Gap-adjustable Au / Ag cascade PS ball array structure and preparation method and application thereof

Abstract

The invention belongs to the technical field of Raman spectrum, and discloses a gap-adjustable Au / Ag cascade PS sphere array structure and a preparation method and application thereof. The preparation method comprises the following steps: heating a first PS ball array structure of which the substrate is PDMS to enlarge gaps among PS balls, fixing with PVP, and removing PDMS to obtain a second PS ball array structure of which the substrate is a PVP film; the preparation method comprises the following steps: replacing a substrate with PDMS, carrying out silver evaporation on the surface of a PS ball, then putting the PS ball into a gold growth solution, and heating for reaction to obtain the Au / Ag cascaded PS ball array structure with the substrate being PDMS. The gaps between the Au / Ag cascaded PS balls in the Au / Ag cascaded PS ball array structure can be regulated and controlled through temperature regulation, and the Au / Ag cascaded PS ball array structure is applied to SERS detection and is wide in application range and high in sensitivity.

Description

Technical Field

[0001] The invention belongs to the technical field of Raman spectroscopy and relates to a gap-adjustable Au / Ag cascade PS ball array structure and a preparation method and application thereof. Background Art

[0002] Surface-Enhanced Raman Scattering (SERS) is an ultra-sensitive detection technology based on the Raman scattering effect. By utilizing the nanoscale roughness of the metal nanostructure surface, SERS can significantly enhance the Raman signal of the target molecule, achieving rapid identification and high-sensitivity detection. Compared with traditional analytical methods such as fluorescence detection or mass spectrometry, SERS has significant advantages: (1) Ultra-high sensitivity: The detection accuracy can even reach the single-molecule level, far exceeding traditional technologies; (2) High resolution: It can distinguish molecules with similar structures and has accurate molecular fingerprint recognition capabilities; (3) Real-time detection: It is suitable for real-time monitoring in dynamic processes and has a fast response speed.

[0003] The improvement of SERS performance mainly depends on the "hotspot" areas on the surface of metal nanostructures, that is, the parts where the local electromagnetic field is significantly enhanced, such as nanotips, nanoholes and nanogaps. These "hotspot" areas can greatly enhance the Raman scattering signals of molecules on the surface.

[0004] In recent years, SERS technology has been widely used in the fields of biomedical diagnosis, food safety testing and environmental monitoring. For example, in early cancer diagnosis, SERS sensors can quickly detect specific biomarkers in blood or tissues; in food safety testing, they can be used to detect pesticide residues or heavy metal pollution; in environmental monitoring, they can achieve rapid screening of pollutants in water and air.

[0005] The intensity of SERS hotspot is closely related to the gap size of the nanostructure. Generally, the smaller the gap size, the more significant the local electromagnetic field enhancement effect. However, too small a gap will cause macromolecules to be difficult to effectively enter the hotspot area due to steric hindrance, affecting the detection sensitivity. Therefore, for macromolecular detection, it is necessary to consider the problem of molecular adsorption to balance molecular accessibility and enhancement efficiency. For example, Wu J et al. prepared a sub-10nm gap Ag / Au plasmon hybrid coupled nanoarray to realize the detection of proteins such as hemoglobin in body fluids, see WU J, "Advancing Protein Detection and Analysis Based on Ag / Au PHCN for Enhanced SERS Sensitivity and Specificity in Biomolecular Diagnostics". Analytical Chemistry, 2024, 96 (39): 15735-45. Teak Lee et al. performed ultrasensitive detection of single functionalized microRNA-155 using gold nanoparticles with a single RNA copy. See Teak Lee, “Single Functionalized pRNA / Gold Nanoparticle for Ultrasensitive MicroRNA Detection Using Electrochemical Surface-Enhanced Raman Spectroscopy”. Advanced Science, 2020, 7(3): 1902477-1902487.

[0006] The enhancement effect can be maximized by dynamically adjusting the substrate gap: the initial large gap allows large molecules to enter freely, and then the gap gradually shrinks to the critical thickness of the tightly compressed adsorbed molecular layer. For example, Gong Weichao can actively adjust the nanostructure gap size by stretching the deformation ability of the metamaterial film, so that molecules can more easily join the hot spot, and finally achieve controllable enhancement of the SERS signal. See Gong Weichao, "Research on dynamic SERS substrate based on flexible and stretchable metal metamaterial film, Zhejiang University, 2019.

[0007] Therefore, developing a SERS detection material with adjustable gap size has become a key issue that needs to be solved urgently. Summary of the invention

[0008] The purpose of the present invention is to at least partially solve the above technical problems, and to provide a gap-adjustable Au / Ag cascade PS ball array structure and its preparation method and application. The structural gap between PS balls in the Au / Ag cascade PS ball array structure can be flexibly adjusted with temperature changes, thereby improving the flexibility, adaptability and sensitivity of SERS detection.

[0009] To solve the above problems, the first aspect of the present invention provides a preparation method of an Au / Ag cascade PS ball array structure with adjustable gap, comprising: heating a first PS ball array structure with a PDMS substrate to a first temperature to expand the gap between each PS ball in the first PS ball array structure, fixing the PS ball array structure with the expanded gap with PVP, removing PDMS, and obtaining a second PS ball array structure with a PVP film substrate; replacing the substrate of the second PS ball array structure with a PVP film substrate with PDMS to obtain a second PS ball array structure with a PDMS substrate; performing silver evaporation on the surface of the PS balls of the second PS ball array structure with a PDMS substrate to obtain a silver-plated PS ball array structure with a PDMS substrate; placing the silver-plated PS ball array structure with a PDMS substrate in a gold growth solution, heating to a second temperature for reaction, and obtaining an Au / Ag cascade PS ball array structure with a PDMS substrate, wherein the gap between each Au / Ag cascade PS ball in the Au / Ag cascade PS ball array structure can be regulated by temperature adjustment.

