Method for manufacturing a surface-enhanced Raman scattering substrate

Abstract

To provide a surface-enhanced Raman scattering substrate having no metal nanoparticles, which comprises an anodic alumina substrate having a three-dimensional pore structure, and a metal nanofilm on the anodic alumina substrate.SOLUTION: A surface-enhanced Raman scattering substrate does not require the use of metal nanoparticles, generates surface plasmon resonance by adopting a metal nanofilm structure, and therefore has excellent uniformity, stability, and reproducibility compared to conventional surface-enhanced Raman scattering substrates containing metal nanoparticles. By using an anodic alumina substrate having a three-dimensional pore structure, the surface-enhanced Raman scattering substrate of the present invention has high sensitivity, high stability, and high reproducibility. The present invention also provides a simple method for manufacturing a substrate that can realize a rapid process and can provide the surface-enhanced Raman scattering substrate with a three-dimensional pore structure.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a surface-enhanced Raman scattering substrate free of metal nanoparticles and a method for manufacturing the same, and more particularly to a surface-enhanced Raman scattering substrate including an anodic alumina substrate having a three-dimensional pore structure and a metal nanofilm on the anodic alumina substrate, and a method for manufacturing the same. [Background technology]

[0002] Surface-enhanced Raman scattering (SERS) is a spectral analysis technique that enables rapid, nondestructive, and noncontact optical analysis of molecular bonds within a specimen. SERS utilizes laser excitation of precious metals attached to micron- or nanoscale surface structures, resulting in surface plasmon resonance. Laser irradiation generates a localized electromagnetic field, which causes charge separation on the localized metal surface, amplifying the Raman signal. Therefore, SERS has been widely applied in analytical science, materials physics, and biomedicine. Due to its high sensitivity, nondestructive nature, and ability to directly test liquid samples, SERS technology is a potentially valuable analytical tool, used in agricultural research, environmental testing, molecular biology testing, and healthcare.

[0003] Conventional SERS technology uses metal nanoparticles, such as silver or gold particles, to enhance Raman signal. However, metal nanoparticles are difficult to control, and their shape and pitch are prone to change when exposed to a laser. As a result, conventional SERS substrates suffer from poor reproducibility and uniformity. China Patent Publication CN102621122A discloses a method for improving SERS detection capabilities by depositing multiple nanometer-sized metal particles at the bottom of the holes in a porous anodic alumina film using electroplating. However, this patented technology is not only complex, but also requires acidic wet etching or destruction of the alumina barrier layer, which may cause environmental pollution. Furthermore, the use of nanometer-sized metal particles results in poor reproducibility and uniformity of SERS substrates.

[0004] Anodic aluminum oxide (AAO) is a porous nanomaterial. Compared with silicon, glass, or polymeric substrates, AAO eliminates the need for complex and costly lithography and etching processes to fabricate SERS substrates.

[0005] According to Taiwan Patent Publication No. I731600, SERS substrates fabricated using AAO substrates can improve SERS detection capabilities because the electric field is enhanced along the nanopores of the AAO. However, although SERS substrates with this two-dimensional AAO substrate have an enhancement effect, their detection capabilities are still insufficient when testing extremely low concentrations of test substances, making their application difficult.

[0006] For these reasons, many studies have been conducted on SERS substrates, but the current industry demands SERS substrates that have a simpler manufacturing process, are lower cost, are contamination-free, have high reproducibility, high uniformity, and can detect even extremely low concentrations of substances (i.e., have higher detection capabilities). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Taiwan Patent Publication No. I731600 Summary of the Invention [Problem to be solved by the invention]

[0008] In view of the above, the present invention aims to provide a metal nanoparticle-free SERS substrate and a manufacturing method thereof, which provides a simpler, lower-cost, and contamination-free process. Furthermore, the SERS substrate manufactured by the above process has an AAO substrate with a three-dimensional pore structure, and can detect even extremely low concentrations of substances with high reproducibility and uniformity. [Means for solving the problem]

[0009] The SERS substrate of the present invention uses a metal nanofilm instead of metal nanoparticles, thereby avoiding changes in the shape and pitch of metal nanoparticles due to laser irradiation, thereby improving the reproducibility and uniformity of the SERS substrate.

[0010] Furthermore, the SERS substrate of the present invention has an AAO substrate with a three-dimensional pore structure. A SERS substrate with extremely high detection capability can be obtained through the following mechanism: Compared with a substrate with a two-dimensional structure, a substrate with a three-dimensional structure has more hot spots distributed three-dimensionally due to the three-dimensional AAO pores, and the hot spots have higher intensity, resulting in higher sensitivity.

