Food inspection system and food inspection method

Abstract

To provide a food inspection system that comprises an extraction facility, a test solution, a laser light source, a Raman spectroscope, and a surface-enhanced Raman scattering substrate having no metal nanoparticles.SOLUTION: A surface-enhanced Raman scattering substrate 3 does not require the use of metal nanoparticles, and therefore has excellent stability and reproducibility compared to conventional surface-enhanced Raman scattering substrates. By using an anodic alumina substrate with a two-dimensional and three-dimensional pore structure, the surface-enhanced Raman scattering substrate has high sensitivity, high stability and high reproducibility, so that it is possible to reduce a food inspection time and save a manufacturing cost, and a food inspection method using a low-cost, rapid, stable and reliable food inspection system is also provided.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a food inspection system and a food inspection method, and more particularly to a food inspection system and a food inspection method including a surface-enhanced Raman scattering substrate, the surface-enhanced Raman scattering substrate including an anodic alumina substrate having a two-dimensional and three-dimensional hole structure, and a metal nanofilm on the anodic alumina substrate. [Background technology]

[0002] As interest in food safety grows, the demand for food safety testing is increasing year by year. However, the current food safety testing market only offers complex and time-consuming testing methods that require expensive traditional mass spectrometers and column cleaning such as liquid chromatography, as well as training for related technicians. As a result, the demand in the food safety testing market is not being met as testing methods that are lower cost, faster, more stable and more reliable have yet to be widely adopted.

[0003] Surface Enhanced Raman Scattering (SERS) has been widely studied in recent years. SERS has characteristics such as high sensitivity, non-destructiveness, and the ability to directly test liquid samples, making it a highly potential analytical and testing tool that is used in agricultural research, food safety, environmental testing, molecular biology testing, and medical health care.

[0004] In conventional SERS technology, silver or gold particles are used as a method of Raman enhancement, but metal nanoparticles are difficult to control because their shape and pitch are easily changed when irradiated with a laser, so conventional SERS substrates have poor reproducibility and uniformity.

[0005] Furthermore, currently, SERS testing technology for pesticides used in agricultural crops has been developed, for example, Taiwan Utility Model Publication No. M619099 discloses a pesticide detection device for agricultural crops, including a SERS substrate on which a metal nanolayer is deposited for adhering pesticides to agricultural crops, and a SERS fixing part for fixing the SERS substrate. However, there is still no SERS-related technology for more complex testing such as food. This is because the structure of the substrate in the prior art is limited to pesticide detection and cannot be applied to various food testing.

[0006] Taiwan Patent Publication No. I604187 discloses a surface-enhanced Raman spectroscopy inspection method including the steps of dropping an extracted and purified sample liquid onto a SERS substrate, adsorbing chemical molecules of the sample liquid onto the SERS substrate, dropping a highly volatile organic solvent onto the SERS substrate onto which the chemical molecules have been adsorbed, dissolving the chemical molecules in the organic solvent and floating up on the surface of the SERS substrate, irradiating the SERS substrate with light to volatilize the organic solvent on the SERS substrate and concentrating the remaining chemical molecules in a concentrated region, and irradiating the concentrated region with a laser to deeply adsorb the chemical molecules in the concentrated region onto the SERS substrate to form a solid-phase spectrum measurement region. However, this technique requires multiple concentration steps using an organic solvent or laser to enhance the SERS signal, which makes the inspection process time-consuming and highly contaminating. In addition, this technique requires multiple concentration steps, which results in a large error in the inspection results, and is highly likely to result in false negatives or false positives, making it unsuitable for highly complex or highly accurate inspections.

[0007] Anodic Aluminum Oxide (AAO) is a porous nanomaterial. Compared with using silicon, glass, or polymeric materials as substrates, AAO eliminates the need for complex and costly lithography and etching processes to fabricate SERS substrates.

[0008] According to Taiwan Patent Publication No. I731600, a SERS substrate made using an AAO substrate can improve the SERS detection ability because the electric field is enhanced along the nanopores of the AAO. However, although the SERS substrate with a two-dimensional AAO substrate has an enhancing effect, the detection ability is still insufficient for testing of extremely low concentrations of test objects, and there are still problems to be solved before it can be applied to highly complex and highly accurate tests such as food testing, and there has been no relevant technological development yet.