[0010] Furthermore, the preparation method further comprises: transferring the Au / Ag cascade PS ball array structure to a new PDMS substrate.

[0011] Furthermore, the first PS ball array structure whose substrate is PDMS is prepared by the following method: self-assembling a layer of periodically densely arranged PS microsphere array on the water surface, and transferring it to the target substrate to form a periodic PS ball template; transferring the PS microsphere array from the target substrate to the PDMS substrate to obtain the first PS ball array structure whose substrate is PDMS.

[0012] Furthermore, the self-assembly of a periodically densely arranged PS microsphere array on the water surface includes: slowly dripping a PS sphere solution into water along a hydrophilically treated slope, so that the PS spheres form a periodically densely arranged PS microsphere array on the water surface under the action of electrostatic force and surface tension.

[0013] Furthermore, the transferring of the PS microsphere array from the target substrate to the PDMS substrate is specifically: using solidified PDMS to adhere the PS microsphere array on the periodic PS ball template.

[0014] Further, the transferring of the PS microsphere array from the target substrate to the PDMS substrate is specifically: heating the PDMS prepolymer until the PDMS is solidified, and immediately using the solidified PDMS to adhere the PS microsphere array on the periodic PS ball template. Preferably, the heating until the PDMS is solidified is specifically: heating to 55°C-65°C and maintaining the temperature until solidification, preferably, it can be maintained for 4h±15min.

[0015] Furthermore, the first temperature is 74°C-76°C.

[0016] Furthermore, the method of fixing the PS ball array structure with enlarged gaps with PVP is specifically as follows: a PVP aqueous solution is added dropwise to the PVP aqueous solution and spread on the PS ball array structure with enlarged gaps, and after the water in the PVP aqueous solution evaporates to form a PVP film, the PVP film with the PS ball array structure with enlarged gaps is torn off, thereby obtaining a second PS ball array structure with the PVP film as the substrate.

[0017] Furthermore, the second temperature is 69° C.-71° C. Preferably, the reaction time is 3 h±5 min.

[0018] To solve the above problems, the second aspect of the present invention provides an Au / Ag cascade PS ball array structure prepared by the above preparation method.

[0019] To solve the above problems, the third aspect of the present invention provides the application of the Au / Ag cascade PS ball array structure with PDMS as the substrate in SERS detection.

[0020] Furthermore, the application is specifically as follows: the Au / Ag cascade PS ball array structure with the PDMS substrate is heated to 70-80°C, the test solution is added dropwise, and after it is dried, the unabsorbed test solution is removed by washing with water, and after drying, the temperature is adjusted to 20-80°C for SERS detection, and then whether the test solution contains the target analyte is determined based on the detection results.

[0021] Furthermore, in the above application, the excitation wavelength of SERS detection is 784.7-785.1 nm.

[0022] To solve the above problems, the fourth aspect of the present invention provides a method for detecting a target analyte using a SERS sensor, wherein the SERS sensor is the Au / Ag cascade PS ball array structure whose substrate is PDMS. The method is: heating the Au / Ag cascade PS ball array structure whose substrate is PDMS to a third temperature, dripping a test solution, and after it dries, washing with water to remove the unabsorbed test solution, and after drying, adjusting the temperature to a fourth temperature to perform SERS detection to determine whether the test solution contains the target analyte or calculate the concentration of the target analyte in the test solution.

[0023] Furthermore, in the above method, the excitation wavelength of SERS detection is 784.7-785.1 nm.

[0024] Furthermore, in the above method, the third temperature is 70-80°C.

[0025] Furthermore, in the above method, the fourth temperature is 20-80°C, preferably 20-60°C.

[0026] Furthermore, in the above method, the target analyte is a compound with a molecular weight of 2-1000, and the fourth temperature is preferably 60-70° C. Preferably, the compound with a molecular weight of 2-1000 is malachite green or crystal violet.

[0027] Furthermore, in the above method, the target analyte is a compound with a molecular weight of 1000-100000, and the fourth temperature is preferably 20-30° C. Preferably, the compound with a molecular weight of 1000-100000 is bovine serum albumin or hemoglobin.

[0028] Furthermore, in the above method, the target analyte is crystal violet, and the method for calculating the concentration of the target analyte in the test solution is: according to the signal intensity of the test solution at 1160cm-1 and the concentration dependence function of crystal violet, the concentration of crystal violet in the test solution is calculated; the concentration dependence function is:

[0029] y=2363.25714x-4269.06667; wherein y is the signal intensity at 1160 cm-1, and x is the concentration of crystal violet in the test solution.

[0030] The correlation coefficient (R2) of the concentration dependence function of the crystal violet is 0.85, showing good linear dependence. The Raman spectrum of the test solution is measured, the Raman signal intensity of the characteristic peak of 1160cm-1 is extracted, and the concentration is inferred by the concentration dependence function to obtain the concentration of crystal violet in the test solution. Preferably, the test solution can be measured multiple times to calculate the average value and standard deviation of the concentration to improve accuracy.

[0031] The Au / Ag cascaded PS ball array structure with adjustable gap and its preparation method and application according to the embodiment of the present invention have at least one of the following advantages:

[0032] 1. The template method is combined with the chemical synthesis method, which has a simple process and low cost.

[0033] The present invention adopts a combination of template method and chemical synthesis method to prepare periodic Au / Ag cascade PS ball array. First, a layer of densely arranged PS microsphere array is self-assembled on the surface of ultrapure water through liquid surface self-assembly technology, and then transferred to the target substrate to form a periodic PS ball template. The template method has a simple preparation process, low cost, and high repeatability. Compared with the traditional high-cost, complex equipment-supported nanostructure preparation method, it significantly reduces the technical threshold and improves the process feasibility.