[0011] Specifically, one aspect of the present invention is an AAO substrate having a three-dimensional pore structure; and a metal nanofilm on the AAO substrate. This is a SERS substrate without metal nanoparticles.

[0012] The three-dimensional pore structure comprises a structure having nano-sized pores in micron-sized or nano-sized depressions, or a structure having nano-sized tips around the nano-sized pores, and the metal film is formed to adhere to the shape of the three-dimensional pore structure.

[0013] In some embodiments, the average pore size of the micron-order depressions in the three-dimensional pore structure is 0.1 to 5 μm, preferably 0.3 to 2 μm, and more preferably 0.5 to 1.5 μm, which allows multiple reflections to occur, and more photons collide inelastically with the probe molecules on the SERS substrate, amplifying the Raman signal.

[0014] In some embodiments, the average roughness of the AAO substrate is 0.1 to 2 μm, preferably 0.4 to 1 μm, and more preferably 0.8 to 1 μm. Compared to two-dimensional substrates that reflect most of the incident light, the roughness of the AAO substrate increases the inelastic collisions between photons and the probe molecules on the SERS substrate, thereby amplifying the Raman signal.

[0015] In some embodiments, the thickness of the metal nanofilm is 6 to 30 nm, preferably 10 to 20 nm. By using a metal nanofilm instead of metal nanoparticles, changes in the shape and pitch of the metal nanoparticles due to laser irradiation can be avoided, improving the uniformity, reproducibility, and uniformity of the SERS substrate. In addition, localized surface plasmon resonance occurs, improving sensitivity to the test object and enabling SERS measurement using an AAO substrate.

[0016] In some embodiments, the nanopores have an average pore spacing of 10 to 300 nm, preferably 10 to 100 nm. By reducing the average pore spacing of the nanopores, the intensity of the hot spots formed by the three-dimensional pore structure of the AAO substrate can be improved, thereby enhancing the Raman signal.

[0017] In some embodiments, the SERS substrate has a detection limit for methylene blue of at least 1×10 -10 This allows it to be applied to industries that require water quality testing and prevent damage from water pollution.

[0018] In some embodiments, the SERS substrate has a detection limit of at least 0.05 ppm for melamine, which can be applied to detect melamine residues since the World Health Organization's recommended residue standard is 1 ppm or less.

[0019] Furthermore, the present invention uses a special electrochemical processing method to fabricate an AAO substrate with a three-dimensional pore structure free of metal nanoparticles, which not only simplifies the process but also avoids the need for complex, costly, and time-consuming acid wet etching and lithography processes in semiconductor manufacturing, thereby reducing environmental pollution.

[0020] Specifically, the present invention involves subjecting an aluminum foil to electrochemical treatment in an electrolyte solution at a specific temperature range, which is 15 to 25°C, preferably 15 to 20°C, to change the surface structure of the aluminum foil and form a three-dimensional pore structure.

[0021] Thus, one aspect of the present invention is (A) a cleaning step of cleaning the aluminum foil; (B) a first electrochemical treatment step in which the aluminum foil is subjected to an electrochemical treatment with a direct current of 5 to 40 V in an electrolyte at 15 to 25°C to change the surface structure of the aluminum foil; (C) a second electrochemical treatment step in which the aluminum foil is further electrochemically treated with a composite pulse square wave to obtain an AAO substrate; (D) a metal plating step of plating a single layer of metal on the surface of the AAO substrate to form the metal nanofilm; This is a method for manufacturing a SERS substrate. The first electrochemical treatment step is performed at 15-25°C to form a three-dimensional pore structure. After two electrochemical treatment steps, the surface structure of the AAO is changed, improving the detection intensity of the SERS substrate.

[0022] Another aspect of the present invention is (A) a first electrochemical treatment step in which an aluminum foil is electrochemically treated with a direct current of 5 to 40 V in an electrolyte at 15 to 25°C to change the surface structure of the aluminum foil; (B) a second electrochemical treatment step, further subjecting the aluminum foil to electrochemical treatment using a composite pulse square wave to obtain an aluminum foil structure having an AAO film; (C) a third electrochemical treatment step, in which the aluminum foil structure with the AAO film is subjected to an electrochemical treatment in a 5 wt% phosphoric acid electrolyte to obtain the AAO substrate; (D) a metal plating step of plating a single layer of metal on the surface of the AAO substrate to form the metal nanofilm; This is a method for manufacturing a SERS substrate. The first electrochemical treatment step is performed at 15-25°C to form a three-dimensional pore structure. The third electrochemical treatment step is a pore-size expansion treatment, which changes the surface structure of the AAO, enlarging the size of the nanopores in the three-dimensional pore structure and reducing the distance between the nanopores. This improves the detection intensity of the SERS substrate and enhances the Raman signal.

