] Based on the same technical concept, the present invention also provides a method for preparing the above-mentioned novel Fe-CN@Au material, comprising the following steps: S1. Preparation of iron single-atom nanoenzymes Fe-CN nanoparticles.

[0009] S2. Disperse the Fe-CN nanoparticles prepared in step S1 in water, add HAuCl4 aqueous solution, and stir at low temperature to obtain a mixture.

[0010] S3. Slowly add the ice-cold NaBH4 solution to the mixture in step S2, and continue stirring at low temperature. After processing, Fe-CN@Au material is obtained.

[0011] In this invention, iron single-atom nanozymes (Fe-CN) are first prepared as the core carrier. Gold nanoparticles are then distributed in situ on the surface of Fe-CN by sodium borohydride reduction, thereby forming Fe-CN@Au composite nanomaterials.

[0012] As an optional implementation, in the preparation method provided by the present invention, the preparation of Fe-CN in step S1 includes the following steps: S11. Add zinc source and iron source to solvent, mix well, add organic ligand, and prepare Fe / ZIF-8 after reaction.

[0013] S12. The Fe / ZIF-8 prepared in step S1 is pyrolyzed in a protective atmosphere to prepare Fe-CN nanoparticles.

[0014] As an optional implementation, in the preparation method provided by the present invention, in step S2, the mass ratio of Fe-CN nanoparticles to HAuCl4 is (0.3-0.5):1.

[0015] Furthermore, a ratio of 0.4:1 is preferred.

[0016] As an optional implementation method, in the preparation method provided by the present invention, the low temperature is 0-10℃.

[0017] As an optional implementation, in the preparation method provided by the present invention, in step S3, the molar ratio of NaBH4 to HAuCl4 is (1.5-2.0):1.

[0018] Furthermore, a ratio of 1.7:1 is preferred.

[0019] As an optional implementation, in the preparation method provided by the present invention, step S3 includes centrifugation, washing and drying.

[0020] Based on the same technical concept, the present invention also provides the application of the above-mentioned novel Fe-CN@Au material or the novel Fe-CN@Au material prepared by the above-mentioned preparation method in detecting the azithromycin or macrolide resistance gene ermB in samples.

[0021] As an optional implementation, in the application provided by the present invention, detection is performed by colorimetry or photothermal quantitative methods.

[0022] As an optional implementation, in the application provided by the present invention, when using colorimetric detection, the change in absorbance value of oxidized 3,3',5,5'-tetramethylbenzidine at 652 nm can be measured by a UV-Vis spectrophotometer to achieve quantitative detection of the ermB gene, which is a resistance gene to azithromycin or macrolides.

[0023] As an optional implementation, in the application provided by the present invention, when using the photothermal quantitative method for detection, the temperature change value of oxidized 3,3',5,5'-tetramethylbenzidine after irradiation under an 808nm laser for 5 minutes can be measured by using an infrared thermal imager to achieve quantitative detection of the ermB resistance gene of azithromycin or macrolides.

[0024] As an optional implementation, in the application provided by the present invention, when detecting azithromycin by colorimetric or photothermal quantitative methods, commercially available streptavidin magnetic beads and aptamers modified with cDNA are used for detection.

[0025] As an optional implementation, in the application provided by the present invention, when detecting the macrolide resistance gene ermB by colorimetric or photothermal quantitative methods, the CRISPR / Cas12a system is used for detection.

[0026] As an optional implementation, in the application provided by this invention, the detection of azithromycin or macrolide resistance gene ermB in a sample via a smartphone includes the following steps: adding a buffer solution containing H2O2 and 3,3',5,5'-tetramethylbenzidine to a transparent plate; adding the novel Fe-CN@Au material described above or the novel Fe-CN@Au material prepared by the above preparation method to the transparent plate; subsequently adding a solution containing the azithromycin or macrolide resistance gene ermB; capturing images using a smartphone and identifying RGB values ​​using smartphone software to quantitatively analyze the azithromycin or macrolide resistance gene ermB.