[0034] 2. Synthesis of highly active metal nanostructures, with significant SERS enhancement effect

[0035] The present invention uses a chemical synthesis method to generate a highly active metal nanostructure on a PS ball array, and uses an in-situ substitution method to synthesize an Au / Ag nanocomposite structure. Through the cascade coupling effect of the composite structure, the SERS enhancement effect is effectively improved, and a large-area, uniform and stable cascade metal nanoparticle array structure is obtained. Compared with traditional nanostructures, this method increases the active site density of the nanostructure and enhances the signal amplification effect, so that it has higher sensitivity and stronger anti-interference ability in practical applications.

[0036] 3. The flexible adjustment characteristics of the PDMS substrate enable dynamic adjustment of the structural gap

[0037] The present invention prepares the Au / Ag cascade PS ball array on a polydimethylsiloxane (PDMS) substrate, and utilizes the flexibility and high thermal expansion coefficient of PDMS to dynamically adjust the structural gap between the PS balls with temperature changes. This adjustable characteristic not only improves the flexibility and adaptability of SERS detection, but also helps to optimize the enhancement effect of nanostructures in different detection environments, meet a variety of practical application requirements, and has significant advantages in the detection of complex and biologically active samples.

[0038] 4. Maximizing the enhancement effect can be achieved by dynamically adjusting the substrate gap: In the initial stage, the larger gap provides sufficient freedom for macromolecules to enter and adsorb on the substrate surface smoothly. When the substrate gradually adjusts the gap width and the gap gradually shrinks to a critical thickness close to the molecular layer, the adsorbed molecules are tightly compressed, thereby significantly improving the local field enhancement effect. This process not only optimizes the interaction between molecules and substrates, but also effectively enhances the signal response, achieving more efficient detection and analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] These and / or other aspects and advantages of the present invention will become apparent and readily understood from the following description of preferred embodiments in conjunction with the accompanying drawings, in which:

[0040] Figure 1 A flow chart of the preparation of an Au / Ag cascade PS ball array with adjustable gap provided in an embodiment of the present invention;

[0041] Figure 2 This is a SEM image of a PS ball array with enlarged gaps after silver plating in Example 1 of the present invention;

[0042] Figure 3 The SEM images of the Au / Ag cascade structure at different temperatures (room temperature, 60°C, 70°C, 80°C) in Example 3;

[0043] Figure 4 In Example 4, 2 μl of 10 -5 SERS spectra of M after crystal violet aqueous solution detected at different temperatures;

[0044] Figure 5 The Raman spectra after adding different concentrations of crystal violet aqueous solution in Example 5;

[0045] Figure 6 The SERS spectra of malachite green aqueous solutions with different concentrations added dropwise in Example 6;

[0046] Figure 7 In order to use the Au / Ag nanocomposite structure and the silver-plated substrate as the SERS active substrate, 2 μl of 10 -5 M concentration of crystal violet aqueous solution, Raman spectrum measured at 60℃;

[0047] Figure 8 The SERS spectra at 50 different positions obtained in Example 8 (Figure a) and the signal intensity statistical analysis diagram of the characteristic peak of crystal violet 1161cm-1 (Figure b);

[0048] Fig. 9 The SERS spectra detected at different temperatures in Example 9;

[0049] Fig.10 The Raman spectra obtained by detecting the aqueous solutions of BSA with different concentrations in Example 10;

[0050] Fig.11 The Raman spectra obtained by detecting HGB aqueous solutions with different concentrations in Example 11. DETAILED DESCRIPTION

[0051] The following examples and accompanying drawings further illustrate the technical solution of the present invention. The following description of the embodiments of the present invention with reference to the accompanying drawings is intended to explain the overall inventive concept of the present invention, and should not be construed as a limitation of the present invention.

[0052] In a typical embodiment of the present application, a preparation method of an Au / Ag cascade PS ball array structure with adjustable gap is provided, comprising: heating a first PS ball array structure with a PDMS substrate to a first temperature to enlarge the gap between each PS ball in the first PS ball array structure, fixing the PS ball array structure with the enlarged gap with PVP, removing PDMS, and obtaining a second PS ball array structure with a PVP film substrate; S200. replacing the substrate of the second PS ball array structure with a PVP film substrate with PDMS to obtain a second PS ball array structure with a PDMS substrate; performing silver evaporation on the surface of the PS balls of the second PS ball array structure with a PDMS substrate to obtain a silver-plated PS ball array structure with a PDMS substrate; placing the silver-plated PS ball array structure with a PDMS substrate in a gold growth solution, heating to a second temperature for reaction, and obtaining an Au / Ag cascade PS ball array structure with a PDMS substrate, wherein the gap between each Au / Ag cascade PS ball in the Au / Ag cascade PS ball array structure can be regulated by temperature adjustment.

[0053] In the preparation method provided in the present application, after the first PS ball array structure with a PDMS substrate is heated to a first temperature, the PDMS expands to a certain extent, thereby expanding the gap between each PS ball fixed on the PDMS, and then the PS ball array structure with the expanded gap is immediately fixed with PVP, and then the PDMS is removed to obtain a second PS ball array structure with a PVP film as the substrate. Compared with the first PS ball array structure, the gap between each PS ball in the second PS ball array structure is enlarged. At this time, the substrate of the second PS ball array structure is replaced with PDMS, and then silver is evaporated, and then gold is grown on the surface of the silver to obtain an Au / Ag cascade PS ball array structure. Since the substrate of the Au / Ag cascade PS ball array structure is PDMS, the flexibility and high thermal expansion coefficient of PDMS can be used to adjust the gap between each Au / Ag cascade PS ball by temperature adjustment. Exemplarily, the gap between each Au / Ag cascade PS ball can be changed in the range of 0nm-20nm by adjusting the temperature to 20°C-80°C.

[0054] In a preferred embodiment of the present invention, a method for preparing an Au / Ag cascade PS ball array structure with adjustable gap is provided, and the preparation process is as follows: Figure 1 As shown, steps S100-S600 are included.