[0023] Another aspect of the present invention is (A) a first electrochemical treatment step in which an aluminum foil is electrochemically treated with a direct current of 5 to 40 V in an electrolyte at −20 to 20° C. to change the surface structure of the aluminum foil; (B) a second electrochemical treatment step, further subjecting the aluminum foil to electrochemical treatment using a composite pulse square wave to obtain an aluminum foil structure having an AAO film; (C) a third electrochemical treatment step, in which the aluminum foil structure with the AAO film is electrochemically treated in a mixed electrolyte of perchloric acid and ethanol to separate the AAO film from the aluminum foil, thereby obtaining the AAO substrate with nano-sized tips; (D) a metal plating step of plating a single layer of metal on the surface of the AAO substrate to form the metal nanofilm; This is a method for manufacturing a SERS substrate. The three electrochemical treatment steps change the surface structure of the AAO, improving the detection intensity of the SERS substrate. The third electrochemical treatment step results in an AAO substrate with a three-dimensional pore structure with nano-sized tips, which enhances the three-dimensionality of the three-dimensional pore structure in the AAO substrate, thereby enhancing the Raman signal.

[0024] Another aspect of the present invention is (A) a cleaning step of cleaning the aluminum foil; (B) a first electrochemical treatment step in which the aluminum foil is subjected to an electrochemical treatment with a direct current of 5 to 40 V in an electrolyte at 15 to 25°C to change the surface structure of the aluminum foil; (C) a second electrochemical treatment step, further subjecting the aluminum foil to electrochemical treatment using a composite pulse square wave to obtain an aluminum foil structure having an AAO; (D) a step of removing the AAO film, in which the aluminum foil structure having the AAO film is placed in a mixed solution of phosphoric acid and chromic acid, and the AAO film is removed to obtain an aluminum foil having a surface structure; (E) a third electrochemical treatment step in which the aluminum foil having the surface structure is electrochemically treated with a composite pulse square wave to obtain an AAO substrate; (F) a metal plating step of plating a single layer of metal on the surface of the AAO substrate to form the metal nanofilm; This is a method for manufacturing a SERS substrate. The AAO film removal process and the third electrochemical treatment process both modify the morphology of the AAO substrate with a three-dimensional pore structure and improve the three-dimensionality of the AAO substrate's three-dimensional pore structure, thereby enhancing the Raman signal. Furthermore, performing the first electrochemical treatment at 15-25°C changes the surface structure of the aluminum foil, resulting in an AAO substrate with a three-dimensional pore structure, thereby improving the detection intensity of the SERS substrate.

[0025] In some embodiments, in the (C) second electrochemical treatment step, the aluminum foil is further subjected to electrochemical treatment for 3 to 5 minutes using a composite pulse square wave under the following conditions: Conditions: 0 to 40°C; phosphoric acid, oxalic acid, sulfuric acid, or a composition thereof is used as the electrolyte; the positive value of the pulse amplitude is 100 to 200 V, the negative value is -2 to -8 V, and the time ratio of the positive and negative pulses is 1:1 to 1:4. This step allows the formation of AAO and improves SERS detection capability. The voltage used varies depending on the type of acid. Phosphoric acid: The positive value is 100 to 200 V, and the negative value is -2 to -8 V. Oxalic acid: The positive value is 100 to 150 V, and the negative value is -2 to -8 V. Sulfuric acid: The positive value is 10 to 60 V, and the negative value is -2 to -4 V. [Effects of the Invention]

[0026] By using a metal nanofilm, the SERS substrate of the present invention forms a structure that generates surface plasmon resonance, preventing changes in the shape and pitch of metal nanoparticles due to laser irradiation and improving the stability, reproducibility, and uniformity of the SERS substrate.

[0027] The SERS substrate of the present invention is an AAO substrate with a three-dimensional pore structure obtained through a special electrochemical process. This allows multiple reflections to occur when a laser is irradiated, generating more photons that collide inelastically with the probe molecules on the SERS substrate, thereby enhancing the intensity of the Raman signal. This overcomes the drawback of the two-dimensional AAO substrate, which has a poor sensitivity.

[0028] The method for manufacturing a SERS substrate of the present invention overcomes the drawbacks of conventional SERS substrate manufacturing, such as high cost and time required, and allows for rapid and low-cost manufacturing. Therefore, the AAO substrate can be disposed of immediately after inspection without being reused. Furthermore, the simplified process is extremely advantageous for mass production.

[0029] The SERS substrate of the present invention has high sensitivity and stability, making it suitable for industries requiring water quality testing, such as sewage treatment and ecosystem protection. After collecting a sample in the field, it can be directly dropped onto the SERS substrate and allowed to settle. After drying, precise SERS testing can be performed to analyze the types of substances contained in the sample and whether they meet international standards.

