Preparation method of Au-CeO2@4MPBA Janus structure nanoparticle array type SERS substrate

Abstract

The application is suitable for the technical field of biosensors, and provides a preparation method of an Au-CeO2@4MPBA Janus structure nanoparticle array type SERS substrate, which comprises the following steps: step 1, synthesis of Au-CeO2@4MPBA Janus structure nanoparticles; step 2, assembly of a nanoparticle array SERS substrate; and step 3, construction of a diagnosis and treatment integrated platform. The method enables the relatively simple nanoparticles to have high-strength SERS hot spots through a metal-semiconductor heterojunction structure. The 4- MPBA multifunctional small molecule is modified on the surface of the nanoparticles, and the small molecule has three functions of regulating the particle spacing, providing Raman signals as a signal molecule, and serving as a bacterial capture molecule. The prepared SERS detection platform exhibits considerable enzyme activity and photothermal performance, so that the SERS biosensor can remove the captured bacteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and in particular relates to a method for preparing a nanoparticle array type SERS substrate with an Au-CeO2@4MPBA Janus structure. Background Art

[0002] SERS biosensors are highly sensitive detection tools that combine plasmonic nanomaterials with Raman spectroscopy. SERS significantly amplifies molecular Raman signals through the localized surface plasmon resonance (LSPR) effect of noble metal nanoparticles. This enhancement can be precisely tuned by designing the nanoparticle morphology and interparticle spacing, particularly by combining sharp tip structures with nanoscale gaps to generate strong localized electromagnetic fields. The abundance of nanoparticles and interparticle gaps in noble metal nanoparticle arrays makes them ideal SERS substrates.

[0003] In the design of SERS biosensing platforms, the performance of SERS substrates is highly dependent on the sharpness and spacing control of the noble metal nanostructures. Therefore, the preparation of SERS substrates is currently too difficult, requiring complex nanomorphology control during the synthesis and preparation process, making the preparation method difficult and conflicting with the simplicity and large-scale preparation required for practical applications. Therefore, in the preparation process of noble metal nanoparticle arrays, there are three problems:

[0004] 1. Intensity of a single hotspot: To maximize the density of noble metal nanoparticles, simple noble metal nanostructures such as nanospheres, nanorods, and nanocubes are often used. In these cases, the SERS intensity of the nanoparticles themselves is poor, and the substrate performance is entirely dependent on the interparticle gaps. While research has shown that metal-semiconductor heterojunction interfaces generate very strong SERS hotspots, the overlying semiconductor material can shield these hotspots from effective utilization.

[0005] 2. Large-scale control of interparticle spacing: The optimal interparticle gap in SERS substrates is typically around 1 nm. Currently, the method for large-scale and precise control of nanogaps is to modify the nanoparticle surface with amphiphilic ligands such as polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG) during the array self-assembly process. Without proper surface functionalization, nanoparticles tend to aggregate uncontrollably during self-assembly, resulting in reduced hotspot intensity, reduced hotspot density, and impaired signal uniformity. However, a technical disadvantage of this strategy is that the surface ligands typically occupy the position of the nanogap that produces the strongest electromagnetic enhancement. This results in Raman noise from the ligand itself and reduced efficiency of subsequent SERS substrate functionalization.

[0006] 3. Existing SERS biosensors for bacteria lack simultaneous bactericidal capabilities, making them incapable of integrated, instant diagnosis and treatment of infectious pathogens. Furthermore, the diversity of bacterial species in the real environment makes traditional label-free SERS detection susceptible to interference from saliva components, resulting in insufficient specificity. Summary of the Invention

[0007] The purpose of the embodiments of the present invention is to provide a method for preparing a nanoparticle array type SERS substrate with an Au-CeO2@4MPBA Janus structure, aiming to solve the problems raised in the above background technology.

[0008] The embodiment of the present invention is implemented as follows: a method for preparing a nanoparticle array type SERS substrate with an Au-CeO2@4MPBA Janus structure comprises the following steps:

[0009] Step 1: Synthesis of Au-CeO2@4MPBA Janus-structured nanoparticles (Janus structure is a double-sided structure. In the field of nanomaterials, Janus structure refers to a single particle or material surface that is divided into two or more regions with different physical / chemical properties);

[0010] Step 2: Assembly of nanoparticle array SERS substrate;

[0011] Step 3: Construction of an integrated diagnosis and treatment platform.