[0055] S100. Preparation of periodic PS sphere template.

[0056] S200. Transferring the PS microsphere array on the periodic PS sphere template to the PDMS substrate to obtain a first PS sphere array structure with the PDMS substrate.

[0057] S300. The first PS ball array structure whose substrate is PDMS is heated to a first temperature to enlarge the gaps between the PS balls in the first PS ball array structure, the PS ball array structure with enlarged gaps is fixed with PVP, and the PDMS is removed to obtain a second PS ball array structure whose substrate is a PVP film.

[0058] S400. Replace the substrate of the second PS ball array structure whose substrate is a PVP film with PDMS to obtain a second PS ball array structure whose substrate is a PDMS.

[0059] S500. Perform silver evaporation on the surface of the PS balls of the second PS ball array structure whose substrate is PDMS, to obtain a silver-plated PS ball array structure whose substrate is PDMS.

[0060] S600. Place the silver-plated PS ball array structure whose substrate is PDMS in a gold growth solution, heat it to a second temperature for reaction, and obtain an Au / Ag cascade PS ball array structure whose substrate is PDMS. The gap between each Au / Ag cascade PS ball in the Au / Ag cascade PS ball array structure can be controlled by temperature regulation.

[0061] In a preferred embodiment of the present invention, step S100 specifically comprises: self-assembling a layer of periodically densely arranged PS microsphere arrays on the water surface, and transferring it to a target substrate (eg, a hydrophilically treated silicon wafer) to form a periodic PS sphere template.

[0062] In a preferred embodiment of the present invention, the method for self-assembling a layer of periodically densely arranged PS microsphere array on the water surface is: slowly dripping the PS sphere solution into the water along the hydrophilic treated slope, so that the PS spheres form a periodically densely arranged PS microsphere array on the water surface under the action of electrostatic force and surface tension.

[0063] In a preferred embodiment of the present invention, a method for self-assembling a periodically densely arranged PS microsphere array on a water surface is as follows: deionized water is injected into a culture dish, a hydrophilic treated glass slide is placed on the edge of the culture dish, and a PS sphere solution is slowly dripped along the glass slide, so that the PS spheres form a dense PS microsphere array under the action of electrostatic force and surface tension.

[0064] In order to enhance the surface tension of water, in a preferred embodiment of the present invention, sodium dodecyl sulfate (SDS) is also added to the water.

[0065] In a preferred embodiment of the present invention, the PS sphere solution is obtained by mixing a PS sphere suspension and ethanol. Preferably, the volume ratio of the PS sphere suspension to the ethanol is 30:8. Exemplarily, the PS sphere suspension can be selected from commercial products (300 nm PS spheres are dispersed in deionized water at a ratio of 2.5% (w / v)).

[0066] In a preferred embodiment of the present invention, the method of transferring the PS microsphere array from the target substrate to the PDMS substrate is specifically: heating the PDMS prepolymer until the PDMS is solidified, and then using the solidified PDMS to adhere the PS microsphere array on the periodic PS ball template. Preferably, heating until the PDMS is solidified is specifically: heating to 55°C-65°C and then maintaining for a period of time until the PDMS is solidified. Preferably, it is maintained for 4h±15min.

[0067] In a preferred embodiment of the present invention, in step S300, the first temperature is 74°C-76°C.

[0068] In a preferred embodiment of the present invention, in step S300, fixing the PS ball array structure with enlarged gaps with PVP is specifically as follows: dripping a PVP aqueous solution onto the PVP aqueous solution and spreading it on the PS ball array structure with enlarged gaps, and after the water in the PVP aqueous solution evaporates to form a PVP film, tearing off the PVP film with the PS ball array structure with enlarged gaps, thus obtaining a second PS ball array structure with the PVP film as the substrate. Preferably, the PVP aqueous solution uses a PVP aqueous solution with a concentration of 1wt%.

[0069] In a preferred embodiment of the present invention, step S400 is specifically: dripping PDMS on one side of the structure of the second PS ball array structure whose substrate is a PVP film, heating until the PDMS is solidified, and then washing away the PVP. Since the PDMS substrate is hydrophobic, it is preferably washed with ethanol with better wettability, which can be cleaned more thoroughly. Preferably, the heating until the PDMS is solidified is specifically: heating to 55°C-65°C and maintaining the temperature until the PDMS is solidified. Preferably, the time for maintaining the temperature is 4h±15min.

[0070] In a preferred embodiment of the present invention, in step S500, the process parameters of silver evaporation are: 100nm silver evaporation, rate Vacuum degree 8×10 - 4Pa.

[0071] In a preferred embodiment of the present invention, in step S600, the preparation method of the gold growth solution is: dissolving CTAB and CTAC in water, adding chloroauric acid solution, silver nitrate solution, hydrochloric acid and ascorbic acid solution, and stirring evenly. Preferably, the dosage ratio of CTAB, CTAC, water, chloroauric acid solution, silver nitrate solution, hydrochloric acid and ascorbic acid solution is 80mg: 560mg: 19ml: 1ml: 200ul: 33.5μL: 160μL. Preferably, the concentration of chloroauric acid solution is 10mM, the concentration of silver nitrate solution is 10mM, and the concentration of ascorbic acid solution is 100mM. The second temperature is preferably 69℃-71℃, and the reaction time is 3h±5min. Under this temperature condition, PDMS will expand to a certain extent.

[0072] In a preferred embodiment of the present invention, the preparation method further comprises replacing the substrate of the Au / Ag cascade PS ball array structure whose substrate is PDMS prepared in step 600 with a new PDMS substrate.