[0030] Screening for additives and detecting poor-quality raw materials are important industries for ensuring food safety. By using the SERS substrate of the present invention, it is possible to directly and quickly test the types of ingredients contained in food and the presence or absence of illegal additives. Furthermore, the SERS substrate of the present invention has a low detection limit, allowing for reliable detection of illegal additives. Furthermore, in the agricultural research industry, where pesticide component analysis and variety identification are performed, the SERS substrate of the present invention can be used to detect the type and concentration of pesticides remaining on the surface of vegetables and fruits, greatly contributing to the management of illegal pesticide use and drug residue limits.

[0031] The SERS substrate of the present invention can also be applied in the medical field, where biomolecules such as cancer cells and DNA viruses must be examined. For example, by creating a SERS database of different cell types, it is possible to distinguish the spectral distribution maps of normal cells and cancer cells through statistical analysis and estimate the spectrum of cancer cells. Therefore, the SERS substrate of the present invention can be applied in fields such as drug screening and disease diagnosis. [Brief explanation of the drawings]

[0032] [Figure 1] is an SEM photograph of AAO-R5. [Figure 2] is an SEM photograph of AAO-R15. [Figure 3] is an SEM photograph of AAO-R20. [Figure 4] is a higher magnification SEM photograph of AAO-R20. [Figure 5] 1 shows the SERS spectra of 10-5 M methylene blue measured on the SERS substrates AAO-R5, AAO-R10, AAO-R15, and AAO-R20. [Figure 6] 1 shows the SERS spectra of methylene blue at various concentrations measured on a SERS substrate with AAO-R20. [Figure 7] 1 shows the SERS spectra measured at various concentrations of melamine on a SERS substrate with AAO-R20. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention fabricates a metal nanoparticle-free SERS substrate by performing multiple electrochemical treatments on aluminum foil, particularly by electrochemically treating the aluminum foil in an electrolyte solution within a specific temperature range. The substrate has an AAO substrate with a three-dimensional pore structure, which can have nanopores within micron- or nano-level depressions, leading to high roughness, or nanopores surrounded by nano-level tips. This allows for multiple reflections during laser irradiation, resulting in more photons being generated and colliding inelastically with the probe molecule, thereby enhancing the Raman signal. Furthermore, a single layer of metal is plated on the AAO substrate, resulting in localized surface plasmon resonance. Therefore, the SERS substrate of the present invention exhibits high stability, high reproducibility, excellent uniformity, and high sensitivity.

[0034] The present invention will be described below through examples. The examples of the present invention are intended to be enumerated, and do not limit the scope of the present invention to all specific environments, applications, or special methods described in the examples. Therefore, the description of the examples is intended to be illustrative of the purpose of the present invention, and does not limit the present invention.

[0035] Example 1 [Manufacturing]

[0036] 1. Cleaning process The aluminum foil was ultrasonically cleaned in acetone and deionized water for 5 minutes to remove surface particles and oily dirt, and the surface of the aluminum substrate was rinsed with deionized water and dried with nitrogen gas.

[0037] 2. First electrochemical treatment process Aluminum foils were electrochemically treated for 5 minutes with a 20V DC current in a mixed solution of perchloric acid and ethanol at temperatures of 5°C, 10°C, 15°C, and 20°C, respectively, to obtain aluminum foils R5, R10, R15, and R20 with three-dimensional pore structures and different surface structures. Furthermore, the applicant conducted multiple experiments and found that temperatures above 25°C worsened the uniformity of SERS, and therefore found that 20°C was the preferred temperature.

[0038] 3. Second electrochemical treatment process The aluminum foils were subjected to a second electrochemical treatment using a one-step hybrid pulse anodization (HPA) method (instrument model: Jiehan 5000) at 25°C to obtain AAO substrates designated as AAO-R5, AAO-R10, AAO-R15, and AAO-R20, respectively. The positive and negative pulse amplitudes were 120 V and -4 V, respectively. The electrolyte was 5 wt% phosphoric acid, and the electrochemical treatment time was 180 s.

[0039] 4. Metal plating process A platinum metal film with a thickness of approximately 15 nm was plated on the surface of the AAO substrate with a three-dimensional pore structure using a platinum sputtering device (model number: JEC-3000FC, JEOL) to prepare a SERS substrate for SERS measurement. 〔analysis〕

[0040] 1. Roughness measurement of R5 to R20 using a staircase contour measuring machine (Kosaka Lab, ET3000) The average roughnesses of R5, R10, R15, and R20 were approximately 0.18, 0.48, 0.82, and 0.91 μm, respectively. This indicates that in the first electrochemical treatment process, the higher the electrolyte temperature, the faster the reaction, resulting in localized over-etching, which resulted in more pronounced holes and depressions in the aluminum foil and improved the three-dimensionality of the pore structure. Furthermore, the aluminum foils obtained by the first electrochemical treatment process performed at 15°C and 20°C had significantly higher average roughness than the aluminum foils treated at 5°C and 10°C.