[0009] Therefore, in order to meet the ever-increasing demand for food safety testing, the current industry requires food testing systems and methods that have simple processes, are low-cost, are rapid, and can detect even extremely low concentrations of substances (i.e., have high detection capabilities). [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Taiwan Patent Publication No. I604187 [Patent Document 2] Taiwan Patent Publication No. I731600 Summary of the Invention [Problem to be solved by the invention]

[0011] In view of the above, the present invention aims to provide a food inspection system that can provide a food inspection method that has a simple process, is low-cost, is rapid, and can detect low-concentration substances. In addition, the food inspection system is not limited to pesticide detection. The SERS substrate used in the process is an AAO substrate with two-dimensional and three-dimensional hole structures, so that even extremely low-concentration substances can be detected with high reproducibility and uniformity. [Means for solving the problem]

[0012] The present invention provides a food inspection system that can realize food inspection that is simple, low-cost, rapid, and has a low detection limit. Furthermore, the food inspection system of the present invention can be widely applied to various food safety inspections, not limited to pesticide detection.

[0013] The food inspection system of the present invention includes an extraction device, a test liquid, a laser light source, a Raman spectrometer, and a SERS substrate without metal nanoparticles.

[0014] FIG. 1 is a schematic diagram of the food inspection system of the present invention. Using an extraction device (not shown), the extract extracted with an extractant is filtered to obtain a test liquid 2. A preferably enhancing liquid can be added to the test liquid 2. Next, the test liquid 2 is dropped onto a SERS substrate 3, irradiated with a laser light source 1, and then measured using a Raman spectrometer 4, and finally the results are analyzed. The surface morphology of the SERS substrate 3 is a structure having a two-dimensional plane and a three-dimensional depression, as shown in the side schematic diagram of the surface morphology circled in the lower right of FIG. 1, and does not contain any metal nanoparticles.

[0015] The SERS substrate of the food inspection system of the present invention uses a metal nanofilm instead of metal nanoparticles, thereby avoiding changes in the shape and pitch of the metal nanoparticles due to laser irradiation and improving the reproducibility and uniformity of the SERS substrate.

[0016] In addition, the SERS substrate of the food inspection system of the present invention is composed of an AAO substrate having a two-dimensional and three-dimensional pore structure, or a structure having a nano-tip around a nano-pore (not shown), as shown in the side schematic diagram of the surface morphology circled in the lower right of Figure 1. When a laser is irradiated onto the nano-pore or the area around the nano-pore covered with a metal nanofilm, surface plasmon resonance occurs, improving the sensitivity to the detected substance and enabling SERS measurement using the AAO substrate. This results in a SERS substrate with extremely high detection ability.

[0017] Furthermore, by modifying the nanopores or the morphology of the nanopores, for example by enlarging the pore size to change the pore spacing, or by creating nanotips around the nanopores through special electrochemical processing steps, the magnitude of the Raman signal can be amplified and low concentrations of substances can be detected.

[0018] The present invention produces an AAO substrate free of metal nanoparticles having two-dimensional and three-dimensional hole structures by a production method having a special electrochemical treatment process. This improves the detection intensity of the SERS substrate. Specifically, the present invention changes the surface structure of the aluminum foil by subjecting the aluminum foil to electrochemical treatment in an electrolyte at a specific temperature range, thereby forming a three-dimensional hole structure. The specific temperature range is 15 to 25°C, preferably 15 to 20°C. In addition, the present invention produces an AAO substrate having a three-dimensional hole structure with nano-level tips by a special electrochemical treatment process. This improves the three-dimensionality of the three-dimensional hole structure of the AAO substrate, thereby enhancing the Raman signal.

[0019] Specifically, one aspect of the present invention is The present invention includes an extraction device, a test liquid, a laser light source, a Raman spectrometer, and a SERS substrate without metal nanoparticles; The SERS substrate without metal nanoparticles includes an AAO substrate having two-dimensional and three-dimensional pore structures, and a metal nanofilm that is attached to conform to the shape of the two-dimensional and three-dimensional pore structures, and the three-dimensional pore structure is a structure having nano-sized pores in micron- or nano-sized depressions, or a structure having nano-sized tips around the nano-sized pores; A food inspection system. The food inspection system of the present invention irradiates the test liquid dropped onto a SERS substrate not containing metal nanoparticles with the laser light source, and measures the Raman spectrum of the test liquid irradiated with the laser light source using a Raman spectrometer.