[0073] During the gold growth process, the reaction temperature will cause the PDMS to expand to a certain extent. When it returns to room temperature, the PDMS shrinks. However, since Au / Ag covers the surface of the PS balls, the shrinkage of the PDMS will cause the Au / Ag cascade PS balls to be close together. However, since the gaps in the structure before shrinkage are different, the shrinkage amounts are also different. Even if it is heated and expanded thereafter, the gaps are still the original uneven gaps. In a preferred embodiment of the present application, the above-mentioned preparation method also includes: transferring the Au / Ag cascade PS ball array structure to a new PDMS substrate. After re-casting the new PDMS, since the Au / Ag cascade PS balls are close together from the beginning, the gap size of the Au / Ag cascade PS ball array structure obtained by adjusting the temperature is uniform.

[0074] In a second typical embodiment of the present invention, an Au / Ag cascade PS ball array structure prepared by the above-mentioned preparation method is provided. The Au / Ag cascade PS ball array structure with a PDMS substrate utilizes the flexibility and high thermal expansion coefficient of PDMS so that the structural gap between the PS balls can be dynamically adjusted with temperature changes. This adjustable characteristic not only improves the flexibility and adaptability of SERS detection, but also helps to optimize the enhancement effect of the nanostructure in different detection environments, meet a variety of practical application requirements, and has significant advantages in the detection of complex and biologically active samples.

[0075] In a third typical embodiment of the present invention, the application of the Au / Ag cascade PS ball array structure with a PDMS substrate in SERS detection is provided. In a preferred embodiment of the present invention, the application method is: heating the Au / Ag cascade PS ball array structure with a PDMS substrate to 70-80°C, dripping the test solution, and after drying, washing with water to remove the unabsorbed test solution, after drying, adjusting the temperature to 20-80°C for SERS detection, and then judging whether the test solution contains the target analyte according to the detection result.

[0076] In a preferred embodiment of the present invention, in the above application, the excitation wavelength of SERS detection is 784.7 nm-785.1 nm.

[0077] In a fourth typical embodiment of the present invention, a method for detecting a target analyte using a SERS sensor is provided, wherein the SERS sensor is the Au / Ag cascade PS ball array structure whose substrate is PDMS as mentioned above, and the method is: heating the Au / Ag cascade PS ball array structure whose substrate is PDMS to a third temperature, dripping a test solution, and after it dries, washing with water to remove unabsorbed test solution, and after drying, adjusting the temperature to a fourth temperature to perform SERS detection to determine whether the test solution contains the target analyte or calculate the concentration of the target analyte in the test solution.

[0078] In a preferred embodiment of the present invention, the excitation wavelength of SERS detection is 784.7-785.1 nm.

[0079] In a preferred embodiment of the present invention, the third temperature is 70-80°C.

[0080] In a preferred embodiment of the present invention, the fourth temperature is 20-80°C, preferably 20-60°C.

[0081] In a preferred embodiment of the present invention, the target analyte is a compound with a molecular weight of 2-1000, and the fourth temperature is preferably 60-70° C. Preferably, the compound with a molecular weight of 2-1000 is malachite green or crystal violet.

[0082] Furthermore, in the above method, the target analyte is crystal violet, and the method for calculating the concentration of the target analyte in the test solution is: according to the concentration of the test solution at 1160 cm -1 The concentration of crystal violet in the test solution is calculated based on the signal intensity at and the concentration dependence function of crystal violet; the concentration dependence function is:

[0083] y=2363.25714x-4269.06667; where y is 1160cm -1where x is the signal intensity at , and x is the concentration of crystal violet in the test solution.

[0084] The correlation coefficient (R2) of the concentration dependence function of the crystal violet is 0.85, showing good linear dependence. The Raman spectrum of the solution to be tested was measured and the 1160 cm -1 The Raman signal intensity of the characteristic peak is used to infer the concentration using a concentration-dependent function to obtain the concentration of crystal violet in the test solution. Preferably, the test solution can be measured multiple times to calculate the average value and standard deviation of the concentration to improve accuracy.

[0085] In a preferred embodiment of the present invention, the target analyte is a compound with a molecular weight of 1000-100000, and the fourth temperature is preferably 20-30° C. Preferably, the compound with a molecular weight of 1000-100000 is bovine serum albumin or hemoglobin.

[0086] The present invention will be described in detail below by way of examples. In the following examples, unless otherwise specified, all the raw materials used are commercially available.

[0087] PS sphere suspension: purchased from Nantong Feiyu Biotechnology Co., Ltd. Product information: 300 nm PS spheres are dispersed in deionized water at a ratio of 2.5% (w / v).

[0088] Hexadecyltrimethylammonium bromide (CTAB), hexadecyltrimethylammonium chloride (CTAC), sulfuric acid (H2SO4), hydrogen peroxide H2O2, sodium dodecyl sulfate (SDS), polyvinylpyrrolidone (PVP), polydimethylsiloxane (PDMS), anhydrous ethanol (C2H5OH), acetone (C3H6O), silver nitrate (AgNO3), chloroauric acid (HAuCl4·4H2O), ascorbic acid (AA), and crystal violet (C24H28ClN3) were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0089] Example 1

[0090] 1. Silicon wafer cleaning and hydrophilic treatment

[0091] The cut silicon wafer (5 mm×5 mm) was immersed in deionized water, acetone, ethanol and deionized water for ultrasonic cleaning for 5 minutes in turn, and dried with nitrogen (N2) to obtain a cleaned silicon wafer.

[0092] 98 wt % concentrated sulfuric acid and 30 wt % hydrogen peroxide were mixed in a volume ratio of 7:3, and the cleaned silicon wafer was washed for 10 minutes to make its surface hydrophilic, and then rinsed with deionized water and dried with nitrogen.

[0093] 2. Preparation of PS Sphere Solution

[0094] The PS sphere suspension was mixed with anhydrous ethanol at a volume ratio of 30:8 and stirred evenly by ultrasonication to obtain a PS sphere solution.