[0041] 2. Observation of nanostructures in AAO using a field emission scanning electron microscope (HR-FESEM, JEOL JSM-7001) Figures 1 to 3 are SEM photographs of AAO-R5, AAO-R15, and AAO-R20, respectively. First, in Figure 1, the surface of AAO-R5 had minimal irregularities and the electric field was uniformly distributed. In contrast, as shown in Figure 2, multiple micron-scale pores were clearly observed on the surface of AAO-R15. These micron-scale pores were larger in area and had a higher color contrast (i.e., a more pronounced three-dimensional structure) than those of AAO-R5. In Figure 2, the two circled areas represent micron-scale depressions and nano-scale depressions, respectively. Observation of the micron-scale depressions in the upper circle reveals color contrast, i.e., differences in elevation, and the multiple black dots within the depressions are nanopores. Therefore, it was determined that the micron-scale depressions in the upper circle have a three-dimensional porous structure consisting of several to several dozen nanopores. Observation of the nano-scale depressions in the lower circle also reveals color contrast, i.e., differences in elevation. The multiple black dots within the depressions are nanopores. Therefore, it was determined that the nano-scale depressions in the lower circle also have a three-dimensional porous structure consisting of several to several dozen nanopores. The above three-dimensional porous structure further improved the surface roughness. This phenomenon is more evident in Figure 4, described below.

[0042] When the reaction temperature of the first electrochemical treatment step was further increased to 20°C, the number and size of the micron- and nano-level depressions etched by the etching increased significantly, and a phenomenon of the depressions clustering was observed, as shown in Figure 3. This significantly improved three-dimensionality of the three-dimensional hole structure in the SERS substrate contributed to the occurrence of multiple reflections, which generated more photons that collided inelastically with the probe molecules, thereby enhancing the Raman signal.

[0043] Figure 4 shows a higher-magnification SEM image of AAO-R20. The three-dimensional pore structure of the AAO substrate is more clearly observed in Figure 4, with multiple nanopores (circled in Figure 4) among the micron- and nano-scale depressions. The aluminum foil AAO substrate, with its pronounced pores and depressions, possesses a three-dimensional pore structure that contributes to the occurrence of multiple reflections and scattering, enhancing the Raman signal during testing.

[0044] 3. Measurement of pore spacing using Image J software Using software called Image J, the pore spacing between nanopores in the micron- and nano-level depressions of AAO-R5, AAO-R10, AAO-R15, and AAO-R20 was measured. The pore spacing is defined as the linear distance from the outermost periphery of a nanopore to the outermost periphery of another nanopore. The measurement results showed that the average pore spacing of AAO-R5, AAO-R10, AAO-R15, and AAO-R20 was 100 to 300 nm.

[0045] 4. Raman Spectrum Measurement SERS substrates with AAO-R5, AAO-R10, AAO-R15, and AAO-R20 were used, and 10 -5 The Raman spectra obtained from each measurement were compared, and the results are shown in Figure 5. The measurement results indicated that a surface with high roughness and a three-dimensional pore structure provides a superior detection effect.

[0046] As shown in Figure 5, the Raman spectra measured using the SERS substrates with AAO-R5, AAO-R10, AAO-R15, and AAO-R20 and the Raman spectrum of the control group were similar to those measured using the SERS substrates with AAO-R5, AAO-R10, AAO-R15, and AAO-R20. -1 The signal intensities at 1000 nm and 1000 nm were 4643, 10844, 40122, 48413, and 421, respectively. Therefore, it is clear that AAO-R15 and AAO-R20, which have a pronounced three-dimensional pore structure, can enhance the Raman signal, with the maximum value being more than 100 times that of the control group.

[0047] 5. Detection limit of methylene blue Using the SERS substrate with AAO-R20, the concentration was 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, and 10 -10The results of the SERS spectrum are shown in Figure 6. -10 Even in a methylene blue solution of M, the -1 The SERS substrate with a three-dimensional pore structure exhibited a detection limit of at least 10 for methylene blue. -10 M, and it was found to have extremely high applicability.

[0048] 6. Melamine detection limit Using a SERS substrate with AAO-R20, melamine solutions with concentrations of 50 ppm, 5 ppm, 0.5 ppm, and 0.05 ppm were measured. The SERS spectrum results are shown in Figure 7. From Figure 7, it can be seen that even with a melamine solution of 0.05 ppm, the peak at 710 cm -1 A significant signal was observed in the SERS substrate with a three-dimensional pore structure, and the detection limit for melamine was as low as 0.05 ppm, which is far lower than the 1 ppm residual standard set by the World Health Organization, demonstrating its extremely high applicability.