[0012] Further technical solution, said step 1 comprises the following specific steps:

[0013] Step 1.1: Synthesis of gold nanorods (Au NRs) using CTAB and sodium oleate dual ligands;

[0014] First, prepare the main reaction system, Solution A: Dissolve 7.0 g of hexadecanotrimethylammonium bromide (CTAB) and 1.234 g of sodium oleate in 250 mL of water. Add 18 mL of 4 mM silver nitrate (AgNO₃) and stir at 30°C. After 15 minutes, add 250 mL of 1 mM chloroauric acid (HAuCl₄) and stir for 90 minutes. Twenty-five minutes before the end of this step, prepare Solution B, the gold seed solution. Add 5 mL of 0.5 mM HAuCl₄ to 5 mL of 0.2 M CTAB, followed by 1 mL of 6 mM sodium borohydride (NaBH₄). Stir vigorously for 2 minutes and let stand at room temperature for 30 minutes. Twenty minutes before the end of Solution B, add 1.5 mL of concentrated hydrochloric acid to Solution A. After stirring at low speed for 15 minutes, add 1.25 mL of 64 mM ascorbic acid (AA). After rapid stirring for 30 s, 0.4 mL of solution B was added. After rapid stirring for 30 s, the mixture was stirred at low speed at 30°C for 12 h. After the reaction was completed, the mixture was centrifuged at 7500 rpm for 13 min twice and collected in 45 mL of water. The total volume was approximately 1 mg mL -1 Store at room temperature.

[0015] Step 1.2: Synthesis of Au@CeO2 nanoparticles with Janus structure;

[0016] 2 mL of Au NRs aqueous solution was dispersed in 15 mL of water. 0.3 mL of 0.1 M CTAB solution was then added and stirred at room temperature. 0.13 mL of 0.1 M cerium acetate was added dropwise, stirred for 10 minutes, and then slowly heated to 80°C. The reaction was carried out at 80°C for 4 hours, and the final product was centrifuged at 7000 rpm for 10 minutes twice. 100 μL of 10 -3 M 4-mercaptophenylboronic acid (4MPBA) was added to the Janus structure Au@CeO2 nanoparticle solution and reacted for 4 hours to obtain Au@CeO2@4MPBA nanoparticles for use.

[0017] Further technical solution, said step 2 includes the following specific steps:

[0018] Step 2.1: Preparation of Au@CeO2@4MPBA nanoparticle film;

[0019] Prepare 3 mL of a concentration of 50-400 µg mL -1Au@CeO2@4MPBA nanoparticle aqueous solution. Under a certain temperature range (20-60°C), a certain amount of n-hexane is added dropwise until the water surface is completely covered. Ethanol is then continuously added to the system until a dense metal film is formed at the interface between water and n-hexane, which is the Au@CeO2@4MPBA nanoparticle film.

[0020] Step 2.2: Transfer the Au@CeO2@4MPBA nanoparticle film to the substrate surface to complete the assembly of the nanoparticle array SERS substrate.

[0021] Further technical solution, said step 1 comprises the following specific steps:

[0022] Gold nanorods (Au NRs) were synthesized using a dual ligand system of CTAB and sodium oleate. First, prepare the main reaction system, Solution A: dissolve 7.0 g of hexadecanotrimethylammonium bromide (CTAB) and 1.234 g of sodium oleate in 250 mL of water. Add 18 mL of 4 mM silver nitrate (AgNO3) and stir at 30°C. After 15 minutes, add 250 mL of 1 mM chloroauric acid (HAuCl4) and stir for 90 minutes. Twenty-five minutes before the end of this step, prepare the gold seed solution, Solution B: add 5 mL of 0.5 mM HAuCl4 to 5 mL of 0.2 M CTAB, followed by 1 mL of 6 mM sodium borohydride (NaBH4). Stir vigorously for 2 minutes and let stand at room temperature for 30 minutes. 20 minutes before the end of the reaction of Solution B, add 1.5 mL of concentrated hydrochloric acid to Solution A. After slow stirring for 15 minutes, add 1.25 mL of 64 mM ascorbic acid (AA). After rapid stirring for 30 seconds, add 0.4 mL of Solution B. After rapid stirring for 30 seconds, maintain slow stirring at 30°C for 12 hours. After the reaction is complete, centrifuge at 7500 rpm for 13 minutes twice and collect in 45 mL of water. The total yield is approximately 1 mg mL -1 Store at room temperature. Next, Janus structure Au@CeO2 nanoparticles were further synthesized and prepared by dispersing 2 mL of Au NRs aqueous solution in 15 mL of water. Then, 0.3 mL of 0.1 M CTAB solution was added and stirred at room temperature. 0.13 mL of 0.1 M cerium acetate was added dropwise, stirred for 10 minutes, and then slowly heated to 80°C. The reaction was carried out at 80°C for 4 hours, and the final product was centrifuged at 7000 rpm for 10 minutes twice. 100 μL of 10 -3 M 4-mercaptophenylboronic acid (4MPBA) was added to the Janus structure Au@CeO2 nanoparticle solution and reacted for 4 hours to obtain Au@CeO2@4MPBA nanoparticles for use.

