A gold nanoflower SERS substrate, a preparation method and application thereof

Abstract

The application provides a gold nanoflower SERS substrate and a preparation method and application thereof, the preparation method removes the surface oxide film of an aluminum foil by using an etching solution, and realizes in-situ growth of the gold nanoflower on the surface of the aluminum foil by combining with a spontaneous displacement reaction of chloroauric acid. The preparation process is simple to operate, does not need an external power supply, raw materials are easy to obtain, and the cost is low; in addition, the substrate can be prepared in a large area, and the problems of a traditional SERS substrate, such as complicated process, high cost and difficulty in large-scale production, are effectively solved. The prepared substrate has a multi-stage rough microstructure, a high-density and uniformly-distributed SERS hot spot is formed on the surface, and the Raman signal enhancement effect is excellent. The substrate does not need sample labeling and substrate surface modification, and can quickly complete Raman spectrum detection of pseudomonas aeruginosa; in addition, the five types of carbapenemases can be accurately identified, and the detection process is efficient, and the result is stable and reliable.

Description

Technical Field

[0001] This invention relates to the field of surface-enhanced Raman spectroscopy (SERS), and more particularly to a gold nanoflower SERS substrate, its preparation method, and its application. Background Technology

[0002] Surface-enhanced Raman scattering (SERS) is an in-situ analytical technique that combines non-destructive, rapid, and highly sensitive characteristics (enabling single-molecule detection). Its unique fingerprint spectral region provides rich information on molecular composition and conformation. Leveraging these advantages, SERS technology has shown broad application prospects in various fields such as biomedicine, chemical analysis, food safety, environmental monitoring, and in-situ research.

[0003] It is worth noting that the single-molecule detection performance of SERS technology requires a core prerequisite—the target molecule must be confined to a specific local region of the substrate (this local region is called a "hotspot"). Therefore, a SERS substrate with a high hotspot density is key to realizing its superior performance and has extremely high practical application value. Especially in the field of bacterial detection, SERS technology demonstrates enormous application potential due to its ultra-high sensitivity, narrow-band resolution, and label-free inherent fingerprint recognition capability.

[0004] However, the research and development and fabrication of high-performance SERS substrates still face many challenges: First, the fabrication process is complex, with most existing substrates relying on precise and cumbersome processes such as metal evaporation, photolithography, nanosphere templates, or expensive nanoparticle self-assembly; second, the fabrication cost is high, with commonly used substrates requiring pure metal sheets or high-cost nanoparticles as core raw materials; and third, it is difficult to achieve large-area reproducible fabrication, as existing substrates generally suffer from poor stability and uncontrollable hotspot distribution, which seriously affects the reproducibility of detection results.

[0005] Therefore, developing a metal SERS substrate that is simple to prepare, low in cost, can be fabricated on a large scale, and has a high hot spot density has become an urgent need in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a gold nanoflower SERS substrate, its preparation method and application, to solve the technical problems of existing SERS substrates, such as complex preparation, high cost, difficulty in large-area reproducible preparation, poor substrate stability and uncontrollable hot spots.

[0007] To address the aforementioned technical problems, this invention first provides a method for preparing a gold nanoflower SERS substrate, comprising the following steps: S10, Immerse the aluminum foil paper substrate in the etching solution for etching treatment to remove the aluminum oxide film on the surface of the aluminum foil paper substrate; S20, the etched aluminum foil substrate is immersed in the growth solution to carry out an in-situ growth reaction, forming a gold nanoflower array pattern layer rich in hot spots on the surface of the aluminum foil substrate; the growth solution includes at least HAuCl4. S30: Take out the aluminum foil paper substrate containing the gold nanoflower array pattern layer, and obtain the gold nanoflower SERS substrate after cleaning and drying.

