Surface-enhanced Raman scattering file card, its manufacturing method, and quantitative analysis method using the file card

Abstract

The present invention provides a surface-enhanced Raman scattering file card, a method for producing the same, and a method for quantitative analysis using the file card. The surface-enhanced Raman scattering file card of the present invention contains the relative scattering cross section of the "surface-enhanced Raman scattering" of a selected molecule and the relative scattering factor of the "surface-enhanced Raman scattering" of the selected molecule and a reference molecule. The surface-enhanced Raman scattering file card usable for quantitative analysis constructed according to the present invention can be used to construct a quantitative analysis database in the SERS field, and the quantitative analysis method of the present invention provides guidelines for performing quantitative analysis using the SERS file card in various cases.

Description

[Technical Field]

[0001] The present invention relates to a surface-enhanced Raman scattering file card, a method for manufacturing the same, and a quantitative analysis method using the file card, which are in the field of material analysis. [Background technology]

[0002] Surface-enhanced Raman scattering (SERS) is an inelastic scattering spectroscopy technique with ultrahigh sensitivity and fingerprint identification. Furthermore, due to the fast acquisition speed of SERS spectra, simple sample preparation, and the advancement of miniaturized detection equipment, SERS spectroscopy is expected to have a wide range of applications in fields such as environmental pollutant detection, food additive detection, pesticide residue testing, biochemical detection, pharmaceutical analysis, and health screening. Since the discovery of the SERS phenomenon in 1974, SERS spectroscopy has developed over nearly half a century. In SERS spectroscopy, SERS substrates are an important carrier that generates a large Raman scattering signal enhancement from target substances. With the advancement of research, various SERS substrates have been designed and applied for detection and analysis, including traditional noble metal SERS substrates made of gold, silver, copper, and their alloys, semiconductor SERS substrates, and flexible SERS substrates. Although semiconductor SERS substrates have also been developed as the understanding of the SERS mechanism has deepened, traditional noble metal SERS substrates remain an essential and irreplaceable enhancement carrier in SERS applications. Furthermore, the development of flexible SERS substrates and the integration of SERS spectroscopy with other technologies, such as atomic force microscopy and electrochemistry, have further expanded the scope and application of SERS spectroscopy. As a result, SERS spectroscopy has made great strides in the detection of trace substances. Although significant progress has been made in the design, manufacturing, and detection analysis of SERS substrates, improving the stability, uniformity, and reproducibility of SERS substrates, as well as reducing lot-to-lot variations, remain important research directions in this field. Because SERS technology possesses ultrahigh sensitivity, the stability, uniformity, reproducibility, lot-to-lot variations, and even slight variations in measurement conditions of SERS substrates all have a significant impact on semi-quantitative and quantitative analysis using SERS spectroscopy.

[0003] Currently, various quantitative analysis methods have been developed and established in the field of quantitative analysis using SERS spectroscopy. While linear, logarithmic, or power-law functional relationships between the absolute intensity of SERS spectra and the concentration of target substances are frequently used for quantitative analysis, these methods require high substrate uniformity, reproducibility, lot-to-lot variations, and control of measurement conditions, making them unsuitable for general use. Therefore, in order to address the adverse effects of SERS spectral intensity variations on quantitative analysis, researchers have attempted to develop novel quantitative analysis methods that combine the relative intensity information of SERS spectra with these functional relationships. Quantitative analysis using relative intensity information of SERS spectra includes methods such as modifying the surface of a SERS substrate with internal standard reference molecules, constructing a core-shell SERS substrate containing internal standard molecules in the shell layer, and correcting for SERS spectral intensity variations of target substances using the characteristic Raman scattering peaks of the internal standard reference molecules or the Raman scattering peaks of the SERS substrate substrate. While these methods have partially eliminated the impact of absolute SERS spectral intensity variations on quantitative analysis, they are still susceptible to lot-to-lot variations and have poor portability. Currently, there is no in-depth discussion on the physical essence behind the quantitative analysis method based on the relative intensity of SERS spectra, so no general semi-quantitative or quantitative analysis method has been established in the field of SERS spectrum quantitative analysis. In other analytical evaluation techniques, relative factors are commonly used as semi-quantitative or quantitative analysis methods. For example, in X-ray diffraction technology and X-ray photoelectron spectroscopy technology, the relative sensitivity factors of each phase and element can be used to quantitatively analyze the phases and surface elements of a sample.

[0004] Research has shown that, at a specific laser wavelength, given the material of the SERS substrate, the system consisting of the molecule and the SERS substrate has a stable relative SERS scattering cross section, i.e., a stable relative scattering cross section between each SERS characteristic peak within the same molecule, and a stable relative SERS scattering factor between each molecule, i.e., a stable relative scattering cross section between selected SERS characteristic peaks in each molecule. The two parameters, relative SERS scattering cross section and relative SERS scattering factor, are related to the laser wavelength and the material of the SERS substrate, not the geometric shape of the nanostructure of the SERS substrate. Furthermore, normalization of the SERS spectrum eliminates fluctuations in the absolute intensity of the SERS spectrum, and the intramolecular and intermolecular scattering cross sections are both represented by the relative SERS scattering cross section and relative SERS scattering factor. A SERS file card can be constructed using the relative SERS scattering cross section within the same molecule, the relative SERS scattering factor between each molecule, and the normalized SERS spectrum. The files contain quantitative SERS spectral information of molecules, and can be used to build a SERS file card database similar to the powder diffraction file card used in X-ray diffraction (XRD) techniques, providing a basis for quantitative and semi-quantitative analysis of SERS spectra.

[0005] However, the SERS file card database is currently in development, and there is no systematic and comprehensive guide for its application in quantitative analysis. To better utilize the functions of SERS file cards in the field of quantitative analysis and promote the development of SERS spectrum quantitative analysis technology, a quantitative analysis method using SERS file cards is needed. Summary of the Invention [Problem to be solved by the invention]

[0006] To address the above-mentioned issues, the present inventors have based their research on fundamental physics concepts and, by referring to general-purpose quantitative analysis methods in technologies such as X-ray diffraction and X-ray photoelectron spectroscopy, provided a highly versatile surface-enhanced Raman scattering file card that can be used for semi-quantitative and quantitative analysis, as well as a method for manufacturing the same. Another object of the present invention is to provide a guide for performing quantitative analysis using a SERS file card in various cases in order to expand the range of applications of SERS spectra in quantitative analysis, as well as a quantitative analysis method using the SERS file card, which can serve as a reference for developing general quantitative analysis methods and quantitative analysis software using the SERS file card. [Means for solving the problem]

[0007] In order to solve the above problem, according to one embodiment of the present invention, The relative scattering cross section of the "surface-enhanced Raman scattering" (relative SERS scattering cross section) of the selected molecule, A surface-enhanced Raman scattering file card is provided that contains the relative scattering factors (relative SERS scattering factors) of the "surface-enhanced Raman scattering" of a selected molecule and a reference molecule.

[0008] According to the surface-enhanced Raman scattering file card of the present invention, the file card includes the material of the surface-enhanced Raman scattering substrate and the measurement wavelength.

[0009] According to the surface-enhanced Raman scattering file card according to the present invention, the file card includes a selected molecule and a selected reference peak of a reference molecule.

[0010] According to the surface-enhanced Raman scattering file card described in the present invention, said file card contains normalized surface-enhanced Raman scattering spectra of selected molecules.

[0011] The present invention further provides a method for producing a surface-enhanced Raman scattering file card according to the present invention, comprising the steps of: a step of measuring the "surface-enhanced Raman scattering" spectrum of the selected molecule, selecting the characteristic peak with the strongest peak intensity in the spectrum as a reference peak, and calculating the relative values ​​of the peak intensities of the other characteristic peaks and the reference peak to obtain a relative scattering cross section of "surface-enhanced Raman scattering"; a step of measuring the "surface-enhanced Raman scattering" spectra of the selected molecule and the reference molecule, respectively, selecting the characteristic peaks with the strongest peak intensities in the spectra of the selected molecule and the reference molecule as reference peaks of the selected molecule and the reference molecule, respectively, measuring the "surface-enhanced Raman scattering" spectrum of a mixture of the selected molecule and the reference molecule, calculating the relative values ​​of the peak intensities of the reference peaks of the selected molecule and the reference molecule, and obtaining a relative scattering factor of "surface-enhanced Raman scattering"; The manufacturing method includes:

[0012] According to the manufacturing method described in the present invention, the characteristic peak with the strongest peak intensity in the spectrum is selected as a reference peak, and then the intensity value of the reference peak is set to 100, and the peak intensities of the other characteristic peaks are normalized, and the peak intensities of the other characteristic peaks after normalization are used as the relative scattering cross section of "surface-enhanced Raman scattering."