[0020] In some embodiments, the average pore size of the depressions in the three-dimensional hole structure is 0.1 to 5 μm, preferably 0.3 to 2 μm, and more preferably 0.5 to 1.5 μm, which can cause multiple reflections and enhance the Raman signal by inelastically colliding more photons with the probe molecules on the SERS substrate.

[0021] In some embodiments, the average roughness of the AAO substrate is 0.1-2 μm, preferably 0.4-1 μm, and more preferably 0.8-1 μm. Compared to a simple two-dimensional substrate that reflects most of the incident light, the roughness of the AAO substrate can increase the inelastic collisions between photons and the tip molecules on the SERS substrate, thereby enhancing the Raman signal.

[0022] In some embodiments, the wavelength of the laser light source is 400 to 850 nm, preferably 500 to 800 nm, and more preferably 532 to 785 nm, which makes it easier to check the measurement results.

[0023] In some embodiments, the average pore spacing of the nanopores is 10 to 300 nm, preferably 10 to 100 nm. The pore size enlargement treatment reduces the average pore spacing of the nanopores, improving the spatiality of the three-dimensional pore structure in the AAO substrate, thereby enhancing the Raman signal.

[0024] In some embodiments, the test liquid further comprises an enhancing liquid, the enhancing liquid being at least one selected from the group consisting of aqueous solutions of sodium chloride, lithium chloride, magnesium chloride, and ammonium chloride.

[0025] In some embodiments, the food inspection system of the present invention further includes an extractant, which is at least one selected from the group consisting of pure water, acetone, methanol, acetonitrile, n-hexane, and an acetonitrile solution containing acetic acid, thereby improving the intensity of the measured signal.

[0026] In some embodiments, the detection limit of the SERS substrate for β agonists is 10 ppb, and the residue standard recommended by Taiwanese law is 10 ppb or less, so it can be applied to detect residues of β agonists.

[0027] Further, another aspect of the present invention is a food inspection method shown in the flowchart of FIG. An extraction step (S1) of crushing a food product and extracting the food product using an extractant to obtain an extract; an enhancement step (S2) of filtering the extract and mixing it with an enhancement solution to obtain a test solution; A dropping step (S3) of dropping the test liquid onto a surface-enhanced Raman scattering substrate without metal nanoparticles; A laser irradiation step (S4) of irradiating the test solution on the surface-enhanced Raman scattering substrate without the metal nanoparticles with a laser; A result analysis step (S5) of analyzing the measurement results of the Raman spectrometer; The surface-enhanced Raman scattering substrate without metal nanoparticles includes an anodic alumina substrate having a two-dimensional and three-dimensional pore structure, and a metal nanofilm attached to the two-dimensional and three-dimensional pore structure in accordance with the shape of the two-dimensional and three-dimensional pore structure, and the three-dimensional pore structure is a structure having nano-sized pores in micron- or nano-sized depressions, or a structure having nano-sized tips around the nano-sized pores; A food inspection method. Conventional methods for enhancing SERS signals by repeatedly concentrating using organic solvents or lasers take a long time to test and are highly polluting, whereas the food testing method of the present invention enhances signals by mixing with an enhancing liquid, eliminating the need for multiple laser irradiations for concentration, significantly shortening detection time, improving accuracy, and eliminating contamination.

[0028] In some embodiments, the enhancing liquid is at least one selected from the group consisting of aqueous solutions of sodium chloride, lithium chloride, magnesium chloride, and ammonium chloride.

[0029] In some embodiments, the extractant is at least one selected from the group consisting of pure water, acetone, methanol, acetonitrile, n-hexane, and acetonitrile solution containing acetic acid, which can improve the signal enhancement effect and make the detection limit and detection effect more prominent.

[0030] Another aspect of the present invention is to use a SERS substrate without metal nanoparticles for food inspection. By using a metal nanofilm instead of metal nanoparticles, changes in the shape and pitch of metal nanoparticles due to laser irradiation can be avoided, and the reproducibility and uniformity of the SERS substrate of the present invention can be improved. Furthermore, since the SERS substrate of the present invention is an AAO substrate having two-dimensional and three-dimensional hole structures, when laser irradiation is performed around the nano-level holes covered with a metal nanofilm, surface plasmon resonance occurs, and at the same time, by changing the shape of the nano-level holes, for example by changing the hole spacing or shape, or by generating nano-level tips around the nano-level holes, the intensity of the Raman signal can be improved and low-concentration substances can be detected. As a result, the SERS substrate without metal nanoparticles of the present invention can be applied to food inspection. Effect of the Invention

[0031] The food inspection system and method of the present invention uses a SERS substrate without metal nanoparticles, which avoids changes in the shape and pitch of metal nanoparticles caused by laser irradiation, and improves the stability, reproducibility and uniformity of the SERS substrate, thereby improving the stability of the inspection system.