[0023] Further technical solution, said step 2 includes the following specific steps:

[0024] Prepare 3 mL of a concentration of 50-400 µg mL -1 An aqueous solution of Au@CeO2@4MPBA nanoparticles was prepared. Within a certain temperature range (20-60°C), a certain amount of n-hexane was added dropwise until the water surface was completely covered. Ethanol was then continuously added to the system until a dense metal film formed at the interface between the water and n-hexane. Next, the Au@CeO2@4MPBA nanoparticle film was salvaged and transferred to the substrate surface.

[0025] According to a further technical solution, in step 2, the substrate may be a silicon wafer, thermoplastic PU, PET, non-woven fabric, or copper mesh, etc. Among them, hydrophobic materials such as silicon wafer, thermoplastic PU, PET, and copper mesh need to be treated with ozone before use to enhance their hydrophilicity.

[0026] The method for preparing the Au-CeO2@4MPBA Janus structure nanoparticle array type SERS substrate provided in the embodiment of the present invention has the following beneficial effects:

[0027] (1) The strong SERS hotspot at the metal-semiconductor interface is exposed and effectively utilized by the semi-coating structure of CeO2 on one side of the gold nanorod nanoparticles, thereby improving the intensity of a single hotspot on the SERS substrate.

[0028] (2) By modifying the surface of Janus structured nanoparticles with 4-mercaptophenylboronic acid (4MPBA) small molecules to uniformly regulate the interparticle spacing, the 4MPBA small molecule surface ligand can increase the nanoparticle density per unit area during the self-assembly process, so that a larger proportion of the interparticle spacing is adjusted to about 1.1 nm, thereby improving the overall intensity of the hot spots in the nanoparticle gaps; the 4MPBA small molecule itself has strong Raman activity and can act as a signal molecule, avoiding the additional surface ligands occupying the interparticle position and causing performance degradation; at the same time, the 4MPBA small molecule has an affinity for bacterial surface glycoproteins and can play a role in bacterial capture in biosensors targeting bacteria.

[0029] (3) During the bacterial detection process, the designed ratiometric SERS sensing strategy can perform sensing by comparing the ratio of the SERS tag signal on the bacterial surface to the reference signal on the substrate surface, thereby reducing the interference of the external environment, equipment, and substrate heterogeneity on the sensing results; at the same time, the photothermal performance and peroxidase activity of the SERS substrate can effectively remove the captured bacteria, and has the integrated detection-treatment function. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The synthesis process of Janus structured nanoparticles;

[0031] Figure 2 Figure 1 shows the assembly process of the nanoparticle array SERS substrate, where a shows the process of assembling nanoparticles into a single-layer dense film through the water-oil interface self-assembly method, b shows the size, appearance, and color of the nanoparticle film prepared on the TPU film surface, and c shows the flexibility of the nanoparticle film prepared on the TPU film surface.

[0032] Figure 3 The process of building an integrated diagnosis and treatment platform;

[0033] Figure 4 Transmission electron microscopy (TEM) images and elemental energy spectrum images of Au@CeO2 nanoparticles, where A is the TEM image of Au@CeO2 nanoparticles, and B, C, and D are elemental energy spectrum images of Au@CeO2 nanoparticles.