[0013] According to the manufacturing method described in the present invention, a selected molecule and a reference molecule are mixed at different molar ratios, and the "surface-enhanced Raman scattering" spectrum at each molar ratio is measured. The characteristic peaks with the strongest peak intensities in the spectra of the selected molecule and the reference molecule are selected as the reference peaks of the selected molecule and the reference molecule, respectively. The intensity ratio of the reference peaks of the selected molecule and the reference molecule is calculated, and the linear regression coefficient between the intensity ratio and the molar ratio is calculated by the least squares regression method, which is used as the relative scattering factor of the "surface-enhanced Raman scattering" between the molecules.

[0014] According to the manufacturing method described in the present invention, the intensity ratio is in the range of 0.1 to 10.

[0015] According to the manufacturing method described in the present invention, when measuring the "surface-enhanced Raman scattering" spectrum, the molecules to be measured are dropped as a solution onto the surface of the substrate, the solvent is allowed to dry naturally, the spectrum is measured, and the fluorescent background signal is subtracted to obtain the "surface-enhanced Raman scattering" spectrum.

[0016] According to the manufacturing method described in the present invention, the total concentration of the solution is 10 -8 ~10 -5 mol / L, and the average in-plane volume range of the drop amount is 0.1 to 5 μL / mm 2 is.

[0017] The present invention further comprises: 1) measuring the surface-enhanced Raman scattering spectrum (SERS spectrum) of the analysis target based on the measurement wavelength and substrate material of the surface-enhanced Raman scattering file card (SERS file card) described in the present invention; 2) selecting 1 to n characteristic peaks for molecule i and 1 to m characteristic peaks for molecule j for different molecules, and solving the following equations I: A quantitative analysis method using a SERS file card containing the above is provided.

[0018]

number

[0019] The present invention further comprises: 1) measuring the surface-enhanced Raman scattering spectrum (SERS spectrum) of the analysis target based on the measurement wavelength and substrate material of the surface-enhanced Raman scattering file card (SERS file card) described in the present invention; 2) selecting 1 to p spectral intervals of the SERS spectrum for n molecules and solving them using the following equation group II: A quantitative analysis method using a SERS file card containing the above is provided.

[0020]

number

[0021] The present invention further comprises: 1) measuring the surface-enhanced Raman scattering spectrum (SERS spectrum) of the analysis target based on the measurement wavelength and substrate material of the surface-enhanced Raman scattering file card (SERS file card) described in the present invention; 2) for n molecules, selecting a complete spectral interval of the SERS spectrum, or a partial spectral interval of the SERS spectrum as the complete spectral interval, and solving it by the following equation group III: A quantitative analysis method using a SERS file card containing the above is provided.

[0022]

number

[0023] According to the quantitative analysis method using the SERS file card of the present invention, the SERS file card has the same laser wavelength and substrate material as the SERS spectrum, and the background is subtracted from the SERS spectrum.

[0024] According to the quantitative analysis method using SERS file card of the present invention, the relative SERS scattering factor of a molecule can be obtained from the SERS file card of the molecule, or calculated by indirect transfer from the SERS file card of other molecules. The calculation of the relative SERS scattering factor of the molecule from the SERS file card of other molecules is realized by the following chain transfer formula IV:

[0025]

number

[0026] According to the quantitative analysis method using the SERS file card described in the present invention, the number of transmissions of the chain transfer formula IV is 6 or less. [Effects of the Invention]

[0027] The present invention defines and measures two physical quantities, the relative scattering cross section and relative scattering factor, of intramolecular "surface-enhanced Raman scattering." This allows each parameter to be used universally across the same type of surface-enhanced Raman scattering substrates with the same surface properties but different geometric forms. This eliminates the effects on quantitative analysis of factors such as uniformity of the surface-enhanced Raman scattering substrate, lot-to-lot differences, fluctuations in measurement conditions, and changes in geometric form. This can be used to build a quantitative analysis database in the SERS field, making SERS technology easy to use for various quantitative analyses, just as X-ray diffraction technology has a standard powder diffraction card database. This creates a surface-enhanced Raman scattering file card that can be used for quantitative analysis, which has a wide range of application prospects and plays an important fundamental role in the field of quantitative analysis of trace molecules.

[0028] The quantitative analysis method using SERS file card provided by the present invention can combine SERS file card with various algorithms in various cases to perform quantitative analysis of the content and concentration of selected molecules, which effectively expands the general application range of SERS technology in quantitative analysis of trace substances, and also simplifies the quantitative analysis work using SERS technology, and has broad application prospects and an important fundamental role in the field of quantitative analysis of trace molecules. [Brief explanation of the drawings]

[0029] The drawings, which are incorporated in and constitute a part of this specification, illustrate exemplary embodiments, configurations and aspects of the invention and together with the description, serve to explain the principles of the invention.

[0030] [Figure 1] FIG. 1 shows reflectance spectra and SEM images of a 490 nm silver nanorod structure substrate, a 700 nm silver nanorod structure substrate, and a V-shaped silver nanorod structure substrate with an arm length of 350 nm. [Figure 2] FIG. 2 shows the SERS spectrum of 4-MBA molecules on the surface of a silver nanorod structure substrate at 490 nm for each laser power and the normalized SERS spectrum from which the fluorescent background signal has been subtracted. [Figure 3]FIG. 3 shows the SERS spectra of two molecules, 4-MBA and 2-MPY, measured on the surface of a silver SERS substrate of each structure, and the normalized SERS spectra from which the fluorescent background signal has been subtracted. [Figure 4] FIG. 4 shows the SERS spectra and normalized SERS spectra from which the background has been subtracted for mixed solutions of 4-MBA and 2-MPY with molecular ratios of 9:1 and 1:9 on the surface of silver SERS substrates of each structure. [Figure 5] FIG. 5 shows the intermolecular relative SERS scattering factors of 4-MBA and 2-MPY measured by the mixed drop method. [Figure 6] FIG. 6 shows a SERS file card of the 2-MPY molecule constructed by the measured relative intramolecular scattering cross section of 2-MPY and the relative intermolecular SERS scattering factors of 2-MPY and 4-MBA. [Figure 7] FIG. 7 is a flow chart of the quantitative analysis method using the SERS file card in each case. [Figure 8] FIG. 8 is a diagram showing the quantitative analysis results of Example 1. [Figure 9] FIG. 9 shows the results of the quantitative analysis in Example 2. [Figure 10] FIG. 10 shows the results of the quantitative analysis in Example 3. [Figure 11] FIG. 11 shows the SERS spectrum obtained in Card Production Example 4, the relationship between the intensity ratio of characteristic peaks and the molecular number ratio, and the constructed SERS file card. [Figure 12] FIG. 12 shows the results of the quantitative analysis in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0031]

[0023] Exemplary embodiments, configurations, and aspects of the present invention will now be described in detail with reference to the drawings, in which like reference numerals indicate functionally identical or similar elements. While various aspects of the embodiments are shown in the drawings, the drawings are not drawn to scale unless otherwise noted.

[0032] The word "exemplary" as used herein means "serving as an example, instance, or illustration." Any embodiment described herein as "exemplary" is not to be construed as superior or preferred over other embodiments.

[0033] Furthermore, in order to better explain the present invention, numerous specific details are described in the following embodiments. It should be understood by those skilled in the art that the present invention can be practiced without certain specific details. In other instances, methods, means, elements, and circuits well known to those skilled in the art are not described in detail in order to clarify the gist of the present invention.

[0034] [Surface-enhanced Raman scattering file card and its manufacturing method] Definitions and derivations of physical quantities The SERS process includes the process of molecular adsorption onto the surface of the SERS substrate, the process of molecular Raman scattering, and the process of molecular Raman scattering enhancement by the SERS substrate. The measured SERS characteristic peak intensity (I peak,i ) is the laser power (L λ ), number of molecules (N m ), the SERS characteristic peak scattering cross section of the molecule at the surface of the substrate (σ SERS,peak,i ) and is expressed by the following formula (1-1).

[0035]

number

[0036] The same molecule usually has multiple Raman scattering characteristic peaks. In the SERS spectrum, the same molecule also has multiple SERS characteristic peaks. SERS,peak,r ) as a reference, (1-1) can be converted into the following equation (1-2),

[0037]

number

number

[0038] The inventors have

[0039]

number

[0040]

number

[0041] The relative SERS scattering cross section is mathematically equivalent to normalizing the SERS spectrum of a molecule using one selected SERS characteristic peak, which preserves the intensity relationship between each of the molecule's SERS characteristic peaks while eliminating absolute intensity fluctuations.

[0042] Based on the above definitions, the following formulas (1-5) and (1-6) hold true for two molecules, M1 and M2, respectively.

[0043]

number

[0044]

number

[0045]

number

[0046]

number

[0047]

number

[0048]

number

[0049]

number

[0050] As is clear from equation (1-10), the relative intermolecular content can be determined from the two defined physical quantities, the intramolecular relative SERS scattering cross section (RCS) and the intermolecular relative SERS scattering factor (RSF), as well as the SERS spectrum. As is clear from equation (1-11), the number of molecules M2 can be determined by combining the two defined physical quantities, RCS and RSF, with the SERS spectrum and the number of molecules M1.