[0008] Specifically, the above-mentioned preparation method can complete the preparation of gold nanoflower SERS substrates through only three core steps: etching, in-situ growth, and cleaning and drying. The process is simple and easy to operate, without the need for complex and precise processes and expensive equipment such as metal evaporation and photolithography. Low-cost aluminum foil is used as the substrate, and a simple growth solution containing only chloroauric acid is used. The raw materials are readily available and the preparation cost is greatly reduced. The in-situ growth method can achieve large-area reproducible preparation of the substrate based on the aluminum foil substrate. At the same time, a gold nanoflower array pattern layer rich in hot spots is formed on the substrate surface, which effectively ensures that the substrate has excellent surface-enhanced Raman scattering performance. From the aspects of process, cost, large-scale preparation and detection performance, it specifically solves the technical shortcomings of existing SERS substrates.

[0009] Preferably, before step S10, the aluminum foil substrate is further subjected to ultrasonic cleaning in sequence with acetone, isopropanol, ethanol and deionized water.

[0010] Specifically, the pretreatment scheme involves sequentially performing graded ultrasonic cleaning of the aluminum foil substrate with acetone, isopropanol, ethanol, and deionized water. This thoroughly removes oil, dust, and organic and inorganic impurities adhering to the surface of the aluminum foil substrate, preventing impurities from interfering with subsequent etching and in-situ growth reactions. This effectively improves the uniformity of etching on the surface of the aluminum foil substrate, thereby ensuring the uniform and stable growth of the gold nanoflower array pattern layer.

[0011] Preferably, in step S10, the concentration of the etching solution is 0.08–0.12 mol / L, and the etching solution is a NaOH solution or a KOH solution; the etching time is 1–3 min.

[0012] Specifically, the alkaline etching solution is readily available, inexpensive, and easy to process, enabling gentle and controllable etching. While efficiently and thoroughly removing the aluminum oxide film on the surface of the aluminum foil substrate, it effectively avoids excessive etching that could damage the aluminum foil substrate itself, ensuring uniform etching on the surface of the aluminum foil substrate and providing a clean and regular reaction interface for the subsequent in-situ growth of gold nanoflowers.

[0013] Preferably, in step S20, the in-situ growth reaction time is 25–35 min, and the in-situ growth temperature is 20–30 °C.

[0014] Specifically, the above-mentioned in-situ growth process uses a mild temperature range and a suitable reaction time, which can avoid problems such as excessively high temperature causing the gold nanoflower growth rate to be too fast, particle agglomeration, and disordered distribution of surface hot spots. It can also prevent defects such as incomplete growth of gold nanoflowers and insufficient hot spot density of the substrate due to insufficient reaction time. It can precisely control the growth process of the gold nanoflower array pattern layer, so that a uniform and dense gold nanoflower structure with regular hot spot distribution is formed on the substrate surface. At the same time, the mild process conditions do not require additional complex temperature control equipment, further simplifying the preparation process and reducing the preparation cost.

[0015] Preferably, the growth solution is a 0.5–4 mmol / L HAuCl4 solution, and the size and surface roughness of the gold nanoflower array pattern layer both increase with the increase of the molar concentration of the HAuCl4 solution.

[0016] Specifically, this preferred method precisely limits the concentration of chloroauric acid in the growth solution to the range of 0.5–4 mmol / L, providing stable and controllable raw material concentration conditions for the in-situ growth of the gold nanoflower array patterned layer, significantly improving the process repeatability and batch stability of substrate preparation. Simultaneously, it clarifies the controllable regulation of the increase in size and surface roughness of the gold nanoflower array patterned layer with increasing chloroauric acid molar concentration, allowing for customized control of the substrate's microstructure through simple adjustments to the raw material concentration. Furthermore, with the simultaneous increase in the size and surface roughness of the gold nanoflower array patterned layer, the number and density of hotspots on the surface of the gold nanoflower SERS substrate increase simultaneously, significantly enhancing the signal amplification effect of surface-enhanced Raman scattering and effectively improving the detection sensitivity of the gold nanoflower SERS substrate.

[0017] Preferably, the growth solution is a mixture of 0.5–4 mmol / L HAuCl4 solution and a metal cation solution. The metal cations in the metal cation solution cannot be deposited in situ as elemental metals on the surface of the aluminum foil substrate through a displacement reaction with chloroauric acid.