[0027] Based on the same technical concept, the present invention also provides the application of the above-mentioned novel Fe-CN@Au material or the novel Fe-CN@Au material prepared by the above-mentioned preparation method in the degradation of antibiotics, wherein the degradation technology is a Fenton-like degradation technology.

[0028] As an optional implementation, in the applications provided by the present invention, the antibiotic includes azithromycin, erythromycin, tetracycline, or ciprofloxacin.

[0029] The detection principle of this invention is as follows: The novel Fe-CN@Au material prepared in this invention possesses peroxidase-like activity that can oxidize colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to blue oxidized TMB (oxTMB), enabling colorimetric detection. Oxidized TMB exhibits a strong photothermal effect; by measuring the temperature change of oxidized TMB over a certain time, photothermal detection can be achieved. The colorimetric and photothermal signals can corroborate each other, thereby improving the accuracy and precision of detection. This material, combined with commercially available magnetic beads, aptamers, and the CRISPR / Cas12a system, enables rapid and sensitive detection of azithromycin AZM and its related resistance gene ermB. Furthermore, the high peroxidase-like activity of Fe-CN@Au can be applied to the Fenton-like degradation of antibiotics.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The Fe-CN@Au material used in this invention has superior ligation ability with thiol-modified DNA compared to Fe-CN alone. After the Fe-CN@Au material is modified into an aptamer, it can be used in conjunction with commercially available magnetic beads to modify cDNA and CRISPR / Cas12a to detect trace amounts of AZM and the antibiotic resistance gene ermB.

[0031] (2) The novel Fe-CN@Au material prepared by this invention can realize the quantitative detection of AZM and ermB in water samples through a dual-mode detection strategy of colorimetric method or photothermal quantitative method. It is simple, fast, low-cost, and highly sensitive, and can significantly improve the accuracy of detection. For example, it can be applied to the quantitative detection of azithromycin and macrolide resistance genes in the fields of food safety and water environment.

[0032] (3) The visualization detection system of the present invention can directly add the sample to be tested into a 24-well plate for reaction, and then take a picture with a mobile phone under LED white light to read the RGB value. Only a small amount of sample is needed to quickly, conveniently and accurately quantitatively detect the target substances in the water sample. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 SEM image of the Fe-CN@Au material prepared in Example 1; Figure 2 TEM image of the Fe-CN@Au material prepared in Example 1; Figure 3 Scanning transmission electron microscopy images and elemental mapping diagrams of C, N, O, Fe, and Au for the Fe-CN@Au material prepared in Example 1; Figure 4 The XPS analysis results are for the Fe-CN@Au material prepared in Example 1; Figure 5 The UV-Vis spectrum (A), photothermal curve (B), and corresponding linear relationship (CD) of the Fe-CN@Au material prepared in Example 1 for detecting AZM are shown. Figure 6 The CIE chromaticity diagram and corresponding linear relationship diagram of the Fe-CN@Au material prepared in Example 1 are shown in Figure A, where the CIE chromaticity diagram of AZM is shown and BC is shown as the corresponding linear relationship diagram; D is shown and the CIE chromaticity diagram of ermB is shown and EF is shown as the corresponding linear relationship diagram. Figure 7 The graph shows the change in the concentration of azithromycin degraded by the Fe-CN@Au material prepared in Example 1 over time. Detailed Implementation

[0035] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0036] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0037] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0038] Example 1 The preparation of novel Fe-CN@Au materials includes the following steps: (1) Synthesis of Fe-CN: Zn(NO3)2∙6H2O (6.4258 g) and iron acetylacetone (Fe(acac)3, 0.9534 g) were added to 90 mL of methanol (solution A), and sonicated for 20 minutes. Then, under sonication, the above solution A was rapidly added to 60 mL of methanol solution containing 2-methylimidazole (6.966 g) (solution B). After sonication for 5 minutes, the solution was placed at room temperature for 20 hours. The prepared sample was centrifuged at 11000 rpm for 10 min, washed three times with methanol, and finally dried in vacuum overnight to obtain Fe / ZIF-8. The Fe / ZIF-8 precursor was heated in flowing argon at 950℃ for 5 min. -1 The mixture was pyrolyzed at a heating rate of 3 hours and then cooled to room temperature to obtain Fe-CN.