[0034] Figure 5 The electric field simulation results of Au@CeO2 nanoparticles and Au NRs, as well as the performance comparison of SERS substrates composed of different nanoparticles, where A is the FDTD electric field simulation result of Au@CeO2 nanoparticles, B is the electric field simulation result of Au NRs, and C and D are the performance comparisons of SERS substrates composed of different nanoparticles;

[0035] Figure 6 Figure 3 is the scanning electron microscopy characterization of the array self-assembly in the absence and presence of 4MPBA ligands on the gold nanorod surface, as well as the statistical results of nanoparticle density and interparticle spacing. A is the TEM image of the Au film, B is the TEM image of the Au@4MPBA film, C is the statistical result of the number of nanoparticles per unit area in the TEM images of the Au film and the Au@4MPBA film, D is the TEM image of the nanoparticle spacing in the Au film, E is the TEM image of the nanoparticle spacing in the Au@4MPBA film, and F is the statistical result of the nanoparticle spacing in the TEM images of the Au film and the Au@4MPBA film.

[0036] Figure 7 Figure 3. Bacterial detection results of an integrated diagnostic and therapeutic platform for Escherichia coli constructed on an Au@CeO2@4MPBA nanoparticle array. Figure A shows the working principle of the integrated diagnostic and therapeutic platform; Figures B and C show the Raman spectra and linear relationships of the platform after testing different concentrations of E. coli; Figure D shows the test results of the platform's specificity for detecting E. coli; and Figure E shows the test results of the platform's ability to distinguish between live and dead bacteria.

[0037] Figure 8Comparison of the peroxidase-like activity and photothermal properties of different nanoparticles, as well as the photothermal performance of Au@CeO2@4MPBA film in dry and aqueous environments. Figure A shows the comparison of the peroxidase-like activity of different nanoparticles, Figure B shows the comparison of the photothermal properties of different nanoparticles, and Figure C shows the photothermal performance of Au@CeO2@4MPBA film in dry and aqueous environments.

[0038] Figure 9 Figure 3. Bacterial sterilization results of the integrated diagnosis and treatment platform for Escherichia coli constructed on the Au@CeO2@4MPBA nanoparticle array. A is a picture of the Escherichia coli colonies in the control and treatment groups on the agar plate, B is the corresponding statistical data of the colony count, the nucleic acid and protein leakage of Escherichia coli in the control and treatment groups, and C is the live / dead staining of Escherichia coli in the control and treatment groups. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0040] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0041] Example 1, as Figure 1-Figure 3 As shown (where, for Figure 2 In terms of Figure 2 a is the process of assembling nanoparticles into a single-layer dense film through water-oil interface self-assembly method. Figure 2 b is a physical picture of the size, appearance and color of the nanoparticle film prepared on the surface of the TPU film. Figure 2 c is a physical picture of the flexibility of the nanoparticle film prepared on the surface of the TPU film), the preparation method of the Au-CeO2@4MPBA Janus structure nanoparticle array type SERS substrate provided by the present invention includes the following steps:

[0042] Step 1: Synthesis of Au-CeO2@4MPBA Janus structured nanoparticles;

[0043] Gold nanorods (Au NRs) were synthesized using a dual ligand system of CTAB and sodium oleate. First, prepare the main reaction system, Solution A: dissolve 7.0 g of hexadecanotrimethylammonium bromide (CTAB) and 1.234 g of sodium oleate in 250 mL of water. Add 18 mL of 4 mM silver nitrate (AgNO3) and stir at 30°C. After 15 minutes, add 250 mL of 1 mM chloroauric acid (HAuCl4) and stir for 90 minutes. Twenty-five minutes before the end of this step, prepare the gold seed solution, Solution B: add 5 mL of 0.5 mM HAuCl4 to 5 mL of 0.2 M CTAB, followed by 1 mL of 6 mM sodium borohydride (NaBH4). Stir vigorously for 2 minutes and let stand at room temperature for 30 minutes. 20 minutes before the end of the reaction of Solution B, add 1.5 mL of concentrated hydrochloric acid to Solution A. After slow stirring for 15 minutes, add 1.25 mL of 64 mM ascorbic acid (AA). After rapid stirring for 30 seconds, add 0.4 mL of Solution B. After rapid stirring for 30 seconds, maintain slow stirring at 30°C for 12 hours. After the reaction is complete, centrifuge at 7500 rpm for 13 minutes twice and collect in 45 mL of water. The total yield is approximately 1 mg mL -1 Store at room temperature. Next, Janus structure Au@CeO2 nanoparticles were further synthesized and prepared by dispersing 2 mL of Au NRs aqueous solution in 15 mL of water. Then, 0.3 mL of 0.1 M CTAB solution was added and stirred at room temperature. 0.13 mL of 0.1 M cerium acetate was added dropwise, stirred for 10 minutes, and then slowly heated to 80°C. The reaction was carried out at 80°C for 4 hours, and the final product was centrifuged at 7000 rpm for 10 minutes twice. 100 μL of 10 -3 M 4-mercaptophenylboronic acid (4MPBA) was added to the Janus structure Au@CeO2 nanoparticle solution and reacted for 4 hours to obtain Au@CeO2@4MPBA nanoparticles for use.

