WO2024099394A1

WO2024099394A1 - Surface-enhanced raman scattering file card and manufacturing method therefor, and method for performing quantitative analysis by using file card

Info

Publication number: WO2024099394A1
Application number: PCT/CN2023/130764
Authority: WO
Inventors: 张政军; 赵丰通; 王炜鹏
Original assignee: 清华大学
Priority date: 2022-11-09
Filing date: 2023-11-09
Publication date: 2024-05-16

Abstract

A surface-enhanced Raman scattering (SERS) file card and a manufacturing method therefor, and a method for performing quantitative analysis by using the file card. The SERS file card comprises: a relative scattering cross section of "SERS" of a selected molecule; and a relative scattering factor of "SERS" of the selected molecule and a reference molecule. The constructed SERS file card applicable to quantitative analysis can be used to construct a quantitative analysis database in the field of SERS, and the method for performing quantitative analysis provides guidance for the implementation of quantitative analysis using the SERS file card in different conditions.

Description

A surface enhanced Raman scattering file card, a method for making the same, and a method for quantitative analysis using the file card

Technical Field

The invention relates to a surface enhanced Raman scattering file card, a manufacturing method thereof and a method for quantitative analysis using the file card, and belongs to the field of material analysis.

Background technique

Surface enhanced Raman scattering (SERS) is an inelastic scattering spectroscopy technique with ultra-high sensitivity and fingerprint recognition. At the same time, due to the fast acquisition speed of SERS spectroscopy, simple sample preparation, and continuous miniaturization of detection equipment, SERS spectroscopy has broad application prospects in the fields of environmental pollutant detection, food additive detection, pesticide residue detection, biochemical detection, pharmaceutical analysis and health screening. Since the discovery of the SERS phenomenon in 1974, SERS spectroscopy technology has developed for nearly half a century. For SERS spectroscopy technology, SERS substrates are the key carriers that enable the target to produce huge Raman scattering signal enhancement. With the continuous in-depth development of research, a variety of SERS substrates have been designed and developed and applied to detection and analysis, including traditional precious metal SERS substrates composed of gold, silver, copper and their alloys, semiconductor SERS substrates, flexible SERS substrates, etc. With the continuous deepening of the understanding of the SERS mechanism, semiconductor SERS substrates are also developing continuously, but traditional precious metal SERS substrates are still an irreplaceable and important enhancement carrier in SERS application technology. At the same time, the development of flexible SERS substrates and the integration of other technologies such as atomic force microscopy and electrochemical technology with SERS spectroscopy are also continuously expanding the application scenarios and scope of SERS spectroscopy. This has made great progress in the detection of trace substances in SERS spectroscopy. Although great progress has been made in the design, preparation, detection and analysis of SERS substrates, how to improve the stability, uniformity, and repeatability of SERS substrates and how to reduce the differences between different batches of SERS substrates are still important research directions in this field. SERS technology has ultra-high sensitivity, so the stability, uniformity, repeatability, batch differences of SERS substrates and slight fluctuations in test conditions will have a significant impact on semi-quantitative and quantitative analysis based on SERS spectroscopy.

At present, in the field of quantitative analysis based on SERS spectroscopy, a variety of specific The linear, logarithmic, power-law and other functional relationships between the absolute intensity of SERS spectra and the concentration of the target are also often used for quantitative analysis. However, these methods have high requirements on the uniformity, repeatability, batch-to-batch variability and control of test conditions of the substrate, and are not universal. Therefore, researchers have tried to combine the relative intensity information of SERS spectra with the above functional relationship to develop new quantitative analysis methods, aiming to overcome the adverse effects of SERS spectrum intensity fluctuations on quantitative analysis. The use of the relative intensity information of SERS spectra for quantitative analysis includes modifying the internal standard reference molecule on the surface of the SERS substrate, constructing a core-shell SERS substrate containing the internal standard molecule in the shell layer, and correcting the intensity fluctuation of the target SERS spectrum using the Raman scattering characteristic peak of the internal standard reference molecule or the Raman scattering characteristic peak of the SERS substrate. These methods have overcome the influence of the absolute intensity fluctuation of SERS spectra on quantitative analysis to a certain extent, but they are still affected by batch-to-batch variability and have poor portability. Since the physical nature behind the quantitative analysis method based on the relative intensity of SERS spectra is not discussed in depth, universal semi-quantitative and quantitative analysis methods have not yet been established in the field of SERS spectrum quantitative analysis. In other analytical characterization techniques, relative factors are commonly used methods for semi-quantitative and quantitative analysis. For example, in X-ray diffraction and X-ray photoelectron spectroscopy, the relative sensitivity factors of different phases and elements can be used for quantitative analysis of phases and sample surface elements.

The study found that under a certain laser wavelength, when the SERS substrate material is fixed, the system composed of the molecule and the SERS substrate has a stable relative SERS scattering cross section, that is, different SERS characteristic peaks within the same molecule have a stable relative scattering cross section, and different molecules also have a stable relative SERS scattering factor, that is, selected SERS characteristic peaks between different molecules have a stable relative scattering cross section. 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 but have nothing to do with the geometric morphology of the SERS substrate nanostructure. In addition, the normalization of the SERS spectrum can remove the absolute intensity fluctuation of the SERS spectrum, and the scattering cross section within the molecule and the scattering ability between molecules are both expressed by the relative SERS scattering cross section and relative SERS scattering factor. Based on the relative SERS scattering cross section within the same molecule, the relative SERS scattering factor between different molecules, and the normalized SERS spectrum, a SERS file card can be constructed. Since the file contains the quantitative SERS spectrum information of the molecule, it can be used to construct a SERS file card database similar to the powder diffraction file card in the X-ray diffraction (XRD) technology, which is based on the quantitative analysis of SERS spectra. and semi-quantitative analysis.

However, the SERS file card database is currently under development, and there is no systematic and comprehensive quantitative analysis application guidance. In order to better play the role of SERS file cards in the field of quantitative analysis and promote the development of SERS spectral quantitative analysis technology, a method for quantitative analysis using SERS file cards is urgently needed.