[0051] As is clear from the above definitions and discussions by the inventors, the molecular content and number can be directly quantitatively analyzed by the two parameters, the relative SERS scattering cross section (RCS) and the relative SERS scattering factor (RSF) of a molecule. The following describes how to measure these two physical quantities.

[0052] Below, we will explain the definitions, measurement methods, and specific measurement processes for two physical quantities, the intramolecular relative SERS scattering cross section (RCS) and the intermolecular relative SERS scattering factor (RSF), and then discuss their properties.

[0053] Measurement process and properties of two physical quantities Regarding the relative SERS scattering cross section (RCS) between Raman vibrational modes within the same molecule, as is clear from equation (1-4), a certain SERS characteristic peak can be selected as a reference peak, and then the relative SERS scattering cross section of other SERS characteristic peaks can be calculated. (Specific procedure) A molecular solution of a certain concentration and volume is dropped onto the surface of a SERS substrate, the solvent is allowed to dry naturally, and the SERS spectrum is measured. Background signals such as fluorescence are subtracted from the measured SERS spectrum, and the strongest characteristic peak in the SERS spectrum is selected as the reference peak, and the spectrum is normalized. Here, normalization refers to setting the intensity of the strongest characteristic peak in the molecule's SERS spectrum at 100, and calculating the relative intensities of any other SERS characteristic peaks to determine the relative SERS scattering cross section of each SERS characteristic peak within the molecule.

[0054] As can be seen from equation (1-7), the relative SERS scattering factor (RSF) between molecules is calculated by selecting a molecule as a reference and calculating the intensity ratio between the reference peak of the SERS spectrum of the other molecule and the reference peak of the selected molecule. Slight changes in factors such as the stability, uniformity, reproducibility, lot-to-lot variation, and measurement conditions of the SERS substrate will result in corresponding changes in the intensity and shape of the SERS spectrum. Therefore, when measuring the relative SERS scattering factor, it is necessary to match all factors in the measurement of two molecules. To meet this requirement, a mixed solution of two molecules at a constant concentration and volume must be dropped onto the surface of the SERS substrate, ensuring that the total molecular coverage is less than one monolayer. This allows for simultaneous measurement of the mixed SERS spectrum of two molecules in the same spot, effectively avoiding the adverse effects of measurement conditions, substrate lot-to-lot variation, uniformity, and reproducibility, while also reducing mutual influence between molecules. After the dropped solvent has dried, the SERS spectrum of the mixed solution at each ratio is measured, and the ratio of the SERS reference peak intensities of the two molecules in the SERS spectrum of the mixed solution is calculated. The reference peak intensity ratio is then divided by the ratio of the number of the two molecules in the mixed solution to calculate the relative SERS scattering factor between the two molecules.

[0055] Verification of the relative scattering cross section of "surface-enhanced Raman scattering" First, we verified the effectiveness of the relative scattering cross section of "surface-enhanced Raman scattering." We provided silver SERS substrates with various structures, and their SEM images are shown in Figures 1(b) to 1(d). These are a 490 nm silver nanorod structure substrate, a 700 nm silver nanorod structure substrate, and a V-shaped silver nanorod structure substrate with an arm length of 350 nm, respectively. The reflectance spectra of the three nanostructured SERS substrates are shown in Figure 1(a). As is clear from Figure 1(a), the three structures have different optical properties, resulting in significant differences in reflectance at each wavelength, with distinct valley values ​​and valley shapes in the reflectance curves.

[0056] Using the 490 nm silver nanorod structure substrate shown in Figure 1(b), the SERS spectrum of 4-mercaptobenzoic acid (4-MBA) molecules was measured at each laser output. The SERS spectrum of 4-MBA molecules measured at each laser output is shown in Figure 2(a). The spectral intensity differs depending on the laser output. After background subtraction from the SERS spectrum measured at each laser output, normalization was performed and the peak intensity at 1074 cm was obtained. -1 The SERS characteristic peak of is selected as the reference peak, and its intensity is set to 100, resulting in the normalized SERS spectrum shown in Figure 2(b). As is clear from the figure, the normalized SERS spectra of the 4-MBA molecule at each output almost completely overlap.

[0057] Similarly, the SERS spectra of 4-MBA molecules obtained under the same measurement conditions for each of the silver SERS substrates are shown in Figure 3(a). As is clear from the figure, the enhancement effect of each SERS substrate structure is different, and there is a large difference in their intensities. After subtracting the background from the spectrum in Figure 3(a), the peak at 1074 cm -1 The SERS characteristic peak of 1002 cm was normalized using the reference peak, and the results shown in Figure 3(b) were obtained. As can be seen from the figure, the normalized SERS spectra of the 4-MBA molecule measured on the SERS substrates of each nanostructure almost completely overlapped. Similarly, for the 2-mercaptopyridine (2-MPY) molecule, Figure 3(c) shows the SERS spectrum of the 2-MPY molecule measured on the silver SERS substrates of each structure. After background subtraction, the SERS spectrum of the 2-MPY molecule measured on the silver SERS substrates of each structure was obtained. -1 The SERS characteristic peak of was used as the reference peak for normalization, and the results shown in Figure 3(d) were obtained. The normalized spectra were almost completely overlapped.

[0058] As is clear from the above discussion, the relative intramolecular SERS scattering cross section is an intrinsic characteristic parameter of the system consisting of the molecule and the SERS substrate material, and is negligible in influence of changes in the measurement laser power and changes in the geometrical form of the nanostructures in the SERS substrate made of the same material.

[0059] Verification of the relative scattering factor of "surface-enhanced Raman scattering" To determine the relative SERS scattering factor between molecules, it is necessary to measure the SERS spectrum of a mixed solution of the two molecules at various ratios. First, 4-MBA molecules and 2-MPY molecules are mixed at various ratios, dropped onto the surface of each silver SERS substrate structure, and allowed to dry naturally. After that, the mixed SERS spectrum of the two molecules at various ratios is measured. Figure 4 shows the SERS spectra of the mixed 4-MBA and 2-MPY molecules at a mixing ratio of 1:9 and 9:1. As shown in Figure 4(a), when the mixing ratio of 4-MBA molecules to 2-MPY molecules is 1:9, there is a large difference in the SERS spectral intensity measured on the silver SERS substrate of each structure. The background-subtracted SERS spectrum is measured at 1002 cm. -1 The results shown in Figure 4(b) were obtained by normalizing the SERS spectrum measured on the silver SERS substrates with the three structures. The SERS spectra were almost completely overlapped. Similarly, as shown in Figure 4(c), when the mixing ratio of 4-MBA molecules to 2-MPY molecules was 9:1, there was a large difference in the SERS spectral intensity measured on the silver SERS substrates with each structure. The background-subtracted SERS spectrum was normalized to the peak at 1074 cm. -1 The results shown in Figure 4(d) were obtained by normalizing the SERS spectrum measured with the three silver SERS substrate structures, and the SERS spectra almost completely overlapped. This indicates that when the SERS substrate material is the same, the geometric shape of the nanostructure of the SERS substrate has little effect on the measurement results of the mixed SERS spectrum. Therefore, the measured relative SERS scattering factor between molecules is an intrinsic characteristic parameter of the system consisting of the molecule and the SERS substrate.

[0060] The intramolecular relative SERS scattering cross section and intermolecular relative SERS scattering factor are intrinsic characteristic parameters of the system consisting of molecules and SERS substrates, and can be used for data file construction and quantitative analysis. As can be seen from Figures 4(c) and 4(d), when the molecular ratio of 4-MBA to 2-MPY is 9:1, the spectrum is dominated by the 4-MBA signal. In this case, small fluctuations in signal and noise have a significant impact on the measurement of the relative SERS scattering factor between the two molecules. To avoid this negative impact on the accuracy of the measurement of the relative SERS scattering factor, it is necessary to calculate the relative SERS scattering cross section between the two molecules when the signal intensity difference between the two molecules is small. To avoid this adverse effect, we first prepared five mixed solutions of 4-MBA and 2-MPY at molecular weight ratios of 1:9, 2:8, 3:7, 4:6, and 5:5. Each of these mixed solutions was dropped onto the surface of each nanostructured SERS substrate and allowed to air dry. The resulting SERS spectra were then measured, and the intensity ratios of selected reference peaks for each of the two molecules were calculated. The intensity ratios and molecular weight ratios are shown in Figure 5. Figure 5 also shows the results for the three silver SERS substrate structures. As can be seen from the figure, there was no significant difference between the three measurements. A linear fit of the measurements revealed that the slope represents the relative SERS scattering factor between the two molecules. The fitting results also revealed no significant differences between the relative SERS scattering factors of 4-MBA and 2-MPY molecules measured on the three SERS substrate structures. These relative SERS scattering factors are considered to be consistent within the experimental error range.

[0061] From the above, the intramolecular relative SERS scattering cross section and the intermolecular relative SERS scattering factor are intrinsic characteristic parameters of the system composed of molecules and SERS substrate materials, and are independent of the geometric form of the SERS substrate. They can be used as general parameters for constructing SERS profile cards and quantitative analysis.