[0039] (2) Synthesis of Fe-CN@Au: First, 15 mg of Fe-CN nanoparticles were dispersed in 150 mL of deionized water. Then, the mixture was sonicated. Subsequently, 3 mL of a 12 mg / mL HAuCl4 aqueous solution was added to the above solution, and the mixture was magnetically stirred at 0°C for 1 hour. Then, a 0.2 M ice-cold NaBH4 solution was prepared, and 0.9 mL of this solution was slowly added to the mixture of Fe-CN nanoparticles and HAuCl4 (completed within 4 minutes). The reaction was continued to be stirred at 0°C for 15 minutes. The solution color changed from light brown to dark purple. The prepared Fe-CN@Au nanomaterials were centrifuged at 11000 rpm for 10 min and washed three times with ethanol. Finally, the product was dried under vacuum at 50°C.

[0040] Example 2 The preparation of novel Fe-CN@Au materials includes the following steps: (1) Synthesis of Fe-CN: Zn(NO3)2∙6H2O (6.4258 g) and iron acetylacetone (Fe(acac)3, 0.9534 g) were added to 90 mL of methanol (solution A), and sonicated for 20 minutes. Then, under sonication, the above solution A was rapidly added to 60 mL of methanol solution containing 2-methylimidazole (6.966 g) (solution B). After sonication for 5 minutes, the solution was placed at room temperature for 20 hours. The prepared sample was centrifuged at 11000 rpm for 10 min, washed three times with methanol, and finally dried in vacuum overnight to obtain Fe / ZIF-8. The Fe / ZIF-8 precursor was heated in flowing argon at 950℃ for 5 min. -1 The mixture was pyrolyzed at a heating rate of 3 hours and then cooled to room temperature to obtain Fe-CN.

[0041] (2) Synthesis of Fe-CN@Au: First, 15 mg of Fe-CN nanoparticles were dispersed in 150 mL of deionized water. Then, the mixture was sonicated. Subsequently, 2.5 mL of a 12 mg / mL HAuCl4 aqueous solution was added to the above solution, and the mixture was magnetically stirred at 5 °C for 1 hour. Then, a 0.15 M ice-cold NaBH4 solution was prepared, and 0.9 mL of this solution was slowly added to the mixture of Fe-CN nanoparticles and HAuCl4 (completed within 4 minutes). The reaction was continued to be stirred at 5 °C for 15 minutes. The solution color changed from light brown to dark purple. The prepared Fe-CN@Au nanomaterials were centrifuged at 11000 rpm for 10 min and washed three times with ethanol. Finally, the product was dried under vacuum at 50 °C.

[0042] Example 3 The preparation of novel Fe-CN@Au materials includes the following steps: (1) Synthesis of Fe-CN: Zn(NO3)2∙6H2O (6.4258 g) and iron acetylacetone (Fe(acac)3, 0.9534 g) were added to 90 mL of methanol (solution A), and sonicated for 20 minutes. Then, under sonication, the above solution A was rapidly added to 60 mL of methanol solution containing 2-methylimidazole (6.966 g) (solution B). After sonication for 5 minutes, the solution was placed at room temperature for 20 hours. The prepared sample was centrifuged at 11000 rpm for 10 min, washed three times with methanol, and finally dried in vacuum overnight to obtain Fe / ZIF-8. The Fe / ZIF-8 precursor was heated in flowing argon at 950℃ for 5 min. -1 The mixture was pyrolyzed at a heating rate of 3 hours and then cooled to room temperature to obtain Fe-CN.