[0044] Step 2: Assembly of nanoparticle array SERS substrate;

[0045] Prepare 3 mL of a concentration of 50-400 µg mL -1 An aqueous solution of Au@CeO2@4MPBA nanoparticles was prepared. Within a certain temperature range (20-60°C), a certain amount of n-hexane was added dropwise until the water surface was completely covered. Ethanol was then continuously added dropwise until a dense metal film, the Au@CeO2@4MPBA nanoparticle film, formed at the interface between the water and n-hexane. Next, the Au@CeO2@4MPBA nanoparticle film was salvaged and transferred to a substrate surface.

[0046] Step 3: Construction of an integrated diagnosis and treatment platform;

[0047] The Au@CeO2@4MPBA Janus nanoparticle arrays are loaded with 4MPBA surface ligands, small molecules that can capture bacteria. Specific detection of captured bacteria is achieved through specific exosomes and gold-silver core-shell SERS tags, with quantitative detection achieved by comparing the signal from the SERS tag to the signal from the substrate 4MPBA. Furthermore, the Au@CeO2@4MPBA nanoparticle arrays exhibit significant photothermal properties and peroxidase activity, enabling the removal of captured bacteria.

[0048] As a preferred embodiment of the present invention, in step 2, the substrate can be a silicon wafer, thermoplastic PU, PET, non-woven fabric, or copper mesh, etc. Among them, hydrophobic materials such as silicon wafer, thermoplastic PU, PET, and copper mesh need to be ozone treated before use to improve their hydrophilicity.

[0049] Example 2, as Figure 4 As shown in the figure (where A is a transmission electron microscope image of Au@CeO2 nanoparticles, and B, C, and D are elemental energy spectrum images of Au@CeO2 nanoparticles), Janus structured nanoparticles are obtained by unilaterally coating CeO2 on one end of a gold nanorod. Nanoparticles with this structure can partially expose the strong hot spot at the metal-semiconductor interface to the external environment, thereby increasing the hot spot intensity of a single particle, as shown in the figure. Figure 5 As shown in the figure (where A is the FDTD electric field simulation result of Au@CeO2 nanoparticles, B is the electric field simulation result of AuNRs, and C and D are performance comparisons of SERS substrates composed of different nanoparticles). Through FDTD simulation and experimental verification, Janus structured nanoparticles can generate a stronger local electric field. The signal intensity of the array SERS substrate prepared using such nanoparticles is an order of magnitude higher than that of the array SERS substrate prepared using gold nanorods, the SERS substrate composed of nanoparticles completely coated on the surface of Au NRs with CeO2 (T-Au@CeO2), and the SERS substrate composed of nanoparticles completely coated on the surface of Au NRs with three times the amount of CeO2 (T-Au@3CeO2).

[0050] In Example 3, a SERS substrate array was prepared by modifying the surface of Janus structured nanoparticles with 4MPBA small molecules as surface ligands. Scanning electron microscopy characterization of the array after self-assembly in the absence and presence of 4MPBA ligands on the gold nanorod surface, as well as statistical results of nanoparticle density and interparticle spacing, verified the regulatory effect of 4-MPBA surface ligands on the nanoparticle spacing. Figure 6As shown in the figures (A is a TEM image of an Au film, B is a TEM image of an Au@4MPBA film, C is a statistical result of the number of nanoparticles per unit area in the TEM images of the Au and Au@4MPBA films, D is a TEM image of the spacing between nanoparticles in the Au film, E is a TEM image of the spacing between nanoparticles in the Au@4MPBA film, and F is a statistical result of the spacing between nanoparticles in the TEM images of the Au and Au@4MPBA films), the TEM images reveal a significant increase in the density of parallel Au NRs on the Au@4MPBA film. Statistical results indicate that the Au@4MPBA film has a higher density and more uniform distribution of particles per square micron. Further analysis revealed that the average spacing between adjacent Au NRs on the Au NR film is 0.75 nm, and direct contact between nanoparticles is observed. In contrast, the spacing on the Au@4MPBA film is approximately 1.15 nm. Furthermore, TEM images clearly show a uniform 4MPBA-modified layer covering the Au NRs on the Au@4MPBA film. Compared to the Au NRs film, the average spacing between adjacent Au NRs on the Au@4MPBA film is larger. The distance between adjacent particles increases from 0.75 nm to 1.15 nm, which not only avoids hotspot failures caused by electron tunneling but also maintains a close enough distance to generate strong electromagnetic field coupling, optimizing the hotspot distribution.