[0032] In order to solve the problems of the conventional SERS measurement substrate using metal nanoparticles, such as poor stability and uniformity, and the poor sensitivity of the two-dimensional AAO substrate, the food inspection system and method of the present invention use a SERS substrate with two-dimensional and three-dimensional hole structure, so that when laser is irradiated around the nano-holes covered with a metal nanofilm, surface plasmon resonance occurs, and at the same time, the morphology of the nano-holes can be changed, for example, by changing the hole spacing or shape, or by generating nano-tips around the nano-holes, and the intensity of the Raman signal can be improved, thereby shortening the detection time and improving the detection accuracy.

[0033] The food inspection system and method of the present invention can be produced quickly and at low cost by using a SERS substrate instead of a conventional method using a mass spectrometer, etc. In addition, since the AAO substrate can be disposed of immediately after inspection without being reused, the simplification of the process is extremely advantageous for mass inspection or an increase in the number of samples.

[0034] The food inspection system and method of the present invention uses a SERS substrate and has a lower detection limit, so that it can be used for various food inspections with high complexity.

[0035] In addition, because the food inspection system and method of the present invention contain an enhancing liquid in the test liquid, it is possible to omit the extraction and concentration process that uses organic solvents and lasers multiple times, which simplifies the process, increases accuracy, and prevents food from being contaminated during inspection. [Brief description of the drawings]

[0036] [Figure 1] 1 is a schematic diagram of an inspection system of the present invention. [Diagram 2] 1 is a flowchart of an inspection method of the present invention. [Diagram 3] shows the SERS spectrum of a β-agonist. [Figure 4] is the SERS spectrum of the plasticizer bisphenol A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The contents of the present invention will be described below through the embodiments. The embodiments of the present invention are for the purpose of enumeration, and do not limit the present invention to all specific environments, applications, or special methods described in the embodiments of the present invention. Therefore, the description of the embodiments is only for the purpose of explaining the object of the present invention, and does not limit the present invention.

[0038] Example 1: [Fabrication of surface-enhanced Raman scattering substrates without metal nanoparticles]

[0039] 1. Cleaning process: The aluminum foil was ultrasonically cleaned in acetone and deionized water for 5 min to remove surface particles and oily dirt, and the surface of the aluminum substrate was rinsed with deionized water and dried with nitrogen gas.

[0040] 2. First electrochemical treatment process: The aluminum foil was subjected to electrochemical treatment for 5 minutes in a mixed solution of perchloric acid and ethanol (4:1 to 1:4) at a temperature of 15 to 25°C with a direct current of 5 to 40 V to obtain an aluminum foil with a three-dimensional surface structure.

[0041] 3. Second electrochemical treatment process: The aluminum foil was subjected to the second electrochemical treatment with a composite pulse square wave at 0-40°C to obtain a porous AAO film. The positive value of the pulse amplitude was 100-200V, and the negative value was -2--8V. The time ratio of the positive and negative pulses was 1:1-1:4. The electrolyte was 5wt% phosphoric acid, and the electrochemical treatment time was 180s.

[0042] 4. The third electrochemical treatment process: Next, the composite pulse square wave was stopped, and the reaction was continued in the electrolyte for 0 to 60 min to obtain the AAO substrate.

[0043] 5. Metal plating process: 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.

[0044] In this example, the first electrochemical treatment step was performed at 15 to 25°C to form a three-dimensional pore structure having nano-sized pores in micron-sized or nano-sized depressions. In addition, the pore size enlargement treatment performed in the third electrochemical treatment step allowed the morphology of the AAO substrate with a three-dimensional pore structure to be further adjusted. The pore size enlargement treatment enlarges the size of the nano-sized pores in the three-dimensional pore structure and reduces the distance between the nano-sized pores. This improves 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 enlargement treatment was measured using software called Image J. The pore spacing is defined as the linear distance from the outermost periphery of a nanopore to the outermost periphery of another nanopore. As a result of the measurement, the average pore spacing was 10 to 100 nm.