[0095] 3. Liquid surface self-assembly of PS sphere film (PS microsphere array)

[0096] 100 mL of deionized water was injected into the culture dish, and 35 μl of 20 mM sodium dodecyl sulfate (SDS) solution was added to the water surface to enhance the surface tension.

[0097] Place the hydrophilic treated glass slide on the edge of the culture dish, and slowly drip the PS sphere solution along the glass slide, so that the PS spheres form a dense PS sphere film under the action of electrostatic force and surface tension.

[0098] 4. Formation of PS sphere template

[0099] Place the hydrophilic silicon wafer under the PS ball membrane and slowly lift it up to separate the PS ball membrane from the water surface. Wait for the deionized water to dry naturally to form a tightly arranged PS ball template.

[0100] 5. Transfer the PS ball film on the PS ball template to the PDMS substrate

[0101] PDMS and a curing agent were mixed at a mass ratio of 10:1 to obtain a PDMS prepolymer (PDMS curing solution), and the mixture was cured at 60° C. for 4 hours to obtain a cured PDMS.

[0102] The PS ball film on the PS ball template is bonded with solidified PDMS to obtain the first PS ball array structure with the PDMS substrate.

[0103] 6. Heat expansion and fixation to PVP

[0104] The PDMS of the material obtained in step 1 is heated to 75°C to expand the PDMS and enlarge the gaps between the PS balls. Then, 100 μl of a 1 wt% polyvinylpyrrolidone (PVP) aqueous solution is dripped onto one side of the PS ball film. After the water evaporates, the PVP film is torn off to fix the PS ball film to the PVP film. That is, the PS ball film with enlarged gaps is fixed with the PVP film to obtain a PVP film with a PS ball film with larger gaps, that is, a second PS ball array structure with the PVP film as the substrate.

[0105] 7. Re-fix the PS ball membrane with PDMS

[0106] PDMS prepolymer was added dropwise to one side of the PS balls of the second PS ball array structure whose substrate was a PVP film, and the mixture was cured at 60°C for 4 hours. The PVP was washed off with ethanol to obtain PDMS with a PS ball film having large gaps, i.e., the second PS ball array structure whose substrate was a PDMS.

[0107] 8. Silver evaporation

[0108] 100nm silver was evaporated on the surface of PS sphere film at a rate of Vacuum degree 8×10 -4 Pa, and a silver-plated PS ball array was obtained, as shown in the SEM image. Figure 2 shown.

[0109] Example 2

[0110] Prepare gold growth solution: dissolve 80 mg CTAB and 560 mg CTAC in 19 ml water, add 1 ml 10 mM chloroauric acid solution, 200 μl 10 mM silver nitrate solution, 33.5 μl hydrochloric acid, and 160 μl 100 mM ascorbic acid solution to obtain gold growth solution.

[0111] The silver-plated PS ball array was used as a sacrificial template, placed in a gold growth solution, reacted at 70°C for 3 hours, and then washed to obtain an Au / Ag cascade PS ball array structure.

[0112] Example 3

[0113] 100 μL of 1 wt % PVP aqueous solution was dripped onto the surface of the Au / Ag cascade PS ball array prepared in Example 2, and after it dried at room temperature, the PVP film with the Au / Ag cascade PS ball array was torn off.

[0114] PDMS prepolymer and curing agent were mixed in a ratio of 10:1 and added dropwise to the array side of the PVP membrane. The mixture was cured at 60°C for 4 hours and the PVP was removed by washing with ethanol to obtain an Au / Ag cascade PS ball array structure with a gap that could be adjusted with temperature.

[0115] The Au / Ag cascade PS ball array structure prepared in Example 3 was heated to different temperatures for SEM observation. The results are as follows: Figure 3 As shown, a, b, c, and d are SEM images at room temperature, 60°C, 70°C, and 80°C, respectively, demonstrating the dynamic regulation effect of temperature change on the gap between Au / Ag cascade PS sphere particles.

[0116] Example 4

[0117] The Au / Ag cascade PS ball array structure obtained in Example 3 was used as a SERS active substrate and heated at 70°C for 10 minutes to allow the PDMS to undergo thermal expansion. 2 μl of 10 -5M concentration of crystal violet aqueous solution, after drying, gently wash the substrate with deionized water to remove unadsorbed CV, and then blow dry the substrate with nitrogen. The substrate was heated to 60℃, 70℃, 80℃, and 90℃ using a heating plate, and the SERS spectra were detected at different temperatures (25℃, 60℃, 70℃, 80℃, and 90℃). The excitation wavelength of the Raman spectrum was 785nm, the integration time was 10s, and the SERS spectra were as follows Figure 4 When the temperature is 60℃, the strongest SERS signal enhancement performance is shown.

[0118] Example 5

[0119] The Au / Ag cascade PS ball array structure obtained in Example 3 was used as the SERS active substrate. The entire sample was placed on a heating table and heated at 70°C for 10 minutes to cause thermal expansion of the PDMS, thereby temporarily increasing the gap between the Au / Ag structures. 2 μl of crystal violet (CV) aqueous solution with different concentrations (concentrations were 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M), and after drying, the substrate was gently washed with deionized water to remove the unabsorbed CV, and then the substrate was blown dry with nitrogen. The substrate was heated to 60°C using a heating plate and Raman spectroscopy was performed. The excitation wavelength of the Raman spectrum was 785nm, and the integration time was 10s. The SERS spectrum was as follows Figure 5 The structure can be detected at a minimum of 10 -10 M's CV.