[0062] Surface-Enhanced Raman Scattering File Card In accordance with the above considerations, the present invention provides a surface-enhanced Raman scattering file card that includes the relative scattering cross section of the "surface-enhanced Raman scattering" of a selected molecule and the relative scattering factor of the "surface-enhanced Raman scattering" of the selected molecule and a reference molecule.

[0063] According to the surface-enhanced Raman scattering file card of the present invention, the file card includes the material of the surface-enhanced Raman scattering substrate and the measurement wavelength. Since the values ​​of these two physical quantities are related to the measurement wavelength of the laser and the material of the SERS substrate, the above information should be specified in the SERS file.

[0064] According to the surface-enhanced Raman scattering file card according to the present invention, the file card includes a selected molecule and a selected reference peak of a reference molecule.

[0065] According to the surface-enhanced Raman scattering file card of the present invention, the file card contains a normalized surface-enhanced Raman scattering spectrum of a selected molecule. Because the intramolecular relative SERS scattering cross section exhibits different values ​​depending on the Raman shift position, it is necessary to provide a normalized SERS spectrum of the molecule. The normalized SERS spectrum here refers to the relative SERS spectrum when the intensity of a selected SERS reference peak is set to 100. To enable more convenient and straightforward qualitative and quantitative analysis, the main characteristic peaks, relative intensities, and corresponding Raman vibrational modes of the SERS spectrum should be listed on the SERS file card.

[0066] The present invention further provides a database comprising one or more molecular surface-enhanced Raman scattering file cards.

[0067] The constructed SERS file card enables simple quantitative analysis, and the information in the SERS file card can be used for quantitative analysis in a variety of situations. When the SERS characteristic peaks between each molecule can be easily distinguished in the SERS spectrum, the relative intensity ratio between each molecule's characteristic peak can be calculated. Then, quantitative analysis of the relative content between molecules can be achieved using the relative SERS scattering cross section and relative SERS scattering factor in the SERS file card. When the SERS characteristic peaks between each molecule cannot be easily distinguished in the SERS spectrum, the normalized SERS spectrum in the SERS file card is multiplied by the relative SERS scattering factor to obtain the relative SERS spectrum of each molecule. Then, an appropriate algorithm is used to calculate the ratio of the relative SERS spectra of each molecule contained in the SERS spectrum to be analyzed, and this ratio is used as the content ratio of each molecule. For the concentration analysis of the target molecule, first, an appropriate reference molecule is selected based on the SERS file card, then a reference molecule with a known concentration is added to the system to be analyzed, and the SERS spectrum is measured to calculate the relative content ratio of the reference molecule and the target molecule based on the two parameters in the SERS file card. Then, the concentration of the target molecule is determined based on the concentration of the added reference molecule and the calculated relative content ratio.

[0068] table Method for manufacturing surface-enhanced Raman scattering file card The present invention provides a method for producing a surface-enhanced Raman scattering file card according to the present invention, comprising the steps of: a step of measuring the "surface-enhanced Raman scattering" spectrum of the selected molecule, selecting the characteristic peak with the strongest peak intensity in the spectrum as a reference peak, and calculating the relative values ​​of the peak intensities of the other characteristic peaks and the reference peak to obtain a relative scattering cross section of "surface-enhanced Raman scattering"; a step of measuring the "surface-enhanced Raman scattering" spectra of the selected molecule and the reference molecule, respectively, selecting the characteristic peaks with the strongest peak intensities in the spectra of the selected molecule and the reference molecule as reference peaks of the selected molecule and the reference molecule, respectively, measuring the "surface-enhanced Raman scattering" spectrum of a mixture of the selected molecule and the reference molecule, calculating the relative values ​​of the peak intensities of the reference peaks of the selected molecule and the reference molecule, and obtaining a relative scattering factor of "surface-enhanced Raman scattering"; The manufacturing method is characterized by comprising the steps of:

[0069] Measurement of the relative scattering cross section of "surface-enhanced Raman scattering" According to the manufacturing method described in the present invention, the characteristic peak with the strongest peak intensity in the spectrum is selected as a reference peak, and then the intensity value of the reference peak is set to 100, and the peak intensities of the other characteristic peaks are normalized, and the peak intensities of the other characteristic peaks after normalization are used as the relative scattering cross section of "surface-enhanced Raman scattering."

[0070] Regarding the relative scattering cross section (RCS) of the SERS spectrum of a selected molecule, as is clear from formula (1-4), it is preferable to select a certain SERS characteristic peak as a reference peak and calculate the relative SERS scattering cross section of other peaks. (Specific procedure) The spectrum of the selected molecule on a SERS substrate is measured, background signals such as fluorescence are subtracted from the measured SERS spectrum, the characteristic peak with the strongest peak intensity in the SERS spectrum is selected as the reference peak, and then normalized to calculate the relative intensity of any of the other characteristic peaks, which is the relative SERS scattering cross section.

[0071] Measurement of the relative scattering factor of "surface-enhanced Raman scattering" According to the manufacturing method described in the present invention, a selected molecule and a reference molecule are mixed at different molar ratios, and the "surface-enhanced Raman scattering" spectrum at each molar ratio is measured. The characteristic peaks with the strongest peak intensities in the spectra of the selected molecule and the reference molecule are selected as the reference peaks of the selected molecule and the reference molecule, respectively. The intensity ratio of the reference peaks of the selected molecule and the reference molecule is calculated, and the linear regression coefficient between the intensity ratio and the molar ratio is calculated by the least squares regression method, which is used as the relative scattering factor of the "surface-enhanced Raman scattering" between the molecules.

[0072] According to the manufacturing method described in the present invention, the intensity ratio is in the range of 0.1 to 10.

[0073] According to the manufacturing method described in the present invention, when measuring the "surface-enhanced Raman scattering" spectrum, the molecules to be measured are dropped as a solution onto the surface of the substrate, the solvent is allowed to dry naturally, the spectrum is measured, and the fluorescent background signal is subtracted to obtain the "surface-enhanced Raman scattering" spectrum.

[0074] According to the manufacturing method described in the present invention, the total concentration of the solution is 10 -8 ~10 -5 mol / L, and the average in-plane volume range of the drop amount is 0.1 to 5 μL / mm 2 is.

[0075] [Quantitative analysis method using SERS file cards] [Quantitative analysis method using the SERS file card of the first embodiment] I i,p denotes the pth SERS characteristic peak of the ith molecule, and I j,q represents the q-th SERS characteristic peak of the j-th molecule, and RSF i,j denotes the relative SERS scattering factor of the ith molecule with respect to the jth molecule, RSC i,p denotes the relative SERS scattering cross section of the pth SERS characteristic peak of the ith molecule, RSC j,q where X denotes the relative SERS scattering cross section of the q-th SERS characteristic peak of the j-th molecule, and i is the content of molecule i, and X j is the content of molecule j. When quantitative analysis is performed using a single characteristic peak for molecule i and molecule j, the following formula (4-1-1) holds:

[0076]

number

[0077] When selecting multiple characteristic peaks for each molecule, if k characteristic peaks are selected for molecule i and m characteristic peaks are selected for molecule j, the following formula (4-1-2) is established.

[0078]

number

[0079] For n molecules, any i or j belongs to the interval 1 to n, and i is not equal to j. In either case, formula (4-1-2) holds. If the total content of all molecules is 100%, then formula (4-1-3) below holds.

[0080]

number

[0081] When a molecule j with a known concentration is selected or added as a reference, its concentration is C j Then, the concentration of any molecule i can be calculated using the following equation (4-1-4).

[0082]

number

[0083] As can be seen from the above discussion, the content and concentration of each molecule can be quantitatively analyzed using the SERS file card. There is no limit to the number of SERS characteristic peaks selected, and only two parameters, namely, the relative SERS scattering cross section and the relative SERS scattering factor in the SERS file card, are required. These two parameters can be directly obtained from the corresponding SERS file card, or can be indirectly calculated from other SERS file cards.

[0084] From the above, the present invention is 1) measuring the surface-enhanced Raman scattering spectrum (SERS spectrum) of the object to be analyzed based on the wavelength and substrate material of the surface-enhanced Raman scattering file card (SERS file card) according to the present invention; 2) selecting 1 to n characteristic peaks for molecule i and 1 to m characteristic peaks for molecule j for different molecules, and solving the following equations I: A quantitative analysis method using a SERS file card containing the above is provided.

[0085]

number

[0086] For different molecules, choosing one characteristic peak for molecule i and one characteristic peak for molecule j, the formula group I becomes:

[0087]

number

[0088] When the SERS characteristic peaks between molecules can be easily distinguished in the SERS spectrum, it is preferable to carry out quantitative analysis by the method of the first embodiment.

[0089] [Quantitative analysis method using the SERS file card of the second embodiment] The present invention provides 1) measuring the surface-enhanced Raman scattering spectrum (SERS spectrum) of the object to be analyzed based on the wavelength and substrate material of the surface-enhanced Raman scattering file card (SERS file card) according to the present invention; 2) selecting 1 to p spectral intervals of the SERS spectrum for n molecules and solving them using the following equation group II: A quantitative analysis method using a SERS file card containing the above is provided.