[0049] Example 2

[0050] 1. Cleaning process The aluminum foil was ultrasonically cleaned in acetone and deionized water for 5 minutes to remove surface particles and oily dirt, and the surface of the aluminum substrate was rinsed with deionized water and dried with nitrogen gas.

[0051] 2. First electrochemical treatment process The aluminum foil was electrochemically treated for 5 minutes with a direct current of 5 to 40 V in a mixed solution of perchloric acid and ethanol (4:1 to 1:4) at a temperature of 15 to 25°C, to obtain an aluminum foil with a three-dimensional surface structure.

[0052] 3. Second electrochemical treatment process The aluminum foil was subjected to a second electrochemical treatment using a composite pulse square wave at 0 to 40°C to obtain a porous AAO film. The positive pulse amplitude was 100 to 200 V, and the negative pulse amplitude was -2 to -8 V. The time ratio of the positive and negative pulses was 1:1 to 1:4. The electrolyte was 5 wt% phosphoric acid, and the electrochemical treatment time was 180 seconds.

[0053] 4. Third electrochemical treatment process Next, the composite pulse square wave was stopped, and the reaction was continued in the electrolyte for 0 to 60 minutes to obtain the AAO substrate.

[0054] 5. Metal plating process A metal film with a thickness of approximately 6 to 30 nm was plated on the surface of the AAO substrate to prepare a SERS substrate for SERS measurement.

[0055] In this example, a three-dimensional pore structure was formed by performing the first electrochemical treatment at 15–25°C. Furthermore, the morphology of the three-dimensional pore-structured AAO substrate could be further adjusted by performing a pore size expansion treatment in the third electrochemical treatment. The pore size expansion treatment enlarges the size of the nanopores in the three-dimensional pore structure and reduces the distance between the nanopores. This improved the three-dimensionality of the three-dimensional pore structure in the AAO substrate, thereby enhancing the Raman signal. Specifically, the pore spacing between nanopores after the pore size expansion treatment was measured using Image J software. The pore spacing is defined as the linear distance from the outermost periphery of one nanopore to the outermost periphery of another nanopore. The measurement results showed that the average pore spacing was 10–100 nm. This indicates that the pore size expansion treatment in the third electrochemical treatment effectively reduced the average pore spacing from 100–300 nm to 10–100 nm, thereby improving the three-dimensionality of the three-dimensional pore structure of the AAO substrate.

[0056] Implementation Method 3

[0057] 1. Cleaning process The aluminum foil was ultrasonically cleaned in acetone and deionized water for 5 minutes to remove surface particles and oily dirt, and the surface of the aluminum substrate was rinsed with deionized water and dried with nitrogen gas.

[0058] 2. First electrochemical treatment process The aluminum foil was subjected to electrochemical treatment for 5 minutes with a direct current of 5 to 40 V in a mixed solution of perchloric acid and ethanol (4:1 to 1:4) at a temperature of -20 to 20°C, to obtain aluminum foil with an average roughness (Ra) of 0.05 to 1 μm.

[0059] 3. Second electrochemical treatment process The aluminum foil was subjected to a second electrochemical treatment using a composite pulse square wave at 0 to 40°C to obtain a porous AAO film. The positive pulse amplitude was 100 to 150 V and the negative pulse amplitude was -2 to 8 V. The time ratio of the positive and negative pulses was 1:1 to 1:4. The electrolyte was 0.1 to 3 M oxalic acid, and the electrochemical treatment time was 5 minutes. This resulted in an aluminum foil structure with an AAO film.

[0060] 4. Third electrochemical treatment process The aluminum foil structure with the AAO film was placed in a mixed solution of perchloric acid and ethanol (4:1 to 1:4), and a third electrochemical treatment was performed with a direct current of 120 to 180 V for 30 seconds to separate the aluminum foil and the AAO film, obtaining an AAO substrate with a three-dimensional tip.

[0061] 5. Metal plating process A metal film with a thickness of approximately 6 to 30 nm was plated on the surface of the tip of an AAO substrate with a three-dimensional tip to fabricate a SERS substrate for SERS measurement.

[0062] In this example, the AAO substrate with a three-dimensional nano-sized pore structure was obtained through the third electrochemical treatment process, which improved the three-dimensionality of the three-dimensional pore structure in the AAO substrate, thereby improving the detection intensity of the SERS substrate.

[0063] Implementation Method 4

[0064] 1. Cleaning process The aluminum foil was ultrasonically cleaned in acetone and deionized water for 5 minutes to remove surface particles and oily dirt, and the surface of the aluminum substrate was rinsed with deionized water and dried with nitrogen gas.