Summary of the invention

technical problem

In view of the above-mentioned problems, the inventors, starting from the basic physical concepts and drawing on the general quantitative analysis methods in technologies such as X-ray diffraction and X-ray photoelectron spectroscopy, provide a surface enhanced Raman scattering file card with wide versatility that can be used for semi-quantitative and quantitative analysis, and a method for making the same. The present invention also aims to provide a method for quantitative analysis using the SERS file card, to provide guidance for the SERS file card to carry out quantitative analysis under different circumstances, to expand the application scope of SERS spectra in quantitative analysis, and to provide a reference for constructing a general quantitative analysis method and quantitative analysis software based on the SERS file card.

solution

In order to solve the above technical problems, according to one embodiment of the present invention, a surface enhanced Raman scattering file card is provided, the file card comprising:

The relative scattering cross section of “surface enhanced Raman scattering” of the selected molecule (relative SERS scattering cross section); the relative scattering factor of “surface enhanced Raman scattering” of the selected molecule and the reference molecule (relative SERS scattering factor).

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 test wavelength.

According to the surface enhanced Raman scattering file card of the present invention, the file card includes selected reference peaks of selected molecules and reference molecules.

According to the surface enhanced Raman scattering file card of the present invention, the file card comprises a normalized surface enhanced Raman scattering spectrum of a selected molecule.

The present invention also provides a method for manufacturing the surface enhanced Raman scattering file card according to the present invention. The method comprises the following steps:

Determine the "surface enhanced Raman scattering" spectrum of the selected molecule, select the characteristic peak with the strongest peak intensity in the spectrum as the reference peak, calculate the relative value of the peak intensity of other characteristic peaks and the peak intensity of the reference peak, and obtain the relative scattering cross section of the "surface enhanced Raman scattering";

The "surface enhanced Raman scattering" spectra of the selected molecule and the reference molecule are measured respectively, and the characteristic peaks with the strongest peak intensity 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 "surface enhanced Raman scattering" spectrum of the mixture of the selected molecule and the reference molecule is measured, and the relative value of the peak intensity of the reference peak of the selected molecule and the reference molecule is calculated to obtain the relative scattering factor of the "surface enhanced Raman scattering".

According to the production method of the present invention, after selecting the characteristic peak with the strongest peak intensity in the spectrum as the reference peak, the intensity value of the reference peak is set to 100, and the peak intensities of other characteristic peaks are normalized. After normalization, the peak intensities of other characteristic peaks are the relative scattering cross sections of "surface enhanced Raman scattering".

According to the preparation method of the present invention, the selected molecule and the reference molecule are mixed in different molar ratios, and the "surface enhanced Raman scattering" spectra of different molar ratios are measured respectively, and the characteristic peaks with the strongest peak intensity in the spectra of the selected molecule and the reference molecule are respectively selected as the reference peaks of the selected molecule and the reference molecule, and the intensity ratio of the reference peaks of the selected molecule and the reference molecule is calculated. The linear regression coefficient between the intensity ratio and the molar ratio is calculated by the least squares regression method, which is the relative scattering factor of the "surface enhanced Raman scattering" between molecules.

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

According to the production method described in the present invention, when measuring the "surface enhanced Raman scattering" spectrum, the molecule to be tested is dropped onto the surface of the substrate in the form of a solution. After the solvent is naturally dried, its spectrum is tested and the fluorescence background signal is subtracted to obtain the "surface enhanced Raman scattering" spectrum.

According to the preparation method of the present invention, the total concentration of the solution is 10 ^-8 to 10 ^-5 mol/L, and the average in-plane volume range of the dropwise addition amount is 0.1 to 5 μL/mm ² .

The present invention also provides a method for quantitative analysis using a SERS file card, comprising the following steps:

1) Based on the test wavelength of the surface enhanced Raman scattering file card (SERS file card) of the present invention and the material of the substrate, a surface enhanced Raman scattering spectrum (SERS spectrum) to be analyzed is measured;

2) For different molecules, select 1 to n characteristic peaks for molecule i, and select 1 to m characteristic peaks for molecule j, and use the following formula group I to solve:

in,

I _i,p : peak intensity of the pth SERS characteristic peak of the i-th molecule,

I _j,q : peak intensity of the qth SERS characteristic peak of the jth molecule,

RSF _i,j : Relative SERS scattering factor of the i-th molecule relative to the j-th molecule,

For k molecules, any i, j belongs to the interval [1, k], and i is not equal to j;

_Xi : the content of the i-th molecule,

_Ci : the concentration of the i-th molecule,

X _j : the content of the jth molecule,

C _j : concentration of the jth molecule,

RSC _i,p : relative SERS scattering cross section of the pth SERS characteristic peak selected by the i-th molecule,

RSC _j,q : relative SERS scattering cross section of the qth SERS characteristic peak selected by the jth molecule,

l and m are the numbers of selected SERS characteristic peaks, respectively.

2) For n molecules, select spectral intervals 1 to p of the SERS spectrum and use the following formula group II to solve:

in,

_Xi : the content of the i-th molecule,

_Ci : the concentration of the i-th molecule,

X _j : the content of the jth molecule,

C _j : concentration of the jth molecule,

RSF _i,j : relative SERS scattering factor of molecule i relative to molecule j,

Anorm _i,1-p : The integrated area of the SERS spectrum intervals 1 to p selected in the i-th molecule SERS file card,

Anorm _j,1-p : The integrated area of the SERS spectrum intervals 1 to p selected in the j-th molecule SERS file card,

PC _A,i,1-p : The principal component value corresponding to the integrated area normalized SERS spectrum of the i-th molecule in the spectral interval 1 to p,

PC _A,range,1-p : The principal component value corresponding to the integrated area normalized SERS spectrum of the spectrum to be analyzed in the spectral range 1 to p.

2) For n molecules, the complete spectral interval of the SERS spectrum or a partial spectral interval of the SERS spectrum is selected as the complete spectral interval, and the following formula group III is used for solution:

in,

_Xi : the content of the i-th molecule,

_Ci : the concentration of the i-th molecule,

X _j : the content of the jth molecule,

C _j : concentration of the jth molecule,

α: the common proportional coefficient introduced,

Spec _i : the normalized SERS spectrum of the i-th molecule in the SERS file card,

_Xi,M : When i ranges from 1 to n, the vector _consisting of α×RSA _i,j ×Xi,

Spec _i,M : The matrix formed by Spec _i when i ranges from 1 to n.

Spec _mix : SERS spectrum to be analyzed.

According to the method for quantitative analysis using a SERS file card of the present invention, the laser wavelength and substrate material of the SERS file card are the same as the SERS spectrum, and the SERS spectrum is subtracted from the background.