[0018] Specifically, a growth solution is preferably prepared by combining a 0.5–4 mmol / L HAuCl4 solution with a solution containing a specific metal cation (this metal cation cannot be deposited in situ as a metallic element on the surface of the aluminum foil substrate through a displacement reaction with chloroauric acid). This provides a stable gold source for the growth of the gold nanoflower array pattern layer and allows for precise control of the nucleation rate and growth kinetics of the gold nanoflowers through the coordination effect of the metal cation and chloroauric acid, thereby optimizing their microstructure and structural arrangement. At the same time, the raw materials for this growth solution are readily available and easy to prepare, without increasing the complexity of the preparation process or production costs. It can also improve the hot spot distribution on the substrate surface, enhance the uniformity and density of hot spots, strengthen the SERS signal enhancement effect, and thus effectively improve the detection sensitivity and signal stability of the gold nanoflower SERS substrate.

[0019] Preferably, the concentration of the metal cation in the metal cation solution is 0.25–1 mmol / L, and the metal cation includes Na. + Mg 2+ And Al 3+ At least one of them.

[0020] Specifically, the above design can avoid the weak morphology regulation effect caused by excessively low metal cation concentration, and also prevent the problem of excessive nucleation and disordered structural aggregation of gold nanoflowers caused by excessively high concentration. The three types of metal cation raw materials selected are widely available and inexpensive, which will not increase the preparation cost of the substrate. Moreover, they can gently and precisely regulate the nucleation rate and growth path of gold nanoflowers, and further optimize the micromorphology of the gold nanoflower array pattern layer.

[0021] Accordingly, the present invention also provides a gold nanoflower SERS substrate, which is prepared by the above-described method for preparing gold nanoflower SERS substrate.

[0022] Specifically, the gold nanoflower SERS substrate prepared by the above preparation method has a uniform and dense gold nanoflower array pattern layer with a microstructure that can be precisely controlled. It has high-density and well-distributed SERS hot spots, and has excellent Raman signal enhancement effect, excellent detection sensitivity and signal stability. It can effectively avoid detection errors caused by impurity interference and structural defects.

[0023] Accordingly, the present invention also provides an application of the above-mentioned gold nanoflower SERS substrate in label-free bacterial detection and carbapenemase type 5 detection.

[0024] Specifically, this invention applies the aforementioned gold nanoflower SERS substrate to label-free bacterial detection and the detection of five types of carbapenemases. This fully leverages the core advantages of the gold nanoflower SERS substrate: high hotspot density, outstanding Raman signal enhancement, and stable and controllable performance. In label-free bacterial detection, detection can be directly performed based on the characteristic Raman spectrum of the bacteria themselves, eliminating the need for exogenous markers such as fluorescent dyes, antibodies, and nucleic acid probes. This simplifies the detection process and shortens the detection cycle by eliminating cumbersome steps such as labeling, incubation, and washing, while also avoiding interference from exogenous markers and improving the accuracy and repeatability of bacterial detection. For the detection of the five types of carbapenemases, the gold nanoflower SERS substrate can accurately identify the characteristic Raman signals of KPC, NDM, VIM, IMP, and OXA-48 carbapenemases, ensuring both sensitivity and specificity. This application solution effectively overcomes the shortcomings of traditional bacterial and drug-resistant enzyme detection processes, which are cumbersome, time-consuming, and costly. It provides an efficient and reliable new technical solution for rapid screening and accurate diagnosis of multidrug-resistant bacteria in clinical settings, and has significant clinical application and promotion value.

[0025] Preferably, the above application is for label-free SERS detection of Pseudomonas aeruginosa.

[0026] Specifically, this application relies on the three-dimensional rough structure and high-density local electromagnetic hotspots of the gold nanoflower SERS substrate to achieve detection of Pseudomonas aeruginosa without any labeling. This not only simplifies the detection process and reduces operating costs, but also completely preserves the natural biological characteristics of the bacteria and obtains clearly identifiable Raman characteristic peaks.