[0045] Example 2: [Fabrication of surface-enhanced Raman scattering substrates without metal nanoparticles with tips]

[0046] 1. Cleaning process: The aluminum foil was ultrasonically cleaned in acetone and deionized water for 5 min to remove surface particles and oily dirt, and the surface of the aluminum substrate was rinsed with deionized water and dried with nitrogen gas.

[0047] 2. First electrochemical treatment process: The aluminum foil was subjected to electrochemical treatment for 5 minutes in a mixed solution of perchloric acid and ethanol (4:1 to 1:4) at a temperature of -20 to 20°C with a direct current of 5 to 40 V to obtain aluminum foil with an average roughness (Ra) of 0.05 to 1 μm.

[0048] 3. Second electrochemical treatment process: The aluminum foil was subjected to a second electrochemical treatment with a composite pulse square wave at 0-40°C to obtain a porous AAO film. The positive value of the pulse amplitude was 100-150V, and the negative value was -2-8V. The time ratio of the positive and negative pulses was 1:1-1:4. The electrolyte was 0.1-3M oxalic acid, and the electrochemical treatment time was 5 minutes. Thus, an aluminum foil structure with an AAO film was obtained.

[0049] 4. The 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 subjected to a third electrochemical treatment with a direct current of 120 to 180 V for 30 s to separate the aluminum foil and the AAO film, thereby obtaining an AAO substrate with a three-dimensional tip.

[0050] 5. Metal plating process: A metal film with a thickness of approximately 6–30 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.

[0051] In this example, the AAO substrate with a three-dimensional nano-sized hole structure was obtained through the third electrochemical treatment process, which improved the three-dimensionality of the three-dimensional hole structure in the AAO substrate, thereby improving the detection intensity of the SERS substrate.

[0052] Example 3: [Detection of beta agonists in pork] First, pork was boiled and crushed, and extracted using pure water as an extractant according to the standard method listed in "Health and Food No. 1071900960" to obtain an extract. A 0.1wt% sodium chloride augmentation solution was prepared with pure water, and 10ppb, 100ppb, and 1000ppb of beta agonist were added to the pure water to prepare three kinds of beta agonist solutions. Next, the extract, the β-agonist solution, and the enhancement solution were mixed in a volume ratio of 1:1:2 to prepare three test solutions based on the three types of β-agonist solutions. 2 μL of the test solution was dropped onto the SERS substrate without metal nanoparticles prepared in Example 1 and dried. The substrate was irradiated with a laser with a wavelength of 532 nm and measured using a Raman spectrometer.

[0053] The measured Raman spectrum is shown in Figure 3. The detection limits of β-agonists measured by the testing method and testing system of the present invention can detect added β-agonists up to 10 ppb, 100 ppb, and 1000 ppb, which meet the residual standards set by Taiwanese law.

[0054] Example 4: [Detection of bisphenol A, a plasticizer]

[0055] 1. When sodium chloride is added as a strengthening solution First, the plastic food container was heated and bisphenol A was eluted using acetonitrile. The bisphenol A extract was extracted using acetonitrile and n-hexane as the extractant. The extract was then filtered through filter paper to remove plastic residues, and mixed with an enhancement solution of 1.5 wt% sodium chloride to obtain a test solution. The test solution was dropped onto the SERS substrate without metal nanoparticles produced in Example 1, irradiated with a laser with a wavelength of 532 nm, the Raman spectrum was read, and the measurement results were analyzed.

[0056] 2. When lithium chloride is added as an enhancing solution First, the plastic food container was heated and bisphenol A was eluted using acetonitrile. The bisphenol A extract was extracted using acetonitrile and n-hexane as the extractant. The extract was then filtered through filter paper to remove plastic residues, and mixed with an enhancement solution of 1.5 wt% lithium chloride to obtain a test solution. The test solution was dropped onto the SERS substrate without metal nanoparticles produced in Example 1, irradiated with a laser with a wavelength of 532 nm, the Raman spectrum was read, and the measurement results were analyzed.

[0057] 3. When no enhancing solution is added The other steps were repeated except for the step of mixing with the enhancing solution of sodium chloride in "1. Adding sodium chloride as the enhancing solution."