[0120] Example 6

[0121] The Au / Ag cascade PS ball array structure obtained in Example 3 was used as a SERS active substrate. The entire sample was placed on a heating table and heated at 70°C for 10 minutes to cause thermal expansion of the PDMS. 2 μl of malachite green (MG) aqueous solution of different concentrations (100 ppm, 10 ppm, 1 ppm, 0.1 ppm, 0.01 ppm) was added dropwise. After drying, the substrate was gently washed with deionized water to remove the unabsorbed MG, and then the substrate was blown dry with nitrogen. The substrate was heated to 60°C using a heating plate and Raman spectroscopy was performed. The excitation wavelength of the Raman spectrum was 785 nm, the integration time was 10 s, and the SERS spectrum was as shown in FIG. Figure 6 The structure can detect MG at a minimum of 0.01 ppm.

[0122] Malachite green and crystal violet were once common pesticides used in aquaculture. In addition to CV, the base also has excellent detection performance for malachite green and can detect trace residues that meet national standards, showing that the base is not only able to detect CV, but can also detect other molecules and has wide applicability.

[0123] Example 7

[0124] The SERS activities of the Au / Ag nanocomposite structures were compared with those of the silver-coated substrate alone.

[0125] The Au / Ag cascade PS ball array structure obtained in Example 2 was used as a SERS active substrate, and the PS ball array with 100 nm silver vapor deposited on it obtained in Example 1 was used as a SERS active substrate. The entire sample was placed on a heating table and heated at 70°C for 10 minutes to cause thermal expansion of PDMS, thereby temporarily increasing the gap between the structures. 2 μl 10 -5 M concentration of crystal violet aqueous solution, after drying, gently wash the substrate with deionized water to remove unadsorbed CV, and then blow dry the substrate with nitrogen. Heat the substrate to 60 ° C with a heating plate for Raman spectroscopy detection. The signal intensity of the cascade structure and the silver-plated structure is shown in Figure 7 As shown, the SERS signal of the Au / Ag cascade structure is significantly enhanced compared with the silver-plated structure, demonstrating its superior cascade coupling enhancement effect.

[0126] Example 8

[0127] Verify the repeatability and uniformity of the SERS signal.

[0128] The Au / Ag cascade PS ball array structure obtained in Example 3 was used as the SERS active substrate. The entire sample was placed on a heating table and heated at 70°C for 10 minutes to allow the PDMS to expand. 2 μl of 10 -5 M concentration of crystal violet aqueous solution, after drying, gently wash the substrate with deionized water to remove unadsorbed CV, and then blow dry the substrate with nitrogen. Heat the substrate to 60 ° C with a heating plate, and randomly select 50 different positions on the same substrate for Raman spectrum detection. The excitation wavelength of the Raman spectrum is 785nm and the integration time is 10s. The SERS spectrum is as follows Figure 8 (a) and the intensity of the characteristic peak of crystal violet 1160cm-1 was selected for statistical analysis. Figure 8 As shown in (b), the relative standard deviation (RSD) is 7.65%. The RSD is less than 10%, indicating that the structure has good SERS signal stability and repeatability.

[0129] Example 9

[0130] The Au / Ag cascade PS ball array structure obtained in Example 3 was used as the SERS active substrate. The entire sample was placed on a heating table and heated at 70°C for 10 minutes to cause thermal expansion of the PDMS, thereby temporarily increasing the gap between the Au / Ag structures and providing sufficient space for the subsequent penetration of the macromolecular BSA. Subsequently, the substrate was immersed in an aqueous solution of BSA at a concentration of 0.5 mg / mL and incubated at 70°C for 12 hours to ensure that the BSA molecules fully penetrated and were uniformly adsorbed on the active area of ​​the cascade structure. After the incubation, the substrate was gently washed with deionized water to remove the unadsorbed BSA, and then the substrate was blown dry with nitrogen. After taking out the sample, it was naturally cooled to room temperature (25°C), and the SERS spectrum was detected. Then, the substrate was heated to 60°C, 70°C, and 80°C using a heating plate. The SERS spectrum was detected at different temperatures (60°C, 70°C, and 80°C) (the laser wavelength was 785nm and the integration time was 10 seconds), and the SERS spectrum was collected, as shown in FIG. Fig. 9 It can be found that when the temperature is room temperature, PDMS shrinks to the maximum extent, so that the previously enlarged Au / Ag structure gap is restored and compressed to a range matching the thickness of the adsorbed BSA molecular layer, thereby achieving the maximum enhancement effect of the local electromagnetic field.

[0131] Example 10

[0132] The Au / Ag cascade PS ball array structure obtained in Example 3 was used as the SERS active substrate. The entire sample was placed on a heating table and heated at 70°C for 10 minutes to cause thermal expansion of the PDMS, thereby temporarily increasing the gap between the Au / Ag structures and providing sufficient space for the subsequent penetration of the macromolecular BSA. Subsequently, the substrate was immersed in an aqueous solution of BSA with different concentrations (0.5 mg / mL, 50 μg / mL, 5 μg / mL, 0.5 μg / mL, 50 ng / mL, 5 ng / mL) and incubated at 70°C for 12 hours to ensure that the BSA molecules fully penetrated and were uniformly adsorbed on the active area of ​​the cascade structure. After the incubation, the substrate was gently washed with deionized water to remove the unadsorbed BSA, and then the substrate was blown dry with nitrogen. After taking out the sample, it was naturally cooled to room temperature (25°C) and the SERS spectrum was detected. Fig.10 As shown, it can be found that the results show that this method can detect BSA at a minimum concentration of about 5 ng / ml.

[0133] Embodiment 11

[0134] The Au / Ag cascade PS ball array structure obtained in Example 3 was used as the SERS active substrate. The sample was placed on a heating table and heated at 70°C for 10 minutes to cause thermal expansion of the PDMS, thereby temporarily increasing the gap of the Au / Ag structure and providing space for the entry of large HGB molecules. Next, the substrate was immersed in an aqueous solution containing HGB of different concentrations (1 mg / mL, 100 μg / mL, 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL) and incubated at 70°C for 12 hours to ensure that the HGB molecules fully penetrated and adsorbed to the active area of ​​the cascade structure. After the incubation, the substrate was gently washed with deionized water to remove the unadsorbed HGB molecules, and then the substrate was blown dry with nitrogen. After the sample was taken out of the 70°C environment, it naturally returned to room temperature (25°C), and the SERS spectrum was detected, as shown in FIG. Fig.11 The results showed that the method was sensitive and accurate and could detect HGB at a minimum concentration of about 10 ng / ml.