[0043] (2) Synthesis of Fe-CN@Au: First, 15 mg of Fe-CN nanoparticles were dispersed in 150 mL of deionized water. Then, the mixture was sonicated. Subsequently, 4 mL of a 12 mg / mL HAuCl4 aqueous solution was added to the above solution, and the mixture was magnetically stirred at 10 °C for 1 hour. Then, a 0.25 M ice-cold NaBH4 solution was prepared, and 0.9 mL of this solution was slowly added to the mixture of Fe-CN nanoparticles and HAuCl4 (completed within 4 minutes). The reaction was continued to be stirred at 10 °C for 15 minutes. The solution color changed from light brown to dark purple. The prepared Fe-CN@Au nanomaterials were centrifuged at 11000 rpm for 10 min and washed three times with ethanol. Finally, the product was dried under vacuum at 50 °C.

[0044] The Fe-CN@Au material prepared in Example 1 was tested, and its SEM image is shown below. Figure 1 As shown, the TEM image is as follows Figure 2 As shown, this invention demonstrates that Fe-CN@Au material with Fe-CN as the core and gold nanoparticles loaded on the surface was prepared.

[0045] The Fe-CN@Au material was examined using a high-angle annular dark-field scanning transmission electron microscope. The results are as follows: Figure 3 As shown in the HAADF-STEM image, the element mapping image is as follows: Figure 3 The other images shown are elemental mappings for C, N, O, Fe, and Au, illustrating that each element is uniformly distributed in the material.

[0046] To further analyze the chemical composition and surface structure of Fe-CN@Au, X-ray photoelectron spectroscopy (XPS) was used to characterize its elemental composition and electronic states. The full XPS spectrum is shown below. Figure 4 As shown in the figure, the characteristic signals of C, N, O, Fe, and Au are clearly displayed, with obvious peaks appearing in the C 1s, N 1s, O 1s, Fe 2p, and Au 4f regions, respectively. Figure 3 The TEM elemental mapping results were consistent, further confirming the successful loading of iron and gold.

[0047] Example 4 Application of novel Fe-CN@Au materials 1. Specific calorimetry and photothermal detection of azithromycin (AZM) Fe-CN@Au modified aptamers (Fe-CN@Au-Aptamer) and commercially available streptavidin magnetic beads modified with cDNA (SMB-cDNA) were used for the detection of azithromycin (AZM).

[0048] Fe-CN@Au-Aptamer material was prepared by bonding Fe-CN@Au with the aptamer DNA strand of azithromycin via gold-sulfur bonds, as follows: First, Fe-CN@Au was dispersed in deionized water at a concentration of 1 mg / mL. The thiol-modified aptamer was prepared using a buffer solution containing 10 mM Tris-HCl, 1 mM EDTA, 10 mM TCEP, and 0.1 M NaCl. Subsequently, the Fe-CN@Au colloid was mixed with the thiol-modified aptamer to a final aptamer concentration of 10 μM, and the aptamer was immobilized on the Fe-CN@Au surface within 24 h via gold-sulfur bonding. To remove unreacted reagents, the resulting Fe-CN@Au-aptamer was centrifuged at 11,000 rpm for 10 min and then washed with 10 mM phosphate buffer (pH 7.4) containing 0.25 M NaCl.

[0049] The specific detection method is as follows: 300 μL of binding buffer (10 mM Tris-HCl, 1 M NaCl, 6 mM CaCl2, 0.02% Tween 20, pH 7.0), 50 μL of Fe-CN@Au-Aptamer (0.4 mg / mL), and 75 μL of azithromycin at different concentrations (0, 10, 20, 30, 40, 50, 60, 80, 100, 120, 140, 160, 280 pM) were mixed and incubated at 37°C with shaking for 10 minutes. Then, 75 μL of SMB-cDNA (1 mg / mL) was added, and the mixture was further incubated for 40 minutes to capture unbound Fe-CN@Au-Aptamer. After magnetic separation, the supernatant was collected. Subsequently, 500 μL of acetate-sodium acetate buffer (HAC-NaAc, 10 mM, pH 4.5) containing 5 mM H2O2 and 0.5 mM TMB was added. After incubation at room temperature for 5 minutes, quantitative analysis was performed using a UV spectrophotometer. The colorimetrically analyzed solution (0.5 mL) was added to a 500 μL centrifuge tube and then irradiated with an 808 nm laser for 5 minutes. The temperature and photothermal signal of the solution were recorded using an infrared thermal imager.