[0058] The three types of detection results for the plasticizer bisphenol A are shown in Figure 4. Comparing the three types of detection results, all of the detection results when sodium chloride or lithium chloride was added as the enhancing liquid showed that the enhancing liquid contributed to improving the detection sensitivity. Furthermore, the detection results showed that better results could be obtained by using lithium chloride as the enhancing liquid. Therefore, the inspection system and inspection method of the present invention can effectively detect the plasticizer bisphenol A even when different enhancing liquids are used, and can be applied to food safety inspection. [Explanation of symbols]

[0059] 1: Laser light source 2: Test liquid 3: Surface-enhanced Raman scattering substrates without metal nanoparticles 4: Raman spectrometer

Claims

1. The present invention includes an extraction device, a test liquid, a laser light source, a Raman spectrometer, and a surface-enhanced Raman scattering substrate free of metal nanoparticles; The surface-enhanced Raman scattering substrate free of metal nanoparticles includes an anodic alumina substrate having a two-dimensional and three-dimensional pore structure, and a metal nanofilm attached to the two-dimensional and three-dimensional pore structure in accordance with the shape of the two-dimensional and three-dimensional pore structure, the three-dimensional pore structure being a structure having nano-sized pores in micron- or nano-sized depressions, or a structure having nano-sized tips around the nano-sized pores; The laser light source is irradiated to the test liquid dropped on a surface-enhanced Raman scattering substrate not containing the metal nanoparticles, and the Raman spectrometer is used to measure the Raman spectrum of the test liquid irradiated with the laser light source. Food inspection system.

2. 2. The food inspection system of claim 1, wherein the test liquid further contains an enhancing liquid, and the enhancing liquid is at least one selected from the group consisting of aqueous solutions of sodium chloride, lithium chloride, magnesium chloride, and ammonium chloride.

3. 2. The food inspection system of claim 1, further comprising an extractant, the extractant being at least one selected from the group consisting of pure water, acetone, methanol, acetonitrile, n-hexane, and an acetonitrile solution containing acetic acid.

4. 2. The food inspection system of claim 1, wherein the wavelength of the laser light source is 532 to 785 nm.

5. The food inspection system of claim 1 , wherein the average pore diameter of the depressions in the three-dimensional pore structure is 0.1 to 5 μm.

6. The food inspection system of claim 1 , wherein the average roughness of the anodic alumina substrate is 0.1 to 2 μm.

7. The food inspection system according to claim 1 , wherein the average pore spacing of the nano-sized pores is 10 to 300 nm.

8. An extraction step (S1) of crushing a food product and extracting the food product using an extractant to obtain an extract; an enhancement step (S2) of filtering the extract and mixing it with an enhancement solution to obtain a test solution; A dropping step (S3) of dropping the test liquid onto a surface-enhanced Raman scattering substrate not containing metal nanoparticles; A laser irradiation step (S4) of irradiating the test solution on the surface-enhanced Raman scattering substrate without the metal nanoparticles with a laser; A result analysis step (S5) of analyzing the measurement results of the Raman spectrometer; The surface-enhanced Raman scattering substrate without metal nanoparticles includes an anodic alumina substrate having a two-dimensional and three-dimensional pore structure, and a metal nanofilm attached to the two-dimensional and three-dimensional pore structure in accordance with the shape of the two-dimensional and three-dimensional pore structure, and the three-dimensional pore structure is a structure having nano-sized pores in micron- or nano-sized depressions, or a structure having nano-sized tips around the nano-sized pores; Food inspection methods.

9. The food inspection method according to claim 8, wherein the enhancing liquid is at least one selected from the group consisting of aqueous solutions of sodium chloride, lithium chloride, magnesium chloride, and ammonium chloride, and the extractant is at least one selected from the group consisting of pure water, acetone, methanol, acetonitrile, n-hexane, and an acetonitrile solution containing acetic acid.

10. 1. A method of using a metal nanoparticle-free surface-enhanced Raman scattering substrate in food testing, comprising: A method for using the substrate, the substrate comprising an anodic alumina substrate having a two-dimensional and three-dimensional pore structure, and a metal nanofilm attached to conform to the shape of the two-dimensional and three-dimensional pore structure, the three-dimensional pore structure being a structure having nano-sized pores in micron- or nano-sized depressions, or a structure having nano-sized tips around the nano-sized pores.

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