According to the method for quantitative analysis using SERS file cards of the present invention, the relative SERS scattering factor of a molecule is obtained through the SERS file card of the molecule, or is indirectly transferred and calculated through the SERS file cards of other molecules. The relative SERS scattering factor of the molecule is calculated through the SERS file cards of other molecules through the following chain transfer formula IV:
RSF _i,j =RSF _i,p ×RSF _p,q ×RSF _q,r ×RSF _r,s ×RSF _r,t ×RSF _t,j

Wherein, RSF _i,j is the relative SERS scattering factor of molecule i relative to molecule j, RSF _i,p is the relative SERS scattering factor of molecule i relative to molecule p, RSF _p,q is the relative SERS scattering factor of molecule p relative to molecule q, RSF _q,r is the relative SERS scattering factor of molecule q relative to molecule r, RSF _r,s is the relative SERS scattering factor of molecule r relative to molecule s, and RSF _{s,t is the relative SERS} scattering factor of molecule s relative to molecule t. The relative SERS scattering factor, RSF _t,j, is the relative SERS scattering factor of molecule t relative to molecule j.

According to the method for quantitative analysis using the SERS file card of the present invention, the number of transfers of the chain transfer formula IV is ≤6.

Beneficial Effects

The present invention constructs a surface enhanced Raman scattering file card that can be used for quantitative analysis by defining and measuring two physical quantities, the relative scattering cross section and the relative scattering factor of "surface enhanced Raman scattering" within a molecule, wherein each parameter is universal between the same type of surface enhanced Raman scattering substrates with the same surface properties but different geometrical morphologies, and can eliminate the influence of factors such as the uniformity of the surface enhanced Raman scattering substrate, the difference between batches, the fluctuation of the test conditions, the change of the geometrical morphology and the like on the quantitative analysis, and can be used to construct a quantitative analysis database in the field of SERS, and can enable the SERS technology to conveniently carry out various quantitative analyses like the X-ray diffraction technology has a standard powder diffraction card library, and has broad application prospects and an important basic supporting role in the field of quantitative analysis of trace molecules.

The method for quantitative analysis using SERS file cards provided by the present invention can combine SERS file cards with different algorithms in a variety of different situations to carry out quantitative analysis of the content and concentration of selected molecules, effectively expanding the general application scope of SERS technology in the quantitative analysis of trace substances, and at the same time making the quantitative analysis work based on SERS technology simple and convenient, and has broad application prospects and important basic supporting role in the field of quantitative analysis of trace molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate exemplary embodiments, features, and aspects of the invention and, together with the description, serve to explain the principles of the invention.

Figure 1 Reflectivity spectra and SEM images of 90nm silver nanorod structure substrate, 700nm silver nanorod structure substrate, and V-shaped silver nanorod structure substrate with a single arm length of 350nm;

FIG2 shows the SERS spectra of 4-MBA molecules on the surface of a silver nanorod structure substrate at 490 nm under different laser powers and the normalized SERS spectra after deducting the fluorescence background signal;

Figure 3 SERS spectra of 4-MBA and 2-MPY molecules tested on silver SERS substrates with different structures and normalized SERS spectra after deducting the fluorescence background signal;

Figure 4 SERS spectra of mixed solutions of 4-MBA and 2-MPY with a molecular ratio of 9:1 and 1:9 on silver SERS substrates with different structures and normalized SERS spectra after background subtraction;

Fig. 5 Relative SERS scattering factors between 4-MBA and 2-MPY molecules measured by the mixed drop method;

FIG6 is a SERS file card of the 2-MPY molecule constructed based on the measured relative scattering cross section within the 2-MPY molecule and the relative SERS scattering factor between the 2-MPY and 4-MBA molecules;

FIG7 is a flow chart of a method for quantitative analysis using SERS file cards under different conditions;

FIG8 is a diagram showing the results of quantitative analysis of Example 1;

FIG9 is a diagram showing the results of quantitative analysis of Example 2;

FIG10 is a diagram showing the results of quantitative analysis of Example 3;

FIG. 11 shows the SERS spectrum obtained in card making example 4, the relationship between the intensity ratio of characteristic peaks and the ratio of the number of molecules, and the SERS file card established;

FIG. 12 is a diagram showing the results of quantitative analysis of Example 4.

Detailed ways

Various exemplary embodiments, features and aspects of the present invention will be described in detail below with reference to the accompanying drawings. The same reference numerals in the accompanying drawings represent elements with the same or similar functions. Although various aspects of the embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless otherwise specified.

The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

In addition, in order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, components and circuits well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[Surface enhanced Raman scattering file card and its production method]

Definition and derivation of physical quantities

The SERS process includes the adsorption process of molecules on the SERS substrate surface, the Raman scattering process of molecules, and the enhancement process of the SERS substrate on the Raman scattering of molecules. The SERS characteristic peak intensity (I _peak,i ) obtained by the test is proportional to the laser power (L _λ ), the number of molecules (N _m ), and the SERS characteristic peak scattering cross section (σ _SERS,peak,i ) of the molecules on the substrate surface, as shown in the following formula (1-1):
I _peak,i ∝L _λ N _m σ _SERS,peak,i (1-1)

The same molecule generally has multiple Raman scattering characteristic peaks. In the SERS spectrum, the same molecule also has multiple SERS characteristic peaks. For different SERS characteristic peaks of the same molecule, one of the SERS characteristic peaks (σ _SERS,peak,r ) is selected as a reference, and (1-1) can be equivalently transformed into the following formula (1-2):

Then it is transformed into the following formula (1-3):

The inventors found that, now define (1-3) in the formula is the relative SERS cross section (RCS) between different SERS peaks of the same molecule, that is, the following formula (1-4) holds true:

The relative SERS scattering cross section is mathematically equivalent to normalizing the SERS spectrum of a molecule with a selected SERS characteristic peak. This normalization preserves the intensity relationship between different SERS characteristic peaks of the molecule while eliminating the fluctuation of the absolute intensity.

According to the above definition, for two different molecules M1 and M2, the following equations (1-5) and (1-6) are respectively established:

where σ _{SERS,peak,r,M1} and σ _{SERS,peak,r,M2} are the selected SERS characteristic peak scattering cross sections of the two molecules M1 and M2 respectively. Now define the ratio of the two is the relative SERS scattering ability factor (RSF) between molecules, which is shown in the following formula (1-7):

Dividing formula (1-6) by formula (1-5), we can get the following formula (1-8):

Substituting equations (1-4) and (1-7) into equation (1-8), we can obtain equation (1-9):

Where RSF _M2/M1 is the relative SERS scattering factor (RSF) of molecule M2 relative to molecule M1, RCS _M1 and RCS _M2 are the relative SERS scattering cross sections (RCS) of molecules M1 and M2 respectively. Formula (1-9) can be transformed into the following formula (1-10):

According to formula (1-10), quantitative analysis can be carried out to directly calculate the relative content information between molecules. Similarly, formula (1-10) can be transformed into formula (1-11):

According to formula (1-11), the number of molecules of molecule M2 can be directly calculated.