[0027] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention provides a gold nanoflower SERS substrate, its preparation method, and its applications. The preparation method employs an etching solution to remove the oxide film on the aluminum foil surface, combined with a chloroauric acid spontaneous displacement reaction, successfully achieving in-situ growth of gold nanoflowers on the aluminum foil surface. The entire preparation process is simple to operate, requires no external power supply, uses widely available and inexpensive raw materials, and can simultaneously achieve large-area substrate preparation, fundamentally overcoming the technical pain points of traditional SERS substrate preparation processes being cumbersome, costly, and difficult to scale up for mass production. Furthermore, the gold nanoflower SERS substrate prepared using this optimized process possesses a multi-level rough microstructure, and its surface can form high-density and uniformly distributed SERS hotspots, exhibiting excellent Raman signal enhancement capabilities, providing reliable performance support for high-precision detection. In practical testing applications, this gold nanoflower SERS substrate can achieve rapid fingerprint spectral detection of Pseudomonas aeruginosa without labeling the test sample or performing additional surface modifications on the substrate itself. At the same time, it can accurately identify the characteristic signals of the five carbapenemases, efficiently complete the detection of the five drug resistance enzyme indicators, and the detection process is simple and efficient with stable and reliable results. Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the preparation process of the gold nanoflower SERS substrate provided in Example 1 and the process of performing label-free SERS detection on Pseudomonas aeruginosa. Figure 2 The image shows the SEM (Scanning Electron Microscope) pattern of the gold nanoflower SERS substrate prepared using a 0.5 mmol / L HAuCl4 solution as the growth solution in Example 1. Figure 3 This is a SEM image of the gold nanoflower SERS substrate prepared using a 1 mmol / L HAuCl4 solution as the growth solution in Example 1. Figure 4This is the SEM image of the gold nanoflower SERS substrate prepared using a 2 mmol / L HAuCl4 solution as the growth solution in Example 1. Figure 5 This is the SEM image of the gold nanoflower SERS substrate prepared using a 4 mmol / L HAuCl4 solution as the growth solution in Example 1. Figure 6 The SERS spectrum obtained by label-free SERS detection of Pseudomonas aeruginosa on the gold nanoflower SERS substrate prepared with 0.5 mmol / L HAuCl4 solution in Example 1 is shown (with comparison results of blank control group). Figure 7 This is a SEM image of the gold nanoflower SERS substrate prepared in Example 2 using a mixture of 0.5 mmol / L HAuCl4 solution and 0.25 mmol / L NaCl solution as the growth solution. Figure 8 This is a SEM image of the gold nanoflower SERS substrate prepared in Example 2 using a mixture of 0.5 mmol / L HAuCl4 solution and 0.5 mmol / L NaCl solution as the growth solution. Figure 9 This is a SEM image of the gold nanoflower SERS substrate prepared in Example 2 using a mixture of 0.5 mmol / L HAuCl4 solution and 1 mmol / L NaCl solution as the growth solution. Figure 10 This is a schematic diagram illustrating the mechanism of five-type detection of carbapenemase on the gold nanoflower SERS substrate prepared in Example 2. Figure 11 This is a bar graph illustrating the specificity of the gold nanoflower SERS substrate prepared in Example 2 using a mixture of 0.5 mmol / L HAuCl4 solution and 1 mmol / L NaCl solution as the growth solution for NDM-type carbapenemase. Figure 12 The Raman spectra of the gold nanoflower SERS substrate prepared in Example 2 using a mixture of 0.5 mmol / L HAuCl4 solution and 1 mmol / L NaCl solution as the growth solution are used to verify the sensitivity of the system. The Raman signal responses of the system to NDM-type carbapenemase targets are shown. Figure 13This is a graph verifying the linear correlation between the gold nanoflower SERS substrate prepared in Example 2 using a mixture of 0.5 mmol / L HAuCl4 solution and 1 mmol / L NaCl solution as the growth solution and the detection of NDM-type carbapenemase. Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] To address the technical problems existing in current SERS substrates, aluminum, as a reactive metal, can achieve gold deposition through noble metal ion substitution, which is a cheap and efficient preparation approach. However, there is currently a lack of systematic methods for the controllable preparation of gold nanoflowers using this substitution reaction, and no published literature applies this method to label-free SERS detection of bacteria. Therefore, this invention provides a novel and efficient solution: through a technical route of "etching away Al2O3 + HAuCl4 spontaneous substitution reaction," gold nanoflowers (Au nanoflowers) grow rapidly and uniformly on the surface of aluminum foil, successfully preparing a gold nanoflower SERS substrate with high SERS hotspot density.