[0090]

number

[0091] Specifically, S range,1-p represents the first to pth sections selected in the SERS spectrum, and RSF i,j denotes the relative SERS scattering factor of the i-th molecule with respect to the j-th molecule, and Snorm i,1-p Let x denote the corresponding 1st to pth intervals in the normalized SERS spectrum of the i-th molecule, and x i is the content of molecule i, and we use the principal component analysis (PCA) algorithm.

[0092] The PCA algorithm can remove system noise and express spatial positional relationships with a small number of principal components. For the normalized SERS spectrum in the SERS file card, first calculate the total integrated area value of the selected section 1 to p, and then use Anorm i,1-p represents the integrated area values ​​of the 1st to pth sections in the normalized SERS spectrum of the i-th molecule. For n molecules, first, the SERS spectrum of the selected section is divided by the total integrated area of ​​the selected section using equation (4-2-1) to obtain the integrated area-normalized SERS spectrum Snorm A,i,1-p Calculate.

[0093]

number

[0094]

number

[0095]

number

[0096]

number

[0097]

number

[0098]

number

[0099]

number

[0100]

number

[0101]

number

[0102]

number

[0103]

number

[0104]

number

[0105] As can be seen from the above discussion, when it is difficult to distinguish between the characteristic peaks of two or more molecules in the SERS spectrum, it is possible to quantitatively analyze the content and concentration of each molecule by selecting a specific SERS characteristic peak section and combining it with a SERS file card. There is no limit to the number of SERS characteristic peak sections selected, and for the SERS file card, two parameters, the relative SERS scattering factor and the normalized SERS spectrum, must be used. These two parameters can be obtained directly from the corresponding SERS file card or can be calculated by indirect transfer from other SERS file cards.

[0106] [Quantitative analysis method using the SERS file card of the third embodiment] The present invention provides 1) measuring the surface-enhanced Raman scattering spectrum (SERS spectrum) of the object to be analyzed based on the wavelength and substrate material of the surface-enhanced Raman scattering file card (SERS file card) according to the present invention; 2) for n molecules, selecting a complete spectral interval of the SERS spectrum, or a partial spectral interval of the SERS spectrum as the complete spectral interval, and solving it by the following equation group III: A quantitative analysis method using a SERS file card containing the above is provided.

[0107]

number

[0108] Spec mix represents the SERS spectrum to be analyzed, and Spec i denotes the normalized SERS spectrum in the SERS file card of the ith molecule, RSF i,j denotes the relative SERS scattering factor of the ith molecule with respect to the jth molecule, and X i is the content of molecule i, and quantitative analysis is performed by combining the multiple linear regression algorithm with the SERS file card.

[0109] In multiple linear regression algorithms, the estimates of the regression coefficients become more robust as the number of observations increases. In a SERS spectrum, each Raman shift corresponds to one observation, so using the entire SERS spectrum for multiple linear regression is more robust. Spec mix When the intermolecular interactions are not significant, the following relational expression (4-3-1) holds true:

[0110]

number

[0111]

number

[0112]

number

[0113]

number

[0114] As can be seen from the above discussion, when the characteristic peaks of each molecule in the SERS spectrum are not easily distinguishable and are dispersed, it is preferable to combine the measured SERS full spectrum with the SERS file card to quantitatively analyze the content and concentration of each molecule. For the SERS file card, two parameters, namely, the relative SERS scattering factor and the normalized SERS spectrum, need to be used. Similarly, these two parameters can be obtained directly from the corresponding SERS file card, or can be calculated indirectly from other SERS file cards.

[0115] According to the method of the present invention, the SERS file card has the same laser wavelength and substrate material as the SERS spectrum, and the background is subtracted from the SERS spectrum.

[0116] According to the method of the present invention, the relative SERS scattering factor of a molecule can be obtained from the SERS file card of the molecule, or calculated by indirect transfer from the SERS file cards of other molecules, and the calculation of the relative SERS scattering factor of the molecule from the SERS file cards of other molecules can be realized by the following chain transfer formula IV:

[0117]

number

[0118] According to the method of the present invention, the number of transfers of the chain transfer formula IV is 6 or less.

[0119] Card manufacturing example Card manufacturing example 1 Step 1. A 490 nm silver nanorod structure substrate with a purity of 99.99% was produced by electron beam oblique deposition.

[0120] Step 2. Volume 10 μL, concentration 10 -5A 4-mercaptobenzoic acid (4-MBA) molecular solution and a 2-mercaptopyridine (2-MPY) molecular solution were each dropped onto the surface of a 10 mm × 10 mm silver nanorod structure substrate with a diameter of 490 nm. After air drying, the SERS spectrum was measured using a 785 nm laser, and the fluorescent background signal was subtracted from the SERS spectrum.

[0121] Step 3. For the SERS spectrum from which the fluorescent background signal was subtracted in step 2, the 1074 cm of the SERS spectrum of the 4-MBA molecule -1 and 1002 cm in the SERS spectrum of 2-MPY molecules. -1 The characteristic peak of was selected as a reference peak, and its intensity was set to 100. The SERS spectra of the two molecules were normalized to obtain the relative SERS scattering cross section at each Raman shift.

[0122] Step 4. The molecular ratio of the mixed solution is 1:9, 2:8, 3:7, 4:6, 5:5, and the total concentration of the mixed solution is 10 -5 4-MBA molecules and 2-MPY molecules were mixed to obtain M, and 10 μL of each mixed solution was dropped onto the surface of a 5 mm × 5 mm silver nanorod structure substrate with a diameter of 490 nm. After air drying, the SERS spectrum was measured and the fluorescent background signal was subtracted.

[0123] Step 5. The 1074 cm of the 4-MBA molecule in the SERS spectrum from which the fluorescent background signal was subtracted in Step 4. -1 and the characteristic peak of 2-MPY molecule at 1002 cm -1 The intensity ratio of the characteristic peaks of the two compounds was calculated, and the linear regression coefficient of the intensity ratio of the two compounds and the corresponding molar ratio in the mixed solution was calculated by the least squares regression method, which was used as the relative SERS scattering factor between the two compounds.

[0124] Step 6. The substrate material in step 1, the measured wavelength of the laser in step 2, the information of the reference peak in step 3, the normalized SERS spectrum in step 3, the relative SERS scattering cross section in step 3, the relative SERS scattering factor in step 5, and the Raman vibration mode of each SERS characteristic peak were combined as shown in Figure 6 to construct a SERS file card between 2-MPY molecules and 4-MBA molecules.

[0125] The SEM photograph of the 490 nm nanorod SERS substrate used is shown in Figure 1(b), its reflectance spectrum is shown in Figure 1(a), the measured normalized SERS spectra of 4-MBA and 2-MPY molecules are shown in Figures 3(b) and 3(d), and the calculated relative SERS scattering factor of 4-MBA molecules to 2-MPY molecules is 6.7 ± 1.1, shown in Figure 5.

[0126] The relative SERS scattering cross section and relative SERS scattering factor obtained from the above study and measurement can be used to construct the SERS file card shown in Figure 6. This figure is merely an example, and from top to bottom, the SERS file card number, the material and measurement wavelength of the SERS substrate used, the name of the molecule, and the SERS reference peak of the selected molecule are listed. The bottom of the SERS file card lists the relative SERS scattering factor and relative SERS scattering cross section, two parameters that can be used for quantitative analysis, and the middle section lists the normalized SERS spectrum and structural formula of the molecule. To facilitate simpler and more straightforward qualitative and quantitative analysis, the table at the bottom of the SERS file card lists the main SERS characteristic peaks of the 2-MPY molecule, their relative intensities, and the corresponding Raman vibrational modes.

[0127] Card manufacturing example 2 Step 1. A 700 nm silver nanorod structure substrate with a purity of 99.99% was produced by electron beam oblique deposition.

[0128] Step 2. Volume 10 μL, concentration 10 -6A 4-mercaptobenzoic acid (4-MBA) molecular solution and a 2-mercaptopyridine (2-MPY) molecular solution were each dropped onto the surface of a 5 mm × 5 mm silver nanorod structure substrate with a diameter of 700 nm. After air drying, the SERS spectrum was measured using a 785 nm laser, and the fluorescent background signal was subtracted from the SERS spectrum.

[0129] Step 3. For the SERS spectrum from which the fluorescent background signal was subtracted in step 2, the 1074 cm of the SERS spectrum of the 4-MBA molecule -1 and 1002 cm in the SERS spectrum of 2-MPY molecules. -1 The characteristic peak of was selected as a reference peak, and its intensity was set to 100. The SERS spectra of the two molecules were normalized to obtain the relative SERS scattering cross section at each Raman shift.

[0130] Step 4. The molecular ratio of the mixed solution is 1:9, 2:8, 3:7, 4:6, 5:5, and the total concentration of the mixed solution is 10 -6 4-MBA molecules and 2-MPY molecules were mixed to obtain M, and 15 μL of each mixed solution was dropped onto the surface of a 5 mm × 5 mm silver nanorod structure substrate with a diameter of 700 nm. After air drying, the SERS spectrum was measured and the fluorescent background signal was subtracted.