It can be seen from formula (1-10) that the relative content between molecules can be calculated through the two defined physical quantities, the relative SERS scattering cross section (RCS) within the molecule and the relative SERS scattering factor (RSF) between molecules, as well as the SERS spectrum; it can be seen from formula (1-11) that the number of molecules M2 can be solved through the two defined physical quantities RCS and RSF, combined with the SERS spectrum and the number of molecules M1.

According to the above definition and discussion of the inventors, the relative SERS scattering cross section (RCS) and relative SERS scattering factor (RSF) of molecules can be used to directly achieve quantitative analysis of molecular content and molecular number. The measurement methods of these two physical quantities will be described below.

For the two physical quantities, the relative SERS scattering cross section (RCS) within a molecule and the relative SERS scattering factor (RSF) between molecules, the following will start from their definitions, explain their measurement methods and specific measurement processes, and study their properties.

The measurement process and properties of two physical quantities

For the relative SERS scattering cross section (RCS) between different Raman vibration modes in the same molecule, according to formula (1-4), a certain SERS characteristic peak can be selected as the reference peak, and then the relative SERS scattering cross sections of other SERS characteristic peaks can be calculated. Specific operation: a molecular solution of a certain concentration and volume is added to the surface of the SERS substrate, and the SERS spectrum is measured after the solvent is naturally dried. The background signal such as fluorescence is subtracted from the SERS spectrum obtained by the test, and the strongest characteristic peak in the SERS spectrum is selected as the reference peak for spectrum normalization. The normalization here is to take the strongest characteristic peak of the molecular SERS spectrum as the reference peak. The characteristic peak intensity is taken as 100, and the relative intensity of any other SERS characteristic peak is calculated as the relative SERS scattering cross section of each SERS characteristic peak in the molecule.

For the relative SERS scattering factor (RSF) between different molecules, according to formula (1-7), a certain molecule is selected as a reference molecule, and the intensity ratio between the reference peak of the SERS spectrum of other molecules and the reference peak of the selected molecule is calculated, which is the relative SERS scattering factor between molecules. When the stability, uniformity, repeatability, batch-to-batch variability, test conditions and other factors of the SERS substrate change slightly, the intensity and shape of the SERS spectrum will also change accordingly. Therefore, when testing the relative SERS scattering factor, it is necessary to ensure that the various factors of the two molecules tested are consistent. In order to meet the above requirements, a mixed solution of the two molecules of a certain concentration and volume needs to be dripped onto the surface of the SERS substrate, and at the same time, the total molecular coverage must be ensured to be less than a monolayer. In this way, the mixed SERS spectra of the two molecules can be tested simultaneously in the same spot, thereby effectively avoiding the adverse effects of test condition fluctuations, substrate batch-to-batch variability, uniformity and repeatability, and at the same time reducing the interaction between molecules. After the added solvent is dried, the SERS spectra of mixed solutions with different proportions are tested, the SERS reference peak intensities of the two molecules in the SERS spectrum of the mixed solution are ratioed, and the ratio of the reference peak intensities is 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.

Verification of the relative scattering cross section of surface-enhanced Raman scattering

First, to verify the validity of the relative scattering cross section of "surface enhanced Raman scattering", a variety of silver SERS substrates with different structures are provided. Their SEM photos are shown in Figure 1 (b-d), which are 490nm silver nanorod structure substrate, 700nm silver nanorod structure substrate, and V-shaped silver nanorod structure substrate with a single arm length of 350nm. The reflectivity spectra of the three different nanostructured SERS substrates are shown in Figure 1 (a). As can be seen from Figure 1 (a), the three different structures have different optical properties, and there are significant differences in reflectivity at different wavelengths. The valley value and shape of the reflection valley of the reflectivity curve are different.

Using the 490nm silver nanorod structure substrate shown in Figure 1(b), the SERS spectra of 4-mercaptobenzoic acid (4-MBA) molecules were tested at different laser powers. The SERS spectra of 4-MBA molecules tested at different laser powers are shown in Figure 2(a). The spectral intensity is different for different laser powers. The SERS spectra obtained by testing at different laser powers are normalized after deducting the background. The 1074cm ^-1 SERS characteristic peak is selected as the reference peak, and its intensity is taken as 100. The normalized SERS spectrum is shown in Figure 2(b). It can be seen from the figure that the normalized SERS spectra of 4-MBA molecules at different powers are The spectra overlap almost completely.

Similarly, for the silver SERS substrates with different structures, the SERS spectra of 4-MBA molecules obtained under the same test conditions are shown in FIG3(a). It can be seen from the figure that the enhancement effects of SERS substrates with different structures are different, and the intensity difference between them is huge. After deducting the background from the spectrum in FIG3(a), the 1074 cm ^-1 SERS characteristic peak is used as the reference peak for normalization, and the result shown in FIG3(b) can be obtained. It can be seen from the figure that the normalized SERS spectra of 4-MBA molecules obtained by the different nanostructured SERS substrates are almost completely overlapped. Similarly, for 2-thiopyridine (2-MPY) molecules, FIG3(c) is the SERS spectra of 2-MPY molecules obtained by the silver SERS substrates with different structures. After deducting the background, the 1002 cm ^-1 SERS characteristic peak is used as the reference peak for normalization, and the result shown in FIG3(d) can be obtained. The normalized spectra are almost completely overlapped.

From the above analysis, it can be seen that the relative SERS scattering cross section within the molecule, for SERS substrates of the same material, can be ignored due to changes in the test laser power and changes in the geometric morphology of the nanostructure. It is an intrinsic characteristic parameter of the system composed of the molecule and the SERS substrate material.