[0050] Test results as follows Figure 5 As shown, from Figure 5 It can be concluded that as the concentration of AZM increases, the amount of free Fe-CN@Au-aptamer in the solution increases accordingly, leading to a gradual increase in the absorbance of oxTMB at 652 nm. Figure 5As shown in Figure A, within the linear range of 10–280 pM, the absorbance change (ΔA) at 652 nm showed a good linear relationship with the AZM concentration. The calibration equations were ΔA = 0.0119C + 0.0542 (10–120 pM, r = 0.997) and ΔA = 0.002C + 1.2028 (120–280 pM, r = 0.995), respectively. The calculated limit of detection (LOD) was 2.34 pM (S / N = 3). Figure 5 As shown in B.

[0051] As the AZM concentration increased from 20 pM to 280 pM, the amount of oxTMB continued to increase, exhibiting a significant and concentration-dependent temperature-dependent rise, such as... Figure 5 As shown in Figure C. Within the range of 20–280 pM, temperature change (ΔT) showed a linear relationship with AZM concentration, with fitting equations of ΔT = 0.3039C + 2.7706 (20–120 pM, r = 0.997) and ΔT = 0.0504C + 32.378 (120–280 pM, r = 0.995), respectively. The calculated limit of detection (LOD) was 9.17 pM (S / N = 3). Figure 5 As shown in D.

[0052] 2. Specific heat method and photothermal detection of macrolide resistance gene ermB The CRISPR / Cas12a system is used for the detection of the macrolide resistance gene ermB.

[0053] Preparation of the Fe-CN@Au-DNA-SMB signal probe: Fe-CN@Au-aptamer was thoroughly mixed with 1 mL of Tris-HCl coupling buffer (10 mM, pH 8.5, containing 1 mg SMB-cDNA, 150 mM NaCl, and 20 mM MgCl2), and then the mixture was continuously shaken at room temperature for 2 hours. After the reaction was complete, the obtained product Fe-CN@Au-DNA-SMB was collected by magnetic separation and washed repeatedly with Tris-HCl buffer to remove unreacted components and impurities. Finally, the purified Fe-CN@Au-DNA-SMB was resuspended in 1 mL of deionized water and stored at 4 °C for later use.

[0054] The CRISPR / Cas12a system consisted of 2 μL of Cas12a (1 μM), 2 μL of crRNA (1 μM), 2 μL of reaction buffer (10x NEB buffer r2.1), and 12 μL of nuclease-free water. 2 μL of ermB at different concentrations (0, 20, 40, 100, 200, 500, 1000, 2500, 5000, and 12500 pM) and 20 μL of 1.5 mg / mL Fe-CN@Au-DNA-SMB signal probe were added to the reaction system. The reaction was carried out at 37°C with a shaking speed of 1000 rpm for 2 hours. Afterward, the supernatant was separated by centrifugation. Subsequently, 460 μL of acetate-sodium acetate buffer (HAC-NaAc, 10 mM, pH 4.5) containing 5 mM H2O2 and 0.5 mM TMB was added. After incubation at room temperature for 5 minutes, quantitative analysis was performed using a UV spectrophotometer. The colorimetrically analyzed solution (0.5 mL) was added to a 500 μL centrifuge tube and then irradiated with an 808 nm laser for 5 minutes. Temperature and photothermal signals of the solution were recorded using an infrared thermal imager.

[0055] Results: The absorbance at 652 nm increased significantly with increasing ermB concentration. This confirms that increased ermB levels enhance the trans-cleavage activity of Cas12a, leading to the cleavage of more probes, the release of Fe-CN@Au, and further catalyzing the oxidation of TMB to generate blue oxTMB. Subsequently, the analytical performance of the CRISPR / Cas12a-conjugated Fe-CN@Au-DNA-SMB sensor in ermB detection was evaluated. Notably, the absorbance change (ΔA) at 652 nm showed a good linear relationship with the logarithm of ermB concentration in the range of 20 pM to 12.5 nM. The calibration equation was ΔA = 0.4611 lgC - 0.4163 (r = 0.999), and the calculated limit of detection (LOD) was 3.77 pM (S / N = 3). This result is consistent with the colorimetric results. In the range of 40 pM–12.5 nM, ΔT is linearly related to the logarithm of ermB concentration, with the fitted equation being ΔT = 13.495 lgC - 5.9871 (r = 0.997) and the limit of detection (LOD) being 8.46 pM (S / N = 3).