[0065] 2. First electrochemical treatment process The aluminum foil was subjected to electrochemical treatment for 5 minutes with a direct current of 5 to 40 V in a mixed solution of perchloric acid and ethanol (4:1 to 1:4) at a temperature of 15 to 20°C, to obtain aluminum foil with an average roughness (Ra) of 0.05 to 1 μm.

[0066] 3. Second electrochemical treatment process The aluminum foil was subjected to a second electrochemical treatment using a composite pulse square wave at 0 to 40°C to obtain a porous AAO film. The positive pulse amplitude was 100 to 150 V, and the negative pulse amplitude was -2 to 8 V. The time ratio of the positive and negative pulses was 1:1 to 1:4. The electrolyte was 0.1 to 3 M oxalic acid, and the electrochemical treatment time was 1 to 60 minutes. This resulted in an AAO substrate.

[0067] 4. AAO film removal process The AAO substrate was placed in a mixed solution of phosphoric acid and chromic acid (1 to 4 wt% chromic acid and 3 to 10 wt% phosphoric acid), and the AAO film on the AAO substrate was removed, thereby obtaining aluminum foil with a surface structure again.

[0068] 5. Third electrochemical treatment process The aluminum foil was subjected to a third electrochemical treatment using a composite pulse square wave at 0 to 40°C. The positive pulse amplitude was 100 to 150 V, and the negative pulse amplitude was -2 to 8 V. The time ratio of the positive and negative pulses was 1:1 to 1:4. The electrolyte was 0.1 to 3 M oxalic acid, and the electrochemical treatment time was 1 to 60 minutes. This resulted in an AAO substrate with both large and small pores.

[0069] Metal plating: A metal film with a thickness of approximately 6–30 nm was plated on the surface of the AAO substrate to prepare a SERS substrate for SERS measurement.

[0070] In this example, a three-dimensional pore structure was formed by performing the first electrochemical treatment step at 15 to 25° C. Furthermore, both the AAO film removal step and the third electrochemical treatment step adjusted the morphology of the AAO substrate with a three-dimensional pore structure and improved the three-dimensionality of the three-dimensional pore structure of the AAO substrate, thereby enhancing the Raman signal and improving the detection intensity of the SERS substrate.

[0071] From the above, it can be seen from the above description of the implementation methods and embodiments that the present invention has the following breakthroughs and advantages over the prior art:

[0072] By using a metal nanofilm, the SERS substrate of the present invention can avoid changes in the shape and pitch of metal nanoparticles due to laser irradiation, thereby improving the stability, reproducibility, and uniformity of the SERS substrate.

[0073] The SERS substrate of the present invention is an AAO substrate with a three-dimensional pore structure obtained through a special electrochemical treatment process. Specifically, the surface structure of aluminum foil or AAO is altered by electrochemical treatment within a specific temperature range, resulting in the production of an AAO substrate with a three-dimensional pore structure. This allows multiple reflections to occur during laser irradiation, generating more photons that collide inelastically with the probe molecules on the SERS substrate, thereby enhancing the intensity of the Raman signal. This overcomes the drawback of the poor sensitivity of two-dimensional AAO substrates.

[0074] The method for manufacturing a SERS substrate of the present invention overcomes the drawbacks of conventional SERS substrate manufacturing, such as high cost and time required, and allows for rapid and low-cost manufacturing. Therefore, the AAO substrate can be disposed of immediately after inspection without being reused. Furthermore, the simplified process is extremely advantageous for mass production.

[0075] The SERS substrate of the present invention has high sensitivity and stability and can be applied to industries requiring water quality testing, such as wastewater treatment and ecosystem protection, and agricultural research industries that analyze pesticide ingredients and identify varieties, as well as fields such as drug screening and disease diagnosis.

Claims

1. (A) a first electrochemical treatment step in which an aluminum foil is electrochemically treated with a direct current of 5 to 40 V in an electrolyte at 15 to 25°C to change the surface structure of the aluminum foil; (B) a second electrochemical treatment step in which the aluminum foil is further electrochemically treated with a composite pulse square wave to obtain an anodic alumina substrate; (C) a metal plating step of plating a single layer of metal on the surface of the anodic alumina substrate to form a metal nanofilm, The surface-enhanced Raman scattering substrate is a surface-enhanced Raman scattering substrate free of metal nanoparticles, the anodic alumina substrate having a three-dimensional pore structure; the metal nanofilm on the anodic alumina substrate; The three-dimensional pore structure has a structure having nano-sized pores in micron-sized or nano-sized depressions, or a structure having nano-spikes around the nano-sized pores, and the metal nanofilm is formed so as to adhere to the shape of the three-dimensional pore structure. Method for producing a surface-enhanced Raman scattering substrate.