Verification of the relative scattering factor of surface enhanced Raman scattering

The relative SERS scattering factor between molecules requires the determination of the SERS spectra of the mixed solutions of the two molecules in different proportions. First, 4-MBA and 2-MPY molecules are mixed in different proportions, dripped on the surface of the above-mentioned silver SERS substrates with different structures, and then naturally dried to obtain the SERS spectra of the two molecules mixed in different proportions. Figure 4 shows the mixed SERS spectra of the two molecules 4-MBA and 2-MPY with a mixing ratio of 1:9 and 9:1. As shown in Figure 4 (a), the mixing ratio of 4-MBA and 2-MPY molecules is 1:9, and the SERS spectrum intensity obtained by testing on silver SERS substrates with different structures is very different. The SERS spectrum after deducting the background is normalized with the 1002cm ^-1 SERS characteristic peak, and the result shown in Figure 4 (b) can be obtained. The SERS spectra obtained by testing the three silver SERS substrates with different structures are almost completely overlapped. Similarly, as shown in Figure 4(c), the mixing ratio of 4-MBA and 2-MPY molecules is 9:1. The SERS spectrum intensities obtained on the silver SERS substrates with different structures are very different. The SERS spectrum after deducting the background is normalized with the 1074cm ^-1 SERS characteristic peak, and the result shown in Figure 4(d) can be obtained. The SERS spectra obtained by the three silver SERS substrates with different structures are almost completely overlapped. This shows that when the SERS substrate material is the same, the nanostructure of the SERS substrate The geometrical morphology has little influence on the measurement results of the mixed SERS spectrum. The relative SERS scattering factor between molecules measured based on this is the intrinsic characteristic parameter of the system composed of molecules and SERS substrate.

Since the relative SERS scattering cross section within a molecule and the relative SERS scattering factor between molecules are intrinsic characteristic parameters of the system composed of molecules and SERS substrates, they can be used to construct data files and conduct 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 mainly composed of the signal of 4-MBA. In this case, the slight signal and noise fluctuations will have a great impact on the measurement of the relative SERS scattering factor between the two molecules. In order to avoid this situation from having an adverse effect on the measurement accuracy of the relative SERS scattering factor, it is necessary to calculate the relative SERS scattering cross section between the two molecules when the difference in the signal intensity of the two molecules is not large. In order to avoid the above adverse effects, five different ratios of mixed solutions of 4-MBA and 2-MPY were prepared, with the ratio of the number of molecules of 1:9, 2:8, 3:7, 4:6, and 5:5, and the mixed solutions of the five ratios were respectively added to the surface of different nanostructured SERS substrates, and the SERS spectra were tested after natural drying. The intensity ratio of the reference peaks selected by the two molecules in the mixed SERS spectra of the two molecules in each ratio was calculated, and the intensity ratio of the two was plotted against the ratio of the number of molecules. The results are shown in FIG5 . The results obtained by the three different structures of silver SERS substrates are all given in FIG5 . It can be seen from the figure that the three test results are not much different. The measured results are linearly fitted, and the slope is the relative SERS scattering factor between the two molecules. It can be seen from the fitting results that the relative SERS scattering factors between the 4-MBA molecules and the 2-MPY molecules obtained by the three different structures of SERS substrates are not much different. It can be considered that the three relative SERS scattering factors are consistent within the range allowed by the experimental error.

In summary, the relative SERS scattering cross section within a molecule and the relative SERS scattering factor between molecules are intrinsic characteristic parameters of the system composed of molecules and SERS substrate materials. They are insensitive to the geometric morphology of the SERS substrate and can be used as general parameters to construct SERS file cards and conduct quantitative analysis.

Surface Enhanced Raman Scattering File Card

Based on the above analysis, the present invention provides a surface enhanced Raman scattering file card, the file card comprising:

Relative scattering cross sections of “surface enhanced Raman scattering” of selected molecules;

Relative scattering factors of Surface Enhanced Raman Scattering of a selected molecule compared to a reference molecule.

According to the surface enhanced Raman scattering file card of the present invention, the file card includes the material and test wavelength of the surface enhanced Raman scattering substrate. Since the values of the two physical quantities are related to the test wavelength of the laser and the material of the SERS substrate, the above information should be noted in the SERS file.

According to the surface enhanced Raman scattering file card of the present invention, the file card includes the normalized surface enhanced Raman scattering spectrum of the selected molecule. The relative SERS scattering cross section within the molecule has different values at different Raman shifts, so it is necessary to give the normalized SERS spectrum of the molecule, and the normalized SERS spectrum here refers to the relative SERS spectrum when the selected SERS reference peak intensity is taken as 100. In order to carry out qualitative and quantitative analysis more simply and directly, the main characteristic peaks and relative intensities of the SERS spectrum and their corresponding Raman vibration modes should be listed in the SERS file card.

The present invention also provides a database, which comprises surface enhanced Raman scattering file cards of more than one molecule.

Quantitative analysis can be conveniently carried out based on the established SERS file card, and the information in the SERS file card can be used for quantitative analysis in different situations. When the SERS characteristic peaks between different molecules are easy to distinguish in the SERS spectrum, the relative intensity ratio between the characteristic peaks of different molecules can be calculated first, and then the relative SERS scattering cross section and relative SERS scattering factor in the SERS file card can be used to realize the quantitative analysis of the relative content between molecules; when the SERS characteristic peaks between different molecules are not easy to distinguish in the SERS spectrum, the normalized SERS spectrum in the SERS file card can be multiplied by the relative SERS scattering factor to obtain the relative SERS spectra of different molecules, and then a suitable algorithm is used to solve the ratio of the relative SERS spectra of each molecule contained in the SERS spectrum to be analyzed, and this ratio is the content ratio of each molecule; for the concentration analysis of the target molecule, a suitable reference molecule can be first selected according to the SERS file card, and then the reference molecule with a known concentration is added to the system to be analyzed, and by measuring the SERS spectrum, the relative content ratio of the reference molecule and the target molecule is first calculated according to the two parameters in the SERS file card, and then the concentration of the target molecule is solved according to the concentration of the added reference molecule and the calculated relative content ratio.

Method for making surface enhanced Raman scattering file card

The present invention provides a method for manufacturing a surface enhanced Raman scattering file card according to the present invention, characterized in that it comprises the following steps:

Determination of relative scattering cross section of surface enhanced Raman scattering

Preferably, for the relative scattering cross section (RCS) of the SERS spectrum of the selected molecule, according to formula (1-4), a certain SERS characteristic peak can be selected as a reference peak, and the relative SERS scattering cross sections of other peaks can be calculated. Specific operation: measure the spectrum of the selected molecule on the SERS substrate, deduct the background signal such as fluorescence from the SERS spectrum obtained by the test, select the characteristic peak with the strongest peak intensity of the SERS spectrum as the reference peak, perform normalization processing, and calculate the relative intensity of any other characteristic peak, which is the relative SERS scattering cross section.