[0051] In Example 4, an integrated diagnosis and treatment platform for E. coli was constructed on the Au@CeO2@4MPBA nanoparticle array. The SERS substrate, E. coli-specific aptamer and SERS tag worked together through a sandwich strategy to monitor E. coli. Figure 7 As shown (where A is the working principle of the integrated diagnosis and treatment platform, B and C are the Raman spectra and linear relationships after the platform detects different concentrations of Escherichia coli, D is the test result of the platform's detection specificity for Escherichia coli, and E is the test result of the platform's ability to distinguish between live and dead bacteria). First, the 4MPBA on the surface of the substrate uses boric acid groups to bind to cis-diols in LPS or glycoproteins on the bacterial cell wall to non-specifically capture various bacteria. 4MPBA also provides a reference signal from the substrate for the construction of a ratiometric SERS biosensor. Next, the E. coli aptamer modified with thiol groups specifically binds to the bacteria captured by the substrate. When the aptamer successfully forms a specific bond with E. coli, the core-shell structure SERS tag further binds to the modified thiol groups on the aptamer through the Ag-S bond. The Raman signal of DTNB in ​​the tag indirectly reflects the bacterial content. Finally, in the result analysis, the 1079 cm -1 The obvious 4MPBA characteristic peak at 1339 cm was selected as the reference signal, and the DTNB molecule at 1339 cm -1The peak at the 1339 / 1079 peak serves as the target signal, and the ratio of I1339 / I1079 is proportional to the bacterial content to be measured. This ratiometric sensor can effectively offset the effects of environmental factors, laser intensity fluctuations, and substrate inhomogeneity on the test results, improving the reliability and repeatability of the results and better eliminating the interference of background signals, especially in complex samples. The platform has low nonspecific adsorption to bacteria-free samples and has good quantitative ability for Escherichia coli, with a linear range of 6×100~6×104 CFU μL -1 The detection limit was ~1.1 CFU μL -1 The same test on different bacteria showed that the biosensor had excellent specificity for E. coli in the test sample. The aptamer could distinguish between dead E. coli and live E. coli after near-infrared laser treatment. The signal obtained after the captured dead bacteria was significantly weaker than that of live bacteria samples.