[0135] The Au / Ag cascaded PS ball array structure with adjustable gap and its preparation method and application according to the embodiment of the present invention have at least one of the following advantages:

[0136] 1. The template method is combined with the chemical synthesis method, which has a simple process and low cost.

[0137] The present invention adopts a combination of template method and chemical synthesis method to prepare periodic Au / Ag cascade PS ball array. First, a layer of densely arranged PS microsphere array is self-assembled on the surface of ultrapure water through liquid surface self-assembly technology, and then transferred to the target substrate to form a periodic PS ball template. The template method has a simple preparation process, low cost, and high repeatability. Compared with the traditional high-cost, complex equipment-supported nanostructure preparation method, it significantly reduces the technical threshold and improves the process feasibility.

[0138] 2. Synthesis of highly active metal nanostructures, with significant SERS enhancement effect

[0139] The present invention uses a chemical synthesis method to generate a highly active metal nanostructure on a PS ball array, and uses an in-situ substitution method to synthesize an Au / Ag nanocomposite structure. Through the cascade coupling effect of the composite structure, the SERS enhancement effect is effectively improved, and a large-area, uniform and stable cascade metal nanoparticle array structure is obtained. Compared with traditional nanostructures, this method increases the active site density of the nanostructure and enhances the signal amplification effect, so that it has higher sensitivity and stronger anti-interference ability in practical applications.

[0140] 3. The flexible adjustment characteristics of the PDMS substrate enable dynamic adjustment of the structural gap

[0141] The present invention prepares the Au / Ag cascade PS ball array on a polydimethylsiloxane (PDMS) substrate, and utilizes the flexibility and high thermal expansion coefficient of PDMS to dynamically adjust the structural gap between the PS balls with temperature changes. This adjustable characteristic not only improves the flexibility and adaptability of SERS detection, but also helps to optimize the enhancement effect of nanostructures in different detection environments, meet a variety of practical application requirements, and has significant advantages in the detection of complex and biologically active samples.

[0142] 4. Maximizing the enhancement effect can be achieved by dynamically adjusting the substrate gap: In the initial stage, the larger gap provides sufficient freedom for macromolecules to enter and adsorb on the substrate surface smoothly. When the substrate gradually adjusts the gap width and the gap gradually shrinks to a critical thickness close to the molecular layer, the adsorbed molecules are tightly compressed, thereby significantly improving the local field enhancement effect. This process not only optimizes the interaction between molecules and substrates, but also effectively enhances the signal response, achieving more efficient detection and analysis.

[0143] Although some embodiments of the present general inventive concept have been shown and described, it will be appreciated by those skilled in the art that changes may be made to these embodiments without departing from the principles and spirit of the present general inventive concept, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for preparing a gap-adjustable Au / Ag cascade PS ball array structure, characterized in that: The preparation method comprises: The first PS ball array structure with a PDMS substrate is heated to a first temperature to expand the gaps between the PS balls in the first PS ball array structure, the PS ball array structure with the expanded gaps is fixed with PVP, and the PDMS is removed to obtain a second PS ball array structure with a PVP film substrate; The substrate of the second PS ball array structure whose substrate is a PVP film is replaced with PDMS to obtain a second PS ball array structure whose substrate is a PDMS; Performing silver evaporation on the surface of the PS balls of the second PS ball array structure whose substrate is PDMS, to obtain a silver-plated PS ball array structure whose substrate is PDMS; The silver-plated PS ball array structure whose substrate is PDMS is placed in a gold growth liquid, and heated to a second temperature for reaction to obtain an Au / Ag cascade PS ball array structure whose substrate is PDMS. The gaps between the individual Au / Ag cascade PS balls in the Au / Ag cascade PS ball array structure can be controlled by temperature regulation.

2. The preparation method according to claim 1, characterized in that: The preparation method further comprises: The Au / Ag cascade PS ball array structure was transferred to a new PDMS substrate.

3. The preparation method according to claim 1, characterized in that: The first PS ball array structure with the substrate being PDMS is prepared by the following method: A periodic and densely packed PS microsphere array was self-assembled on the water surface and transferred to the target substrate to form a periodic PS sphere template; The PS microsphere array is transferred from the target substrate to a PDMS substrate to obtain a first PS sphere array structure whose substrate is PDMS.

4. The preparation method according to claim 3, characterized in that: The method of self-assembling a periodically densely arranged PS microsphere array on the water surface is specifically as follows: slowly dripping a PS sphere solution into water along a hydrophilic-treated slope, so that the PS spheres form a periodically densely arranged PS microsphere array on the water surface under the action of electrostatic force and surface tension.

5. The preparation method according to claim 3, characterized in that: The transferring of the PS microsphere array from the target substrate to the PDMS substrate comprises: The PS microsphere array on the periodic PS sphere template is bonded with the cured PDMS.

6. The preparation method according to claim 5, characterized in that: The step of heating the PDMS until it solidifies is as follows: heating the PDMS to 55° C.-65° C. and maintaining the temperature until solidification.

7. The preparation method according to claim 1, characterized in that: The first temperature is 74°C-76°C.

8. The preparation method according to claim 1, characterized in that: The second temperature is 69°C-71°C.

9. The Au / Ag cascade PS ball array structure with a PDMS substrate prepared according to the preparation method according to any one of claims 1 to 8.

10. Application of the Au / Ag cascade PS ball array structure with PDMS as the substrate as claimed in claim 9 in SERS detection.

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