Determination of relative scattering factor of surface enhanced Raman scattering

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

【Quantitative analysis method using SERS file cards】

[The first aspect of the method for quantitative analysis using the SERS file card]

I _i,p represents the pth SERS characteristic peak of the i-th molecule, I _j,q represents the qth SERS characteristic peak of the j-th molecule, RSF _i,j represents the relative SERS scattering factor of the i-th molecule relative to the j-th molecule, RSC _i,p represents the relative SERS scattering cross section of the pth SERS characteristic peak of the i-th molecule, RSC _j,q represents the relative SERS scattering cross section of the qth SERS characteristic peak of the j-th molecule, _Xi represents the content of molecule i, and _Xj represents the content of molecule j. For molecules i and j, when a single characteristic peak is used for quantitative analysis, the following formula (4-1-1) holds:

When multiple characteristic peaks of each molecule are selected: k characteristic peaks are selected for molecule i, and m characteristic peaks are selected for molecule j, then the following formula (4-1-2) is established:

For n molecules, any i, j is in the interval 1 to n, and i is not equal to j, and equation (4-1-2) is valid. At the same time, the total content of all molecules is 100%, that is, the following equation (4-1-3) is valid:

When a molecule j with a known concentration is selected or added as a reference, its concentration is set as C _j , then the concentration of any molecule i can be calculated by the following formula (4-1-4):

Based on the above discussion, we can use SERS file cards to analyze the content and Quantitative analysis of concentration, and the number of selected SERS characteristic peaks is not limited, and only two parameters, relative SERS scattering cross section and relative SERS scattering factor in the SERS file card, are needed. These two parameters can be directly obtained from the corresponding SERS file card, and can also be indirectly calculated from other SERS file cards.

Based on the above, the present invention provides a method for quantitative analysis using a SERS file card, comprising the following steps:

1) Based on the wavelength of the surface enhanced Raman scattering file card (SERS file card) of the present invention and the material of the substrate, a surface enhanced Raman scattering spectrum (SERS spectrum) to be analyzed is measured,

in,

_Xi : the content of the i-th molecule,

_Ci : the concentration of the i-th molecule,

X _j : the content of the jth molecule,

C _j : concentration of the jth molecule,

l and m are the numbers of selected SERS characteristic peaks, respectively.

For different molecules, when molecule i selects one characteristic peak and molecule j selects one characteristic peak, the above formula group I is:

Preferably, when the SERS characteristic peaks between molecules are easily distinguishable in the SERS spectrum, the method of the first aspect is selected for quantitative analysis.

[A second aspect of the method for quantitative analysis using SERS file cards]

The present invention provides a method for quantitative analysis using a SERS file card, comprising the following steps:

in,

_Xi : the content of the i-th molecule,

_Ci : the concentration of the i-th molecule,

X _j : the content of the jth molecule,

C _j : concentration of the jth molecule,

Specifically, S _range,1-p represents the selected 1st to pth interval in the SERS spectrum, RSF _i,j represents the relative SERS scattering factor of the ith molecule relative to the jth molecule, Snorm _i,1-p represents the corresponding 1st to pth interval in the normalized SERS spectrum of the ith molecule, _Xi is the content of molecule i, and the principal component analysis (PCA) algorithm is adopted.

For the PCA algorithm, it can filter system noise and represent the spatial position relationship with a few principal components. For the normalized SERS spectrum in the SERS file card, first calculate the total integrated area value of the selected interval 1 to p, and use Anorm _i,1-p to represent the integrated area value of the 1st to pth interval in the normalized SERS spectrum of the i-th molecule. For n molecules, first calculate the integrated area normalized SERS spectrum Snorm _A,i,1-p after dividing the SERS spectrum of the selected interval by the total integrated area of the selected interval by formula (4-2-1):

Similarly, for S _range,1-p , A _range,1-p is used to represent the total integrated area of the selected interval. Then the SERS spectrum of the selected interval is divided by the total integrated area to obtain the integrated area normalized SERS spectrum S _A,range,1-p as shown in formula (4-2-2):

Then, the load matrix L is directly solved according to Snorm _A,i,1-p. The load matrix L is multiplied by the integral area normalized SERS spectrum of the selected interval to obtain the principal components of the SERS spectrum of the selected interval. For _SA,range,1-p , PC _A,range,1-p is used to represent its principal component, and then equation (4-2-3) holds:
PC _A,range,1-p =L×S _A,range,1-p (4-2-3)

Similarly, for Snorm _A,i,1-p , use PC _A,i,1-p to represent its principal component, then equation (4-2-4) holds:
PC _A,i,1-p = L×Snorm _A,i,1-p (4-2-4)

For S _range,1-p , the relationship (4-2-5) holds:

Where _EF represents the proportionality coefficient related to the SERS enhancement factor. Similarly, for A _{range, 1-p} , the relationship (4-2-6) holds true:

Substituting equations (4-2-5) and (4-2-6) into equation (4-2-2), we can obtain equation (4-2-7):

Convert equation (4-2-7) to equation (4-2-8):

Substituting equation (4-2-1) into equation (4-2-8), we get equation (4-2-9):

Multiply the load matrix by equation (4-2-9) and substitute it into equations (4-2-3) and (4-2-4), and we get equation (4-2-10):

Formula (4-2-10) can be converted into formula (4-2-11):

Similar to equation (1-3), the following equation (4-2-12) also holds:

Substitute the calculated PC _A,i,1-p and PC _A,range,1-p , the relative SERS scattering factor RSFi _,j , the integral area Anorm _i,1-p and Anorm _j,1-p of the selected interval in the normalized SERS spectrum into formula (4-2-11), and combine them with formula (2-12) to solve the content _Xi of each molecule. Similarly, when a molecule j with a known concentration is selected or added as a reference, its concentration is set to _Cj , then the concentration of any i molecules can be solved using formula (4-1-4).

According to the above discussion, when the characteristic peaks between two or more molecules in the SERS spectrum are not easily distinguished, In time division, a specific SERS characteristic peak interval can be selected and combined with the SERS file card to carry out quantitative analysis of the content and concentration of each molecule. The number of selected SERS characteristic peak intervals is not limited. For the SERS file card, two parameters, the relative SERS scattering factor and the normalized SERS spectrum, are needed. These two parameters can be obtained directly from the corresponding SERS file card, or indirectly transferred and calculated from other SERS file cards.

[The third aspect of the method for quantitative analysis using the SERS file card]

in,

_Xi : the content of the i-th molecule,

_Ci : the concentration of the i-th molecule,

X _j : the content of the jth molecule,

C _j : concentration of the jth molecule,

α: the common proportional coefficient introduced,

_Xi,M : When i ranges from 1 to n, the vector _consisting of α×RSA _i,j ×Xi,

Spec _i,M : The matrix formed by Spec _i when i ranges from 1 to n.