[0031] The technical solution of the present invention will now be further described with reference to specific embodiments.

[0032] Example 1: Please see Figure 1 Example 1 provides a gold nanoflower SERS substrate and its preparation method. The preparation process of the gold nanoflower SERS substrate specifically includes the following steps: Step (1): Cut commercial aluminum foil to the required size and ultrasonically clean it in acetone, isopropanol, ethanol and deionized water in sequence to thoroughly remove surface contaminants, and then place it in a 40°C oven to dry completely. Step (2): Immerse the cleaned aluminum foil in a 0.1 mol / L sodium hydroxide aqueous solution and etch it at room temperature for 2 minutes to completely remove the natural aluminum oxide film on its surface and expose the highly reactive fresh aluminum surface. Immediately after etching, rinse it repeatedly with deionized water and blow it dry. Step (3): Immerse the etched aluminum foil in HAuCl4 solutions of different molar concentrations (0.5 mmol / L, 1 mmol / L, 2 mmol / L and 4 mmol / L), shake for 2 min, and then shake continuously for 30 min to form a gold nanoflower array pattern layer with "hot spots" on the aluminum foil. Step (4): Take out the aluminum foil paper substrate containing the gold nanoflower array pattern layer, rinse with deionized water and dry to obtain the gold nanoflower SERS substrate.

[0033] Please see Figures 2-5 , Figures 2-5 In Example 1, SEM images of the gold nanoflower SERS substrates prepared using HAuCl4 solutions with concentrations of 0.5 mmol / L, 1 mmol / L, 2 mmol / L, and 4 mmol / L as growth solutions were obtained. Figures 2-5 It can be seen that the morphology of gold nanoflowers on the SERS substrate is regulated by the concentration of HAuCl4. The higher the concentration, the larger the size of the nanoflowers and the higher the surface roughness.

[0034] Please see Figure 1 and Figure 6 In Example 1, a gold nanoflower SERS substrate prepared with 0.5 mmol / L HAuCl4 solution was used to perform label-free SERS detection on Pseudomonas aeruginosa. The specific detection process included: taking a bacterial suspension of P. aeruginosa (with a viable bacterial concentration of 10). 6 ~10 8 (CFU / mL, i.e., colony-forming units per milliliter), was broken up and 5-10 μL was added to the surface of the above-mentioned gold nanoflower SERS substrate; after natural drying, the spectrum was collected using a Raman spectrometer, and the results are as follows: Figure 6 As shown.

[0035] Depend on Figure 6 As can be seen, the red curve represents the average Raman spectrum of *Pseudomonas aeruginosa*, with an overall signal intensity of approximately 17,000 au (dimensionless relative units), which is significantly higher than the black curve representing the PBS (Phosphate Buffered Saline) blank control (intensity generally below 5,000 a.u.), and is also higher at 400 cm⁻¹. -1 600cm -1 800cm -1 1000cm -1 1300cm -1 and 1500cm -1Clear bacterial characteristic peaks were observed at isowavenumbers, corresponding to the specific vibrational modes of intracellular biomolecules such as proteins and nucleic acids; while the PBS blank control showed only weak noise and no obvious characteristic peaks. This indicates that the gold nanoflower SERS substrate has high signal amplification capability, low background interference and excellent detection specificity, and can achieve label-free and accurate identification of Pseudomonas aeruginosa.

[0036] Example 2: Example 2 provides a gold nanoflower SERS substrate and its preparation method. The preparation process of the gold nanoflower SERS substrate specifically includes the following steps: Step (1): Cut commercial aluminum foil to the required size and ultrasonically clean it in acetone, isopropanol, ethanol and deionized water in sequence to thoroughly remove surface contaminants, and then place it in a 40°C oven to dry completely. Step (2): Immerse the cleaned aluminum foil in a 0.1 mol / L sodium hydroxide aqueous solution and etch it at room temperature for 2 minutes to completely remove the natural aluminum oxide film on its surface and expose the highly reactive fresh aluminum surface. Immediately after etching, rinse it repeatedly with deionized water and blow it dry. Step (3) involves immersing the etched aluminum foil in growth solutions with different formulations, shaking for 2 minutes, and then shaking for 30 minutes to form a gold nanoflower array pattern layer with "hot spots" on the aluminum foil. The growth solution in step (3) is prepared by mixing 0.5 mmol / L HAuCl4 solution with 0.25 mmol / L, 0.5 mmol / L, and 1 mmol / L NaCl solutions, respectively. Step (4): Take out the aluminum foil paper substrate containing the gold nanoflower array pattern layer, rinse with deionized water and dry to obtain the gold nanoflower SERS substrate.