2. The method according to claim 1, wherein the average pore size of the depressions in the three-dimensional pore structure is 0.1 to 5 μm.

3. The manufacturing method according to claim 1, wherein the average roughness of the anodic alumina substrate is 0.1 to 2 μm.

4. The method of claim 1, wherein the metal nanofilm has a thickness of 6 to 30 nm.

5. The method according to claim 1, wherein the nanopores have an average pore spacing of 10 to 300 nm.

6. The surface-enhanced Raman scattering substrate has a detection limit of at least 0.05 ppm for melamine, and a detection limit of at least 1×10 for methylene blue. －１０ The method according to any one of claims 1 to 4, wherein M is M.

7. (A) a first electrochemical treatment step in which an aluminum foil is electrochemically treated with a direct current of 5 to 40 V in an electrolyte at 15 to 25°C to change the surface structure of the aluminum foil; (B) a second electrochemical treatment step, further subjecting the aluminum foil to electrochemical treatment using a composite pulse square wave to obtain an aluminum foil structure having an anodic alumina film; (C) a third electrochemical treatment step, in which the aluminum foil structure having the anodic alumina film is subjected to an electrochemical treatment in a 5 wt % phosphoric acid electrolyte to obtain an anodic alumina substrate; (D) a metal plating step of plating a single layer of metal on the surface of the anodic alumina substrate to form a metal nanofilm, The surface-enhanced Raman scattering substrate is a surface-enhanced Raman scattering substrate free of metal nanoparticles, the anodic alumina substrate having a three-dimensional pore structure; the metal nanofilm on the anodic alumina substrate; The three-dimensional pore structure has a structure having nano-sized pores in micron-sized or nano-sized depressions, or a structure having nano-spikes around the nano-sized pores, and the metal nanofilm is formed so as to adhere to the shape of the three-dimensional pore structure. Method for producing a surface-enhanced Raman scattering substrate.

8. (A) a first electrochemical treatment step in which an aluminum foil is electrochemically treated with a direct current of 5 to 40 V in an electrolyte at −20 to 20° C. to change the surface structure of the aluminum foil; (B) a second electrochemical treatment step, further subjecting the aluminum foil to electrochemical treatment using a composite pulse square wave to obtain an aluminum foil structure having an anodic alumina film; (C) a third electrochemical treatment step, in which the aluminum foil structure having the anodic alumina film is subjected to an electrochemical treatment in a mixed electrolyte of perchloric acid and ethanol to separate the anodic alumina film from the aluminum foil, thereby obtaining an anodic alumina substrate having nanospikes; (D) a metal plating step of plating a single layer of metal on the surface of the anodic alumina substrate to form a metal nanofilm, The surface-enhanced Raman scattering substrate is a surface-enhanced Raman scattering substrate free of metal nanoparticles, the anodic alumina substrate having a three-dimensional pore structure; the metal nanofilm on the anodic alumina substrate; The three-dimensional pore structure has a structure having nano-sized pores in micron-sized or nano-sized depressions, or a structure having nano-spikes around the nano-sized pores, and the metal nanofilm is formed so as to adhere to the shape of the three-dimensional pore structure. Method for producing a surface-enhanced Raman scattering substrate.

9. (A) a first electrochemical treatment step in which an aluminum foil is electrochemically treated with a direct current of 5 to 40 V in an electrolyte at 15 to 25°C to change the surface structure of the aluminum foil; (B) a second electrochemical treatment step, further subjecting the aluminum foil to electrochemical treatment using a composite pulse square wave to obtain an aluminum foil structure having an anodic alumina film; (C) a step of removing the anodic alumina film, which includes placing the aluminum foil structure having the anodic alumina film in a mixed solution of phosphoric acid and chromic acid, and removing the anodic alumina film to obtain an aluminum foil having a surface structure; (D) a third electrochemical treatment step in which the aluminum foil having the surface structure is electrochemically treated with a composite pulse square wave to obtain an anodic alumina substrate; (E) a metal plating step of plating a single layer of metal on the surface of the anodic alumina substrate to form a metal nanofilm, The surface-enhanced Raman scattering substrate is a surface-enhanced Raman scattering substrate free of metal nanoparticles, the anodic alumina substrate having a three-dimensional pore structure; the metal nanofilm on the anodic alumina substrate; The three-dimensional pore structure has a structure having nano-sized pores in micron-sized or nano-sized depressions, or a structure having nano-spikes around the nano-sized pores, and the metal nanofilm is formed so as to adhere to the shape of the three-dimensional pore structure. Method for producing a surface-enhanced Raman scattering substrate.

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