Spec _mix : SERS spectrum to be analyzed.

Spec _mix represents the SERS spectrum to be analyzed, Spec _i represents the normalized SERS spectrum in the SERS file card of the i-th molecule, RSF _i,j represents the relative SERS scattering factor of the i-th molecule relative to the j-th molecule, _Xi is the content of molecule i, and the multiple linear regression algorithm is combined with the SERS file card to carry out quantitative analysis.

For the multivariate linear regression algorithm, the estimated value of the regression coefficient becomes more robust as the number of observations increases. For SERS spectra, each Raman shift corresponds to one observation, so the full SERS spectrum is more robust for multivariate linear regression. For Spec _mix , when the interaction between molecules is not significant, the following relationship (4-3-1) holds:

Where α represents the common proportional coefficient caused by changes in test conditions, uniformity of SERS substrate, repeatability, and differences between batches, and μ is a constant term related to noise. According to the principle of least squares method, the estimated values of its parameters can be calculated. For the convenience of expression, Xi _,M represents the column vector represented by α×RSF _i,j ×Xi when _i ranges from 1 to n, and Spec _i,M represents the matrix composed of Spec _i when i ranges from 1 to n. Then, equation (4-3-1) can be expressed as the following equation (4-3-2):
Spec _mix = Spec _i,M × Xi _,M + μ (4-3-2)

According to the principle of least squares method, we can know that the estimated value of Xi _,M is (4-3-3):

Similar to equations (4-1-3) and (4-2-12), the following equation (4-3-4) is also true:

Based on the calculated Xi _,M and formula (4-3-4), the content _Xi of each molecule can be solved; similarly, when a molecule j with a known concentration is selected or added as a reference, its concentration is set to _Cj , then the concentration of any i molecules can be solved using formula (4-1-4).

Preferably, according to the above discussion, when the characteristic peaks of each molecule in the SERS spectrum are difficult to distinguish and are very scattered, the measured SERS full spectrum can be combined with the SERS file card to carry out quantitative analysis of 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, are needed. Similarly, these two parameters can be directly obtained from the corresponding SERS file card, or indirectly calculated from other SERS file cards.

According to the method of the present invention, the laser wavelength and substrate material of the SERS document card are the same as the SERS spectrum, and the SERS spectrum is subtracted from the background.

According to the method of the present invention, the relative SERS scattering factor of a molecule is obtained through the SERS file card of the molecule, or is indirectly transferred and calculated through the SERS file cards of other molecules. The relative SERS scattering factor of the molecule is calculated through the SERS file cards of other molecules through the following chain transfer formula IV:
RSF _i,j =RSF _i,p ×RSF _p,q ×RSF _q,r ×RSF _r,s ×RSF _r,t ×RSF _t,j

Wherein, RSF _i,j is the relative SERS scattering factor of molecule i relative to molecule j, RSF _i,p is the relative SERS scattering factor of molecule i relative to molecule p, RSF _p,q is the relative SERS scattering factor of molecule p relative to molecule q, RSF _q,r is the relative SERS scattering factor of molecule q relative to molecule r, RSF _r,s is the relative SERS scattering factor of molecule r relative to molecule s, RSF _s,t is the relative SERS scattering factor of molecule s relative to molecule t, and RSF _t,j is the relative SERS scattering factor of molecule t relative to molecule j.

According to the method described in the present invention, the number of transmissions of the chain transmission formula IV is ≤6.

Card making example

Card making example 1

Step 1. Prepare a 490nm silver nanorod structure substrate with a purity of 99.99% by using an electron beam tilted deposition method;

Step 2. 10 μL of 4-mercaptobenzoic acid (4-MBA) molecular solution and 10 ^-5 M 2-mercaptopyridine (2-MPY) molecular solution were respectively added dropwise onto the surface of a 10 mm × 10 mm 490 nm silver nanorod structure substrate. After drying naturally, the SERS spectrum was tested using a 785 nm laser, and the fluorescence background signal of the SERS spectrum was subtracted.

Step 3. For the SERS spectrum after deducting the fluorescence background signal in step 2, select the 1074 cm ^-1 characteristic peak of the SERS spectrum of the 4-MBA molecule and the 1002 cm ^-1 characteristic peak of the SERS spectrum of the 2-MPY molecule as reference peaks, take their intensities as 100, and normalize the SERS spectra of the two molecules to obtain the relative SERS scattering cross sections at different Raman shifts;

Step 4. 4-MBA molecules and 2-MPY molecules were mixed respectively, the molecular number ratio of the mixed solution was 1:9, 2:8, 3:7, 4:6, 5:5, and the total concentration of the mixed solution was 10 ^-5 M. 10 μL of each mixed solution was dropped onto the surface of a 490 nm silver nanorod structure substrate with an area of 5 mm × 5 mm. After natural drying, the SERS spectrum was tested and the fluorescence background signal was subtracted;

Step 5. Calculate the 4-MBA molecules in the SERS spectrum after deducting the fluorescence background signal in step 4. The intensity ratio of the characteristic peak of 1074 cm ^-1 and the characteristic peak of 2-MPY molecule 1002 cm ^-1 is used to calculate the linear regression coefficient of the intensity ratio of the two and the molar ratio in the corresponding mixed solution by the least square regression method, which is the relative SERS scattering factor between the two.

Step 6. Combine the substrate material in step 1, the test wavelength of the laser 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 in the form shown in FIG. 6 to establish a SERS file card between the 2-MPY molecule and the 4-MBA molecule.

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

Based on the relative SERS scattering cross section and relative SERS scattering factor obtained from the above discussion and testing, a SERS file card as shown in Figure 6 can be constructed. This figure is just an example. From top to bottom in the figure are the number of the SERS file card, the material and test wavelength of the SERS substrate used, as well as the name of the molecule and the SERS reference peak of the selected molecule. The lower part of the SERS file card continues to give two parameters that can be used for quantitative analysis, the relative SERS scattering factor and the relative SERS scattering cross section, and the normalized SERS spectrum of the molecule and the structural formula of the molecule are listed in the middle. In order to conduct qualitative and quantitative analysis more easily and directly, the table at the bottom of the SERS file card lists the main SERS characteristic peaks of the 2-MPY molecule and their relative intensities, as well as their corresponding Raman vibration modes.