[0056] 3. Detection of AZM and ermB based on Fe-CN@Au smartphones The supernatant from the AZM or ermB colorimetric assay was added to 24-well plates containing 500 μL of acetate-sodium acetate buffer (HAC-NaAc, 10 mM, pH 4.5), which contained 5 mM H2O2 and 0.5 mM TMB. Images were captured using a smartphone, and RGB (red, green, blue) values ​​were identified using Color Grab software. A standard curve was then established to correlate the colorimetric values ​​with the AZM or ermB concentrations for quantitative detection. The solution was subsequently irradiated with an 808 nm laser for 5 minutes. Temperature changes (ΔT) were monitored using an infrared thermal imager, and the correlation between ΔT values ​​and AZM / ermB concentrations was established.

[0057] Test results as follows Figure 6 As shown, the quantitative analysis of AZM ( Figure 6 A and 6B show that as the concentration increases, the solution exhibits a concentration-dependent blue shift from light blue to dark blue, corresponding to a shift in the CIE chromaticity coordinates; within the range of 60–500 pM, the G / R value shows a linear relationship with concentration: G / R = 0.0042C + 0.730 (r = 0.994), with a detection limit of 22.6 pM. Photothermal detection yields complementary linear results: ΔT = 6.540C + 12.736 (r = 0.991), with a detection limit of 37.5 pM. Figure 6 As shown in Figure C, the dual-signal capability of the platform is verified. Parallel analysis of ermB ( Figure 6 Both D and 6E showed excellent performance: as the concentration increased from 0.1 nM to 5 nM, the blue shift in color gradually intensified, and the B / R value showed a linear relationship with the logarithm of the concentration: B / R = 0.4961 lgC + 1.560 (r = 0.992), with a detection limit of 57.3 pM; further validation was achieved through photothermal measurement: ΔT = 6.4442 lgC + 13.520 (r = 0.994), with a detection limit of 83.4 pM. Figure 6 As shown in F. This integrated strategy not only enables the simultaneous detection of antibiotic residues and resistance genes, but also allows for high-precision quantification on-site without the need for laboratory instruments through mutual verification of colorimetric and photothermal dual signals.

[0058] 4. Antibiotics based on Fe-CN@Au degradation 25 mg of Fe-CN@Au was weighed and added to 50 mL of azithromycin (AZM) at a concentration of 50 μg / mL in an acetate-sodium acetate buffer solution (pH 4.5) for the reaction. The mixture was shaken thoroughly for 30 minutes in the dark on a shaker to reach adsorption / desorption equilibrium. Then, 50 μL of 30% H2O2 was added to bring the H2O2 concentration in the water sample to 10 mM. At 25°C, 1 mL of solution was taken every 10 minutes and centrifuged at 11000 rpm for 5 min to separate the solids. The solution was mixed with 1 mL of 13.5 mol / L sulfuric acid until it turned yellow. Quantification was performed at 482 nm. The concentration of AZM was obtained by substituting the values ​​into a standard curve, and the AZM degradation rate was calculated.

[0059] Test results are as follows Figure 7 As shown, from Figure 7 It can be concluded that the Fe-CN@Au nanocomposite material synthesized in this invention exhibits intrinsic peroxidase-like activity, and can catalyze the reaction of AZM with H2O2 like a natural enzyme. When using H2O2 and Fe-CN@Au, the synergistic effect of the two achieves a degradation efficiency of over 90% within 60 minutes.