[0037] Please see Figures 7 to 9 , Figures 7 to 9 This is a SEM image of the gold nanoflower SERS substrate prepared in Example 2 using a mixture of 0.5 mmol / L HAuCl4 solution and 0.25 mmol / L, 0.5 mmol / L, and 1 mmol / L NaCl solutions as the growth solution; wherein, the substrate is composed of... Figures 7 to 9 It can be known that: When 0.5 mmol / L HAuCl4 solution and 0.25 mmol / L NaCl aqueous solution were used as growth solutions to prepare gold nanoflower SERS substrates, the gold nanoflower array pattern was relatively sparse, the size was small, and the branching structure was not well developed. When using 0.5 mmol / L HAuCl4 solution and 0.5 mmol / L NaCl aqueous solution as the growth solution to prepare gold nanoflower SERS substrates, as Na... +With increasing concentration, the growth density of gold nanoflower array patterns increases significantly, and the size increases, forming a two-dimensional flower-like structure with abundant branches and sharp tips. This is also the morphology most conducive to the formation of local electromagnetic hotspots. When using 0.5 mmol / L HAuCl4 solution and 1 mmol / L NaCl aqueous solution as the growth solution to prepare gold nanoflower SERS substrates, as Na... + As the concentration increases further, the density of the gold nanoflower array pattern decreases, the structure becomes more compact, and the number of branches decreases.

[0038] Please see Figure 10 , Figure 10 This is a schematic diagram illustrating the mechanism of five-type detection of carbapenemase using the gold nanoflower SERS substrate prepared in Example 2; Figure 10 It is known that the surface of the gold nanoflower SERS substrate is immobilized with specific capture antibodies against different types of carbapenemases, which can bind to target carbapenemases such as NDM, KPC, IMP, VIM, and OXA-48, respectively. These antibodies then bind to gold nanoparticle probes modified with the corresponding detection antibodies, forming immune complexes. Through the localized surface plasmon resonance coupling effect between the gold nanoflower SERS substrate and the gold nanoparticle probes, the Raman signal can be significantly amplified, thereby achieving highly sensitive and specific parallel detection of five types of carbapenemases.

[0039] Specifically, in Example 2, a gold nanoflower SERS substrate prepared using a mixture of 0.5 mmol / L HAuCl4 solution and 1 mmol / L NaCl solution as the growth solution was used to specifically detect NDM-type carbapenemase. The specific detection process included: First, the prepared gold nanoflower SERS substrate is surface functionalized by immobilizing an NDM-type detection antibody against NDM-type carbapenemase on its surface, ensuring that the NDM-type detection antibody is uniformly and stably bound to the surface of the gold nanoflower SERS substrate.

[0040] Next, 2 μL of the test sample containing NDM-type carbapenemase was dropped onto the surface of the gold nanoflower SERS substrate and incubated at a suitable temperature to allow the NDM-type carbapenemase in the test sample to specifically bind to the NDM-type detection antibody on the gold nanoflower SERS substrate, thus completing the target capture.

[0041] Subsequently, gold nanoparticle probes with NDM-type detection antibodies on their surface were added. The NDM-type detection antibodies on the gold nanoparticle probes specifically bind to the already bound NDM-type carbapenemase to form an immune complex.

[0042] Next, the surface of the gold nanoflower SERS substrate was washed with PBS buffer to remove unbound gold nanoparticle probes and impurities, thereby reducing background interference.