[0131] Step 5. The 1074 cm of the 4-MBA molecule in the SERS spectrum from which the fluorescent background signal was subtracted in Step 4. -1 and the characteristic peak of 2-MPY molecule at 1002 cm -1 The intensity ratio of the characteristic peaks of the two compounds was calculated, and the linear regression coefficient of the intensity ratio of the two compounds and the corresponding molar ratio in the mixed solution was calculated by the least squares regression method, which was used as the relative SERS scattering factor between the two compounds.

[0132] Step 6. The substrate material in step 1, the measured wavelength of the laser in step 2, the information of the reference peak in step 3, the normalized SERS spectrum in step 3, the relative SERS scattering cross section in step 3, the relative SERS scattering factor in step 5, and the Raman vibration mode of each SERS characteristic peak were combined as shown in Figure 6 to construct a SERS file card between 2-MPY molecules and 4-MBA molecules.

[0133] The SEM photograph of the 700 nm nanorod SERS substrate used is shown in Figure 1(c), its reflectance spectrum is shown in Figure 1(a), and the calculated relative scattering factor of the SERS of 4-MBA molecules relative to 2-MPY molecules is 6.3 ± 0.8, shown in Figure 5.

[0134] Card manufacturing example 3 Step 1. A V-shaped silver nanorod structure substrate with a purity of 99.99% and an arm length of 350 nm was fabricated by electron beam oblique deposition.

[0135] Step 2. Volume 15 μL, concentration 10 -6 A 4-mercaptobenzoic acid (4-MBA) molecular solution and a 2-mercaptopyridine (2-MPY) molecular solution were each dropped onto the surface of a 5 mm × 5 mm silver nanorod structure substrate with a diameter of 700 nm. After air drying, the SERS spectrum was measured using a 785 nm laser, and the fluorescent background signal was subtracted from the SERS spectrum.

[0136] Step 3. For the SERS spectrum from which the fluorescent background signal was subtracted in step 2, the 1074 cm of the SERS spectrum of the 4-MBA molecule -1 and 1002 cm in the SERS spectrum of 2-MPY molecules. -1 The characteristic peak of was selected as a reference peak, and its intensity was set to 100. The SERS spectra of the two molecules were normalized to obtain the relative SERS scattering cross section at each Raman shift.

[0137] Step 4. The molecular ratio of the mixed solution is 1:9, 2:8, 3:7, 4:6, 5:5, and the total concentration of the mixed solution is 10 -64-MBA molecules and 2-MPY molecules were mixed to obtain M, and 15 μL of each mixed solution was dropped onto the surface of a V-shaped silver nanorod structure substrate with an area of ​​5 mm × 5 mm and an arm length of 350 nm. After air drying, the SERS spectrum was measured and the fluorescent background signal was subtracted.

[0138] Step 5. The 1074 cm of the 4-MBA molecule in the SERS spectrum from which the fluorescent background signal was subtracted in Step 4. -1 and the characteristic peak of 2-MPY molecule at 1002 cm -1 The intensity ratio of the characteristic peaks of the two compounds was calculated, and the linear regression coefficient of the intensity ratio of the two compounds and the corresponding molar ratio in the mixed solution was calculated by the least squares regression method, which was used as the relative SERS scattering factor between the two compounds.

[0139] Step 6. The substrate material in step 1, the measured wavelength of the laser in step 2, the information of the reference peak in step 3, the normalized SERS spectrum in step 3, the relative SERS scattering cross section in step 3, the relative SERS scattering factor in step 5, and the Raman vibration mode of each SERS characteristic peak were combined as shown in Figure 6 to construct a SERS file card between 2-MPY molecules and 4-MBA molecules.

[0140] The SEM photograph of the V-shaped nanorod SERS substrate with an arm length of 350 nm used is shown in Figure 1(d), its reflectance spectrum is shown in Figure 1(a), and the calculated relative scattering factor of the SERS of 4-MBA molecules to that of 2-MPY molecules is 6.7 ± 0.9 is shown in Figure 5.

[0141] Card manufacturing example 4 Step 1. Gold nanostructure substrates with a purity of 99.99% were produced by electron beam oblique deposition.

[0142] Step 2. Volume 10 μL, concentration 10 -5A 4-mercaptobenzoic acid (4-MBA) molecular solution and a 2-mercaptopyridine (2-MPY) molecular solution were dropped onto the surface of a 5 mm × 5 mm gold nanostructure substrate, respectively, and after air drying, their SERS spectra were measured using a 785 nm laser. The fluorescence background signal was subtracted from the SERS spectra.

[0143] Step 3. For the SERS spectrum from which the fluorescent background signal was subtracted in step 2, the 1076 cm of the SERS spectrum of the 4-MBA molecule -1 and the characteristic peak at 1004 cm in the SERS spectrum of 2-MPY molecules. -1 The characteristic peak of was selected as a reference peak, and its intensity was set to 100. The SERS spectra of the two molecules were normalized to obtain the relative SERS scattering cross section at each Raman shift.

[0144] Step 4. The molecular ratio of the mixed solution is 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and the total concentration of the mixed solution is 10 -6 4-MBA molecules and 2-MPY molecules were mixed to obtain M, and 15 μL of each mixed solution was dropped onto the surface of a 10 mm × 10 mm gold nanostructure substrate. After air drying, the SERS spectrum was measured and the fluorescent background signal was subtracted.

[0145] Step 5. The 1076 cm of the 4-MBA molecule in the SERS spectrum from which the fluorescent background signal was subtracted in Step 4. -1 and the characteristic peak of 2-MPY molecule at 1004 cm -1 The intensity ratio of the characteristic peaks of the two compounds was calculated, and the linear regression coefficient of the intensity ratio of the two compounds and the corresponding molar ratio in the mixed solution was calculated by the least squares regression method, which was used as the relative SERS scattering factor between the two compounds.

[0146] Step 6. A SERS file card of the 2-MPY molecule was constructed using the substrate material in step 1, the laser measurement wavelength in step 2, the reference peak information in step 3, the normalized SERS spectrum in step 3, the relative SERS scattering cross section in step 3, the relative SERS scattering factor in step 5, and the Raman vibration mode of each SERS characteristic peak.

[0147] The nanostructure used was a 420 nm rod-shaped gold SERS substrate. The SERS spectra of the mixed solutions at each ratio are shown in Figure 11(a). The shape of the SERS spectrum changes sequentially depending on the ratio of the two molecules. -1 and the characteristic peak of 2-MPY molecule at 1004 cm -1 The relationship between the intensity ratio of the characteristic peaks and the molecular number ratio is shown in FIG. 11(b), and the constructed SERS file card is shown in FIG. 11(c).

[0148] [Example]

[0149] Example 1 1. A high-purity silver nanostructured SERS substrate was used as a SERS substrate for quantitative analysis, and a mixed solution of 4-mercaptobenzoic acid (4-MBA) molecules and 2-mercaptopyridine (2-MPY) molecules was used as the analytical system.

[0150] 2. Mixed solutions of 4-MBA molecules and 2-MPY molecules were prepared so that the molecular ratios of 4-MBA to 2-MPY in the mixed solutions were 1:9, 3:7, 1:1, 7:3, and 9:1, i.e., the content of 2-MPY molecules was 90%, 70%, 50%, 30%, and 10%, and 2-MPY in the mixed solutions was used as the selected molecule.

[0151] 3. Using the SERS substrate selected in step 1, each mixed solution prepared in step 2 was measured. The laser wavelength was set to 785 nm, the spot was set to 80 μm, and the output was set to 15 mW. 10 μL of the mixed solution was dropped onto the SERS substrate of step 1 with dimensions of 10 mm × 10 mm or less, and after air drying, the SERS spectrum was measured.

[0152] 4. The background was subtracted from the SERS spectrum of each mixed solution obtained in step 3, and the SERS file card between 2-MPY molecules and 4-MBA molecules was searched based on the substrate material in step 1 and the laser wavelength in step 3.

[0153] 5. From the SERS file cards of 2-MPY and 4-MBA molecules, the relative SERS scattering factor of 2-MPY molecules to 4-MBA molecules, 1002 cm -1 Relative SERS scattering cross section of the characteristic peak of 4-MBA molecule at 1074 cm -1 The relative SERS scattering cross sections of the characteristic peaks were obtained.

[0154] 6. The background-subtracted SERS spectrum from step 4 was analyzed at 1074 cm -1 and 1002 cm -1 The characteristic peak intensities of the above were extracted and substituted into equations (4-1-2) and (4-1-3) together with the relative SERS scattering factor obtained from the SERS file card in step 5 to directly calculate the content of 2-MPY molecules.