[0052] Example 5, CeO2 is a material with redox catalytic activity. It can catalyze the decomposition of H2O2 to produce free radicals. These free radicals are highly oxidizing and can destroy bacterial cell walls and internal structures. At the same time, under the irradiation of near-infrared light, gold nanorods generate a large number of hot electrons, which are transferred to the valence band of CeO2, further activating the catalytic activity of CeO2, thereby more effectively generating free radicals. Figure 8 (A is a comparison of the peroxidase-like activity of different nanoparticles, B is a comparison of the photothermal properties of different nanoparticles, and C is the photothermal performance of Au@CeO2@4MPBA film in dry and aqueous environments). Figure 8 As shown in A, the enzyme activity of the Au@CeO2@4MPBA nanoparticle array substrate is significantly stronger than that of the gold nanorod array substrate. Figure 8 In B, under 808 nm near-infrared laser irradiation, the two free nanoparticles have similar photothermal properties. Figure 8 As shown in Figure C, the Au@CeO2@4MPBA film exhibits excellent photothermal properties in both dry and wet environments. When near-infrared light irradiates Au NRs, the gold nanorods absorb light energy and quickly convert it into heat energy due to the LSPR effect. This conversion efficiency is very high, causing the local temperature around the Au NRs to rise rapidly. When the local temperature reaches above 50°C, the thermal death point of bacteria, it can quickly kill nearby bacterial cells. This thermal effect can penetrate the bacterial cell wall and cell membrane, leading to protein denaturation, cell structure destruction and ultimately cell death. The antibacterial ability of the Au@CeO2@4MPBA nanoparticle array film was verified by colony counting. Reference Figure 9(A is a picture of E. coli colonies in the control and treatment groups on agar plates; B is the corresponding statistical data on the number of colonies, the amount of nucleic acid and protein leakage of E. coli in the control and treatment groups; C is the live / dead staining of E. coli in the control and treatment groups. Green or red fluorescence represents live or dead bacteria, respectively. The scale bar in the picture is 100µm.) Figure 9 In A, the number of E. coli decreased by two orders of magnitude after laser light treatment. Figure 9 In B, by comparing the leakage of nucleic acids and proteins from dead bacteria in the supernatant after centrifugation of each group, the values ​​in the treatment group were higher. Figure 9 In C, the bacteria in the control and treatment groups were stained for live and dead bacteria. The red light in the treatment group was stronger, which further verified the antibacterial ability of ACBF.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a nanoparticle array-type SERS substrate with an Au-CeO2@4MPBA Janus structure, characterized in that: The following steps are involved: Step 1: Synthesis of Au-CeO2@4MPBA Janus structured nanoparticles; Step 2: Assembly of nanoparticle array SERS substrate; Step 3: Construction of an integrated diagnosis and treatment platform; The step 1 includes the following specific steps: Step 1.1: Synthesis of Au NRs using CTAB and sodium oleate dual ligands; First, prepare the main reaction system A solution by dissolving 7.0 g CTAB and 1.234 g sodium oleate in 250 mL water. Add 18 mL 4 mM AgNO3 and stir at 30°C for 15 min. Then pour in 250 mL 1 mM HAuCl4 and stir for 90 min to obtain solution A. 25 min before the end of the above steps, prepare the synthetic gold seed solution B by adding 5 mL of 0.5 mM HAuCl4 to 5 mL of 0.2 M CTAB, followed by 1 mL of 6 mM NaBH4, stirring vigorously for 2 min, and standing at room temperature for 30 min to obtain solution B; 20 min before the end of the reaction of Solution B, add 1.5 mL of concentrated hydrochloric acid to Solution A and stir at low speed for 15 min; then add 1.25 mL of 64 mM AA and stir rapidly for 30 s; finally, add 0.4 mL of Solution B and stir rapidly for 30 s, then keep stirring at low speed at 30°C for 12 h; After the reaction, the mixture was centrifuged at 7500 rpm for 13 min twice to obtain Au NRs, which were collected in 45 mL of water and stored at room temperature. Step 1.2: Synthesis of Au@CeO2 nanoparticles with Janus structure; 2 mL of Au NRs aqueous solution was dispersed in 15 mL of water, and then 0.3 mL of 0.1 M CTAB solution was added and stirred at room temperature; 0.13 mL of 0.1 M cerium acetate was added dropwise, stirred for 10 minutes, and then slowly heated to 80°C; the reaction was carried out at 80°C for 4 hours, and the final product was centrifuged at 7000 rpm for 10 minutes twice; 100 μL of 10 -3 M 4MPBA was added to the Janus structure Au@CeO2 nanoparticle solution and reacted for 4 hours to obtain Au@CeO2@4MPBA nanoparticles for standby use.

2. The method for preparing the Au-CeO2@4MPBA Janus structure nanoparticle array type SERS substrate according to claim 1, characterized in that: The step 2 includes the following specific steps: Step 2.1: Preparation of Au@CeO2@4MPBA nanoparticle film; Prepare 3 mL of a concentration of 50-400 µg mL -1 Au@CeO2@4MPBA nanoparticle aqueous solution; n-hexane is added dropwise at a temperature range of 20-60°C until the water surface is completely covered, and ethanol is continuously added dropwise to the system until a dense metal film is formed at the interface between water and n-hexane, namely the Au@CeO2@4MPBA nanoparticle film; Step 2.2: Transfer the Au@CeO2@4MPBA nanoparticle film to the substrate surface to complete the assembly of the nanoparticle array SERS substrate.

3. The method for preparing the Au-CeO2@4MPBA Janus structure nanoparticle array type SERS substrate according to claim 2, characterized in that: In step 2, the substrate is a silicon wafer, thermoplastic PU, PET, non-woven fabric or copper mesh; wherein, hydrophobic materials such as silicon wafer, thermoplastic PU, PET and copper mesh need to be ozone treated before use.

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