[0043] Finally, the processed gold nanoflower SERS substrate was placed under a Raman spectrometer to collect signals. The local surface plasmon resonance coupling effect between the gold nanoflower SERS substrate and the gold nanoparticle probe will significantly amplify the Raman signal. By analyzing the signal intensity, the specific identification and quantitative detection of NDM-type carbapenemase can be achieved.

[0044] Please see Figure 11 , Figure 11 This is a bar graph illustrating the specificity of the gold nanoflower SERS substrate prepared in Example 2 using a mixture of 0.5 mmol / L HAuCl4 solution and 1 mmol / L NaCl solution as the growth solution for NDM-type carbapenemase. Figure 11 It can be seen that the gold nanoflower SERS substrate prepared in Example 2 has extremely high detection specificity for NDM-type carbapenemases: when the target is NDM-type carbapenemase and a corresponding capture antibody is matched, the Raman signal intensity can reach approximately 2.3 × 10⁻⁶. 3 au; while the Raman signal intensity of PBS blank control, BSA (Bovine Serum Albumin) unrelated proteins, other types of carbapenemases such as IMP / KPC / VIM / OXA-48, and mismatch groups with NDM as the target but capturing antibodies of other types of carbapenemases were all close to 0. Figure 11 The probability value P<0.0001 indicates that the signal difference between the positive group and all negative control groups is statistically significant, which fully demonstrates that the gold nanoflower SERS substrate can accurately identify NDM-type carbapenemases without interference from other irrelevant substances or non-target carbapenemases, and has excellent detection specificity.

[0045] Please see Figure 12 , Figure 12 This is a sensitivity verification Raman spectrum of the gold nanoflower SERS substrate prepared in Example 2 using a mixture of 0.5 mmol / L HAuCl4 solution and 1 mmol / L NaCl solution as the growth solution. It shows the Raman signal response of this system to NDM-type carbapenemase targets at concentrations of 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.1 ng / mL, and 0.01 ng / mL. Figure 12It can be seen that the gold nanoflower SERS substrate prepared in Example 2 has excellent detection sensitivity for NDM carbapenemase: as the mass concentration of NDM carbapenemase decreases from 100 ng / mL to 0.01 ng / mL, the Raman signal intensity shows a gradient decreasing trend, with the highest Raman signal intensity in the 100 ng / mL concentration group, approximately 4.5 × 10⁻⁶. 3 Even at a low concentration of 0.01 ng / mL, the system still produced a detectable Raman signal; simultaneously, at 1000 cm⁻¹... -1 1050~1100cm -1 At the characteristic wavenumbers, the characteristic peaks of each concentration group remained clear, and the peak intensity decreased synchronously with the decrease of concentration. This indicates that the gold nanoflower SERS substrate can not only respond to NDM-type carbapenemases as low as 0.01 ng / mL, but also that the signal intensity shows a good positive correlation with the concentration of the target analyte, providing strong support for the realization of highly sensitive quantitative detection of NDM.

[0046] Please see Figure 13 , Figure 13 This is a graph validating the linear correlation between the gold nanoflower SERS substrate prepared in Example 2 (using a mixture of 0.5 mmol / L HAuCl4 solution and 1 mmol / L NaCl solution as the growth solution) and the detection of NDM-type carbapenemase. Figure 13 It can be seen that the gold nanoflower SERS substrate prepared in Example 2 has excellent linear correlation with the detection of NDM-type carbapenemase: at the logarithmic ratio of NDM concentration (lgC / ng) mL -1 When the range is -2 to 2 (corresponding to concentrations of 0.01 ng / mL to 100 ng / mL), the Raman signal intensity shows a good linear positive correlation with the logarithm of the NDM concentration, with the fitted equation being y = 926.03x + 2319 and the correlation coefficient R0. 2 =0.9982 (close to 1), indicating that the detection system can accurately quantify NDM-type carbapenemase within the above concentration range, providing a reliable methodological basis for subsequent quantitative analysis of actual samples.

[0047] Compared with the prior art, the present invention has the following outstanding advantages: First, this invention utilizes an aluminum-gold spontaneous displacement reaction to grow a gold nanoflower array pattern layer with "hot spots" in situ, without the need for additional chemical reducing agents, and the preparation process is simple, safe and environmentally friendly.