[0155] The measured SERS spectrum is shown in Figure 8(a). The spectrum changes sequentially with the amount of 2-MPY molecules. Figure 8(b) shows the results of comparing the amount of 2-MPY molecules calculated from the SERS file card and the formula group in step 6 with the actual amount in the solution in step 2. As is clear from the figure, the two spectra are in good agreement.

[0156] Example 2 1. A high-purity silver nanostructured SERS substrate was used as a SERS substrate for quantitative analysis, and a mixed solution of 4-mercaptobenzoic acid (4-MBA) molecules and 2-mercaptopyridine (2-MPY) molecules was used as the analytical system.

[0157] 2. Mixed solutions of 4-MBA and 2-MPY molecules were prepared so that the molecular ratios of 4-MBA and 2-MPY in the mixed solutions were 1:4, 2:3, 3:2, and 4:1, i.e., the 2-MPY molecule contents were 80%, 60%, 40%, and 20%. 4-MBA molecules were added as the reference substance, and 2-MPY in the mixed solution was used as the selected molecule. The concentration of the added reference molecule, 4-MBA, was 2 × 10 -6 mol / L (M), 4 x 10 -6 M, 6 x 10 -6 M, 8 x 10 -6 I chose M.

[0158] 3. Using the SERS substrate selected in step 1, each mixed solution prepared in step 2 was measured. The laser wavelength was set to 785 nm, the spot was set to 80 μm, and the output was set to 30 mW. 5 μL of the mixed solution was dropped onto the SERS substrate of step 1 with dimensions of 5 mm × 5 mm or less, and after air drying, the SERS spectrum was measured.

[0159] 4. The background was subtracted from the SERS spectrum of each mixed solution obtained in step 3, and the SERS file card between the 2-MPY molecule and the 4-MBA molecule was searched based on the substrate material in step 1 and the laser wavelength in step 3, and the relative SERS scattering factor was calculated by indirect transmission of the molecules.

[0160] 5. From the SERS file cards of 2-MPY molecules and 4-MBA molecules, the relative SERS scattering factor of 2-MPY molecules to 4-MBA molecules was indirectly calculated, and the normalized SERS spectrum of 2-MPY molecules and the normalized SERS spectrum of 4-MBA molecules were obtained.

[0161] 6. From the SERS spectra of 2-MPY and 4-MBA molecules, 900–1250 cm -1 Since the characteristic peaks are most concentrated between 988 cm and 988 cm, the SERS spectrum from which the background was subtracted in step 4 was -1 ~1202cm -1The range of the characteristic peaks was selected and substituted into equations (4-2-11) and (4-2-12) along with the relative SERS scattering factor and normalized SERS spectrum obtained from the SERS file card in step 5 to directly calculate the content of 2-MPY molecules. This was then substituted into equation (4-1-4) along with the concentration of the added reference molecule 4-MBA to calculate the concentration of 2-MPY molecules.

[0162] The measured SERS spectrum is shown in Figure 9(a). The spectrum changes sequentially with the amount of 2-MPY molecules. The spectrum in the selected section was normalized by integral area, and the first principal component calculated is shown in Figure 9(b). The final amount of 2-MPY molecules calculated from the SERS file card and formula group is in good agreement with the actual amount in the solution in step 2, as shown in Figure 9(c). The concentration of 2-MPY molecules calculated from the reference molecule 4-MBA added in step 2 is in good agreement with the actual molecular concentration, as shown in Figure 9(d).

[0163] Example 3 1. A high-purity silver nanostructured SERS substrate was used as a SERS substrate for quantitative analysis, and a mixed solution of 4-mercaptobenzoic acid (4-MBA) molecules and 2-mercaptopyridine (2-MPY) molecules was used as the analytical system.

[0164] 2. Mixed solutions of 4-MBA and 2-MPY molecules were prepared so that the molecular ratios of 4-MBA to 2-MPY in the mixed solutions were 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, i.e., the content of 2-MPY molecules was 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, and 10%. The 4-MBA molecules were used as the added reference molecule, and the concentration of 4-MBA molecules was increased in order from 1×10 to 1×10. -6 M, 2 x 10 -6 M, 3 x 10 -6 M, 4 x 10 -6 M, 5 x 10 -6 M, 6 x 10 -6 M, 7 x 10 -6 M, 8 x 10 -6 M, 9 x 10 -6 I chose M.

[0165] 3. Using the SERS substrate selected in step 1, each mixed solution prepared in step 2 was measured. The laser wavelength was set to 785 nm, the spot was set to 80 μm, and the output was set to 15 mW. 20 μL of the mixed solution was dropped onto the SERS substrate of step 1 with dimensions of 10 mm × 10 mm or less, and after air drying, the SERS spectrum was measured.

[0166] 4. The background was subtracted from the SERS spectrum of each mixed solution obtained in step 3, and the SERS file card between the 2-MPY molecule and the 4-MBA molecule was searched based on the substrate material in step 1 and the laser wavelength in step 3, and the relative SERS scattering factor was calculated by indirect transmission of the molecules.

[0167] 5. From the SERS file cards of 2-MPY molecules and 4-MBA molecules, the relative SERS scattering factor of 2-MPY molecules to 4-MBA molecules was indirectly calculated, and the normalized SERS spectrum of 2-MPY molecules and the normalized SERS spectrum of 4-MBA molecules were obtained.

[0168] 6. The entire spectral range was selected from the SERS spectrum from which the background was subtracted in step 4, and substituted into equations (4-3-3) and (4-3-4) together with the relative SERS scattering factor indirectly calculated from the SERS file card in step 5 to directly calculate the content of 2-MPY molecules. This was then substituted into equation (4-1-4) together with the concentration of the added reference molecule 4-MBA to calculate the concentration of 2-MPY molecules.

[0169] As shown in Figure 10(a), the calculated content of 2-MPY molecules is in good agreement with the actual content of the solution in step 2. As shown in Figure 10(b), the concentration of 2-MPY molecules calculated from equation (4-1-4) and the reference 4-MBA molecules added in step 2 is in good agreement with the actual concentration of 2-MPY molecules.

[0170] Example 4 1. A high-purity gold nanostructured SERS substrate was used as a SERS substrate for quantitative analysis, and a mixed solution of 4-mercaptobenzoic acid (4-MBA) molecules and 2-mercaptopyridine (2-MPY) molecules was used as the analytical system.

[0171] 2. Mixed solutions of 4-MBA and 2-MPY molecules were prepared so that the molecular ratios of 4-MBA to 2-MPY in the mixed solutions were 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, i.e., the content of 2-MPY molecules was 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, and 10%. The 4-MBA molecules were used as the added reference molecule, and the concentration of 4-MBA molecules was increased in order from 1×10 to 1×10. -6 M, 2 x 10 -6 M, 3 x 10 -6 M, 4 x 10 -6 M, 5 x 10 -6 M, 6 x 10 -6 M, 7 x 10 -6 M, 8 x 10 -6 M, 9 x 10 -6 I chose M.

[0172] 3. Using the SERS substrate selected in step 1, each mixed solution prepared in step 2 was measured. The laser wavelength was set to 785 nm, the spot was set to 80 μm, and the output was set to 60 mW. 10 μL of the mixed solution was dropped onto the SERS substrate of step 1 with dimensions of 5 mm × 5 mm or less, and after air drying, the SERS spectrum was measured.

[0173] 4. The background was subtracted from the SERS spectrum of each mixed solution obtained in step 3, and the SERS file card between the 2-MPY molecule and the 4-MBA molecule was searched based on the substrate material in step 1 and the laser wavelength in step 3, and the relative SERS scattering factor was calculated by indirect transmission of the molecules.

[0174] 5. From the SERS file cards of 2-MPY molecules and 4-MBA molecules, the relative SERS scattering factor of 2-MPY molecules to 4-MBA molecules was indirectly calculated, and the normalized SERS spectrum of 2-MPY molecules and the normalized SERS spectrum of 4-MBA molecules were obtained.

[0175] 6. The entire spectral range was selected from the SERS spectrum from which the background was subtracted in step 4, and substituted into equations (4-3-3) and (4-3-4) together with the relative SERS scattering factor indirectly calculated from the SERS file card in step 5 to directly calculate the content of 2-MPY molecules. This was then substituted into equation (4-1-4) together with the concentration of the added reference molecule 4-MBA to calculate the concentration of 2-MPY molecules.

[0176] As shown in Figure 12(a), the calculated content of 2-MPY molecules is in good agreement with the actual content of the solution in step 2. As shown in Figure 12(b), the concentration of 2-MPY molecules calculated from equation (4-1-4) and the reference 4-MBA molecules added in step 2 is in good agreement with the actual concentration of 2-MPY molecules.

[0177] As is clear from a comparison of Figure 10 in Example 3 and Figure 12 in Example 4, although the relative SERS scattering factors of 4-MBA molecules and 2-MPY molecules differ depending on the material, when quantitative analysis is performed using SERS file cards of corresponding materials and laser wavelengths, the accurate content and concentration of the molecules can be obtained in all cases, and the quantitative analysis results obtained with the SERS substrates of each material are consistent.