[0060] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. However, it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A novel Fe-CN@Au material, characterized in that, The Fe-CN@Au material has an iron single-atom nanozyme Fe-CN as its core and gold nanoparticles loaded on its surface.

2. The preparation method of the novel Fe-CN@Au material as described in claim 1, characterized in that, Includes the following steps: S1. Preparation of Fe-CN nanoparticles containing iron single-atom nanozymes; S2. Disperse the Fe-CN nanoparticles prepared in step S1 in water, add HAuCl4 aqueous solution, and stir at low temperature to obtain a mixed solution; S3. Slowly add the ice-cold NaBH4 solution to the mixture in step S2, and continue stirring at low temperature. After processing, Fe-CN@Au material is obtained.

3. The preparation method of the novel Fe-CN@Au material according to claim 1, characterized in that, The preparation of Fe-CN in step S1 includes the following steps: S11. Add zinc source and iron source to solvent, mix well, add organic ligand, and prepare Fe / ZIF-8 after reaction. S12. The Fe / ZIF-8 prepared in step S1 is pyrolyzed in a protective atmosphere to prepare Fe-CN nanoparticles.

4. The preparation method of the novel Fe-CN@Au material according to claim 1, characterized in that, In step S2, the mass ratio of Fe-CN nanoparticles to HAuCl4 is (0.3-0.5):

1.

5. The preparation method of the novel Fe-CN@Au material according to claim 1, characterized in that, The low temperature range is 0-10℃.

6. The preparation method of the novel Fe-CN@Au material according to claim 1, characterized in that, In step S3, the molar ratio of NaBH4 to HAuCl4 is (1.5-2.0):

1.

7. The preparation method of the novel Fe-CN@Au material according to claim 1, characterized in that, In step S3, the processing includes centrifugation, washing, and drying.

8. The application of the novel Fe-CN@Au material as described in claim 1 or the novel Fe-CN@Au material prepared by the preparation method according to any one of claims 2-7 in the detection of azithromycin or macrolide resistance gene ermB in samples.

9. The application according to claim 8, characterized in that, Detection is performed using colorimetric or photothermal quantitative methods.

10. The application according to claim 9, characterized in that, In colorimetric detection, the change in absorbance at 652 nm of oxidized 3,3',5,5'-tetramethylbenzidine can be measured using a UV-Vis spectrophotometer to achieve quantitative detection of the ermB gene, which is associated with resistance to azithromycin or macrolides.

11. The application according to claim 9, characterized in that, In the photothermal quantitative method, the temperature change of oxidized 3,3',5,5'-tetramethylbenzidine after irradiation with an 808nm laser for 5 minutes can be measured using an infrared thermal imager to achieve quantitative detection of the ermB resistance gene for azithromycin or macrolides.

12. The application according to claim 10, characterized in that, When detecting azithromycin using colorimetric or photothermal quantitative methods, commercially available streptavidin magnetic beads and aptamers modified with cDNA are used for detection.

13. The application according to claim 10, characterized in that, When detecting the macrolide resistance gene ermB using colorimetric or photothermal quantitative methods, the CRISPR / Cas12a system is used for detection.

14. The application according to claim 8, characterized in that, The method for detecting azithromycin or macrolide resistance gene ermB in samples using a smartphone includes the following steps: adding a buffer solution containing H2O2 and 3,3',5,5'-tetramethylbenzidine to a transparent plate; adding the novel Fe-CN@Au material as described in claim 1 or the novel Fe-CN@Au material prepared by any one of claims 2-7 to the transparent plate; subsequently adding a solution containing the azithromycin or macrolide resistance gene ermB; capturing images using a smartphone and identifying RGB values ​​using smartphone software to quantitatively analyze the azithromycin or macrolide resistance gene ermB.

15. The application of the novel Fe-CN@Au material as described in claim 1 or the novel Fe-CN@Au material prepared by the preparation method according to any one of claims 2-7 in the degradation of antibiotics, characterized in that, The degradation technology is a Fenton-like degradation technology.

16. The application according to claim 15, characterized in that, The antibiotics include azithromycin, erythromycin, tetracycline, or ciprofloxacin.

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