[0048] Secondly, this invention uses inexpensive aluminum foil as a substrate carrier, which enables large-area mass production and significantly reduces production costs.

[0049] Third, the gold nanoflower SERS substrate grown in situ using a mixture of HAuCl4 solution and metal cation solution as the growth solution has a three-dimensional rough morphology, rich branching structure and sharp tips, and can form high-density local electromagnetic hot spots. Its surface-enhanced Raman scattering enhancement effect is significantly better than that of conventional nanoparticle-coated SERS substrates.

[0050] Fourth, this invention can precisely regulate the growth density of gold nanoflowers by controlling the concentration of chloroauric acid, resulting in high reproducibility of the preparation.

[0051] Fifth, the gold nanoflower SERS substrate of the present invention can achieve label-free detection of Pseudomonas aeruginosa, and can provide clear biological characteristic peaks to meet the needs of rapid biological detection.

[0052] Sixth, the gold nanoflower array pattern layer of the gold nanoflower SERS substrate of the present invention is tightly bonded to the aluminum foil through in-situ growth, and is firmly attached and not easy to fall off, so as to maintain stable detection performance for a long time.

[0053] Seventh, the gold nanoflower SERS substrate of the present invention can support highly specific parallel detection of five types of carbapenemases (NDM, KPC, IMP, VIM, OXA-48). Combined with its excellent SERS enhancement performance and immune recognition system, it can achieve accurate differentiation and quantification of different types of carbapenemases, meeting the actual needs of rapid multi-target detection in clinical practice.

[0054] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0055] The above embodiments merely illustrate implementation methods of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing a gold nanoflower SERS substrate, characterized in that, Includes the following steps: S10, Immerse the aluminum foil paper substrate in an etching solution for etching treatment to remove the aluminum oxide film on the surface of the aluminum foil paper substrate; S20, the etched aluminum foil substrate is immersed in a growth solution for in-situ growth reaction, forming a gold nanoflower array pattern layer rich in hot spots on the surface of the aluminum foil substrate; the growth solution includes at least HAuCl4. S30, the aluminum foil substrate containing the gold nanoflower array pattern layer is removed, and after cleaning and drying, a gold nanoflower SERS substrate is obtained.

2. The method for preparing the gold nanoflower SERS substrate according to claim 1, characterized in that, Before step S10, the process further includes: ultrasonically cleaning the aluminum foil substrate sequentially with acetone, isopropanol, ethanol, and deionized water.

3. The method for preparing the gold nanoflower SERS substrate according to claim 1, characterized in that, In step S10, the concentration of the etching solution is 0.08–0.12 mol / L, and the etching solution is a NaOH solution or a KOH solution; the etching time is 1–3 min.

4. The method for preparing the gold nanoflower SERS substrate according to claim 1, characterized in that, In step S20, the in-situ growth reaction time is 25-35 min, and the in-situ growth temperature is 20-30℃.

5. The method for preparing the gold nanoflower SERS substrate according to claim 4, characterized in that, The growth solution is a 0.5–4 mmol / L HAuCl4 solution, and the size and surface roughness of the gold nanoflower array pattern layer both increase with increasing molar concentration of the HAuCl4 solution.

6. The method for preparing the gold nanoflower SERS substrate according to claim 4, characterized in that, The growth solution is a mixture of 0.5–4 mmol / L HAuCl4 solution and metal cation solution; the metal cations in the metal cation solution cannot be deposited in situ as elemental metals on the surface of the aluminum foil substrate through a displacement reaction with chloroauric acid.

7. The method for preparing the gold nanoflower SERS substrate according to claim 6, characterized in that, The concentration of the metal cation in the metal cation solution is 0.25–1 mmol / L, and the metal cation includes Na. + Mg 2+ And Al 3+ At least one of them.

8. A gold nanoflower SERS substrate, characterized in that, It is prepared by the method for preparing the gold nanoflower SERS substrate as described in any one of claims 1 to 7.

9. An application of the gold nanoflower SERS substrate as described in claim 8 in label-free bacterial detection and carbapenemase five-type number detection.

10. The application according to claim 9, characterized in that, The application is for label-free SERS detection of Pseudomonas aeruginosa.

Images