[0178] As is clear from the above embodiments, the SERS file card provided by the present invention, its construction method, and application method in quantitative analysis define and measure two physical quantities, i.e., the intramolecular relative SERS scattering cross section and the intermolecular relative SERS scattering factor, so that each parameter is versatile among SERS substrates with the same material properties but different geometric forms, and this makes it possible to eliminate the adverse effects on quantitative analysis caused by factors such as uniformity of SERS substrates, lot-to-lot differences, fluctuations in measurement conditions, and changes in geometric form, and can be used to construct a quantitative analysis database in the SERS field.SERS technology can easily perform various quantitative analyses, just as X-ray diffraction technology has a standard powder diffraction file card database.A SERS file card that can be used for quantitative analysis has been constructed, which has a wide application prospect and an important fundamental role in the field of trace molecule quantitative analysis.

[0179] As described above, the quantitative analysis method using the SERS file card according to the present invention has been described by way of example with reference to the drawings. However, it should be understood that those skilled in the art can make various modifications to the quantitative analysis method using the SERS file card according to the present invention without departing from the scope of the present invention, and all modifications are within the scope of the present invention.

Claims

1. the relative scattering cross section of the "surface-enhanced Raman scattering" of the selected molecule; The relative scattering factors of the "surface-enhanced Raman scattering" of the selected molecule and the reference molecule; A surface-enhanced Raman scattering file card including:

2. 2. The surface-enhanced Raman scattering file card according to claim 1, further comprising a material of the surface-enhanced Raman scattering substrate and a measurement wavelength.

3. 3. A surface-enhanced Raman scattering file card according to claim 1 or 2, comprising selected reference peaks of selected molecules and reference molecules.

4. 3. The surface-enhanced Raman scattering file card of claim 1 or 2, comprising normalized surface-enhanced Raman scattering spectra of selected molecules.

5. A method for producing the surface-enhanced Raman scattering file card according to any one of claims 1 to 4, comprising: a step of measuring the "surface-enhanced Raman scattering" spectrum of the selected molecule, selecting the characteristic peak with the strongest peak intensity in the spectrum as a reference peak, and calculating the relative values ​​of the peak intensities of the other characteristic peaks and the reference peak to obtain a relative scattering cross section of "surface-enhanced Raman scattering"; a step of measuring the "surface-enhanced Raman scattering" spectra of the selected molecule and the reference molecule, respectively, selecting the characteristic peaks with the strongest peak intensities in the spectra of the selected molecule and the reference molecule as reference peaks of the selected molecule and the reference molecule, respectively, measuring the "surface-enhanced Raman scattering" spectrum of a mixture of the selected molecule and the reference molecule, calculating the relative values ​​of the peak intensities of the reference peaks of the selected molecule and the reference molecule, and obtaining a relative scattering factor of "surface-enhanced Raman scattering"; The manufacturing method according to claim 1, further comprising:

6. 6. The manufacturing method according to claim 5, wherein the characteristic peak with the strongest peak intensity in the spectrum is selected as a reference peak, and then the intensity value of the reference peak is set to 100, and the peak intensities of the other characteristic peaks are normalized, and the normalized peak intensities of the other characteristic peaks are used as the relative scattering cross section of "surface-enhanced Raman scattering".

7. The manufacturing method according to claim 5 or 6, characterized in that the selected molecule and the reference molecule are mixed at different molar ratios, the "surface-enhanced Raman scattering" spectrum is measured for each molar ratio, the characteristic peaks with the strongest peak intensities in the spectra of the selected molecule and the reference molecule are selected as reference peaks for the selected molecule and the reference molecule, respectively, the intensity ratio of the reference peaks of the selected molecule and the reference molecule is calculated, and a linear regression coefficient between the intensity ratio and the molar ratio is calculated by least squares regression, and this is used as the relative scattering factor of "surface-enhanced Raman scattering" between the molecules.

8. 8. The method according to claim 7, wherein the intensity ratio is in the range of 0.1 to 10.

9. The manufacturing method according to any one of claims 5 to 8, characterized in that, when measuring the "surface-enhanced Raman scattering" spectrum, the molecules to be measured are dropped as a solution onto the surface of the substrate, the solvent is allowed to dry naturally, the spectrum is measured, and a fluorescent background signal is subtracted to obtain the "surface-enhanced Raman scattering" spectrum.

10. The total concentration of the solution is 10 －８ ~10 －５ mol / L, and the average in-plane volume range of the drop amount is 0.1 to 5 μL / mm ２ The method according to claim 9, wherein

11. 1) measuring the surface-enhanced Raman scattering spectrum (SERS spectrum) of the object to be analyzed based on the measurement wavelength of the surface-enhanced Raman scattering file card (SERS file card) according to any one of claims 1 to 4 and the material of the substrate; 2) for different molecules, selecting 1 to n characteristic peaks for molecule i and selecting 1 to m characteristic peaks for molecule j, and solving the following equation group I: A quantitative analysis method using a SERS file card, comprising: [Equation 1] (In the formula, I i,p : the peak intensity of the pth SERS characteristic peak of the ith molecule, I j,q : the peak intensity of the qth SERS characteristic peak of the jth molecule, RSF i,j : the relative SERS scattering factor of the i-th molecule with respect to the j-th molecule, For k molecules, any i, j belong to the interval [1, k] and i is not equal to j; X i : content of the i-th molecule, C i : concentration of the i-th molecule, X j : content of jth molecule, C j : concentration of the jth molecule, RSC i,p : the relative SERS scattering cross section of the selected pth SERS characteristic peak in the ith molecule, RSC j,q : the relative SERS scattering cross section of the qth selected SERS characteristic peak in the jth molecule, l and m are the numbers of the selected SERS characteristic peaks, respectively.

12. 1) measuring the surface-enhanced Raman scattering spectrum (SERS spectrum) of the object to be analyzed based on the wavelength and the material of the substrate of the surface-enhanced Raman scattering file card (SERS file card) according to any one of claims 1 to 4; 2) selecting 1 to p spectral intervals of the SERS spectrum for n molecules and solving them using the following equation group II: A quantitative analysis method using a SERS file card, comprising: [Equation 2] (In the formula, X i : content of the i-th molecule, C i : concentration of the i-th molecule, X j : content of jth molecule, C j : concentration of the jth molecule, RSF i,j : the relative SERS scattering factor of molecule i with respect to molecule j, Anorm i,1-p : the integrated area of ​​1 to p SERS spectrum intervals selected in the SERS file card of the i-th molecule, Anorm j,1-p : the integrated area of ​​1 to p SERS spectrum intervals selected in the SERS file card of the jth molecule, PC A,i,1-p : principal component values ​​corresponding to the integrated area-normalized SERS spectrum of the i-th molecule in the spectral interval from 1 to p, PC A,range,1-p : Principal component value corresponding to the integrated area-normalized SERS spectrum of the spectrum to be analyzed in the spectral interval 1 to p.

13. 1) measuring the surface-enhanced Raman scattering spectrum (SERS spectrum) of the object to be analyzed based on the wavelength and the material of the substrate of the surface-enhanced Raman scattering file card (SERS file card) according to any one of claims 1 to 4; 2) For n molecules, selecting a complete spectral interval of the SERS spectrum, or selecting a partial spectral interval of the SERS spectrum as the complete spectral interval, and solving the following equation group III: A quantitative analysis method using a SERS file card, comprising: [Equation 3] (In the formula, X i : content of the i-th molecule, C i : concentration of the i-th molecule, X j : content of jth molecule, C j : concentration of the jth molecule, RSF i,j : the relative SERS scattering factor of molecule i with respect to molecule j, α: common proportionality coefficient introduced, Spec i : the normalized SERS spectrum in the SERS file card of the i-th molecule, X i,M : α × RSA for i from 1 to n i,j ×X i A vector composed by Spec i,M :Spec for i from 1 to n i A matrix composed by Spec mix : SERS spectrum to be analyzed.

14. The method according to any one of claims 11 to 13, wherein the SERS file card has the same laser wavelength and substrate material as the SERS spectrum, and a background is subtracted from the SERS spectrum.

15. The method according to any one of claims 11 to 13, characterized in that the relative SERS scattering factor of a molecule is obtained from the SERS file card of the molecule or calculated by indirect transfer from the SERS file card of another molecule, and the calculation of the relative SERS scattering factor of the molecule from the SERS file card of another molecule is realized by the following chain transfer formula IV: [Equation 4] (In the formula, RSF i,j is the relative SERS scattering factor of molecule i with respect to molecule j, and RSF i,p is the relative SERS scattering factor of molecule i with respect to molecule p, and RSF p,q is the relative SERS scattering factor of molecule p to molecule q, and RSF q,r is the relative SERS scattering factor of molecule q to molecule r, and RSF r,s is the relative SERS scattering factor of molecule r to molecule s, and RSF s,t is the relative SERS scattering factor of molecule s with respect to molecule t, and RSF t,j is the relative SERS scattering factor of molecule t with respect to molecule j.

16. 16. The method of claim 15, wherein the number of transfers in the chain transfer formula IV is 6 or less.

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