Card making example 2

Step 1. Prepare a 700nm silver nanorod structure substrate with a purity of 99.99% by using an electron beam tilted deposition method;

Step 2. Add 10 μL of 4-mercaptobenzoic acid (4-MBA) molecular solution and 2-mercaptopyridine (2-MPY) molecular solution at a concentration of 10 ^-6 M to the surface of a 700 nm silver nanorod structure substrate with an area of 5 mm × 5 mm, and test its SERS spectrum using a 785 nm laser after drying naturally, and deduct the fluorescence background signal of the SERS spectrum;

Step 4. 4-MBA molecules and 2-MPY molecules were mixed respectively, the molecular number ratio of the mixed solution was 1:9, 2:8, 3:7, 4:6, 5:5, and the total concentration of the mixed solution was 10 ^-6 M. 15 μL of each mixed solution was dropped onto the surface of a 700 nm silver nanorod structure substrate with an area of 5 mm×5 mm, and the SERS spectrum was tested after natural drying, and the fluorescence background signal was subtracted;

Step 5. Calculate the intensity ratio of the characteristic peak of 1074 cm ^-1 of 4-MBA molecule and the characteristic peak of 1002 cm ^-1 of 2-MPY molecule in the SERS spectrum after deducting the fluorescence background signal in step 4, and calculate the linear regression coefficient of the intensity ratio of the two and the molar ratio in the corresponding mixed solution by the least squares regression method, which is the relative SERS scattering factor between the two;

The SEM photograph of the 700 nm nanorod SERS substrate used is shown in FIG1(c), and its reflectivity spectrum is shown in FIG1(a). The calculated relative scattering factor of the SERS of 4-MBA relative to 2-MPY molecules is 6.3±0.8, as shown in FIG5.

Card making example 3

Step 1. Prepare a V-shaped silver nanorod structure substrate with a purity of 99.99% and an arm length of 350 nm by using an electron beam tilted deposition method;

Step 2. Add 15 μL of 4-mercaptobenzoic acid (4-MBA) molecular solution and 2-mercaptopyridine (2-MPY) molecular solution at a concentration of 10 ^-6 M to the surface of a 700 nm silver nanorod structure substrate with an area of 5 mm × 5 mm, and test its SERS spectrum using a 785 nm laser after drying naturally, and deduct the fluorescence background signal of the SERS spectrum;

Step 4. 4-MBA molecules and 2-MPY molecules were mixed respectively, the molecular number ratio of the mixed solution was 1:9, 2:8, 3:7, 4:6, 5:5, and the total concentration of the mixed solution was 10 ^-6 M. 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 natural drying, the SERS spectrum was tested and the fluorescence background signal was subtracted;

The SEM photograph of the 350 nm nanoarm length V-shaped nanorod SERS substrate is shown in Figure 1(d), and its reflectivity spectrum is shown in Figure 1(a). The calculated relative scattering factor of the SERS of 4-MBA relative to the 2-MPY molecule is 6.7±0.9, as shown in Figure 5.

Card making example 4

Step 1. Prepare a gold nanostructure substrate with a purity of 99.99% by using an electron beam tilted deposition method;

Step 2. Add 10 μL of 4-mercaptobenzoic acid (4-MBA) molecular solution and 2-mercaptopyridine (2-MPY) molecular solution at a concentration of 10 ^-5 M to the surface of a gold nanostructure substrate with an area of 5 mm × 5 mm, and after drying naturally, use a 785 nm laser to measure its SERS spectrum, and deduct the fluorescence background signal of the SERS spectrum;

Step 3. For the SERS spectrum after deducting the fluorescence background signal in step 2, select 4-MBA The characteristic peak of 1076 cm ^-1 of the molecular SERS spectrum and the characteristic peak of 1004 cm ^-1 of the 2-MPY molecular SERS spectrum were used as reference peaks, and their intensities were taken as 100. The SERS spectra of the two molecules were normalized to obtain the relative SERS scattering cross sections at different Raman shifts.

Step 4. 4-MBA molecules and 2-MPY molecules were mixed respectively, the molecular number ratio of the mixed solution was 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 was 10 ^-6 M. 15 μL of each mixed solution was dropped onto the surface of a gold nanostructure substrate with an area of 10 mm×10 mm, and the SERS spectrum was tested after natural drying, and the fluorescence background signal was subtracted;

Step 5. Calculate the intensity ratio of the characteristic peak of 1076 cm ^-1 of 4-MBA molecule and the characteristic peak of 1004 cm ^-1 of 2-MPY molecule in the SERS spectrum after deducting the fluorescence background signal in step 4, and calculate the linear regression coefficient of the intensity ratio of the two and the molar ratio in the corresponding mixed solution by the least squares regression method, which is the relative SERS scattering factor between the two;

Step 6. Create a SERS file card for the 2-MPY molecule using the substrate material in step 1, the test wavelength of the laser 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.

The nanostructure used is a 420nm rod-shaped gold SERS substrate. The SERS spectra of mixed solutions with different ratios are shown in Figure 11(a). The shape of the SERS spectrum changes with the ratio of the two molecules. The relationship between the intensity ratio of the characteristic peak of the 1076cm ^-1 of the 4-MBA molecule and the characteristic peak of the 1004cm ^-1 of the 2-MPY molecule and the ratio of the number of molecules is shown in Figure 11(b). The established SERS file card is shown in Figure 11(c).

Example

Example 1

1. A high-purity silver nanostructured SERS substrate was used as the 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 target analysis system;

2. Prepare a mixed solution of 4-MBA and 2-MPY molecules. The ratio of 4-MBA to 2-MPY molecules in the mixed solution is 1:9, 3:7, 1:1, 7:3, 9:1, and the corresponding content of 2-MPY molecules is 90%, 70%, 50%, 30%, 10%, and 2-MPY in the mixed solution is selected. Determine the molecule;

3. Use the SERS substrate selected in step 1 to measure the different mixed solutions prepared in step 2, select a laser wavelength of 785nm, a spot of 80μm, and a power of 15mW, and drop 10μL of the mixed solution onto the SERS substrate in step 1 with a size not exceeding 10mm×10mm, and test the SERS spectrum after it is naturally dried;

4. Subtract the background from the SERS spectra of the different mixed solutions tested in step 3, and find the SERS file card between the 2-MPY molecule and the 4-MBA molecule according to the substrate material in step 1 and the laser wavelength in step 3;

5. According to the SERS file cards of 2-MPY molecules and 4-MBA molecules, obtain the relative SERS scattering factor of 2-MPY molecules relative to 4-MBA molecules, the relative SERS scattering cross section of the characteristic peak of 2-MPY molecules at 1002 cm ^-1 , and the relative SERS scattering cross section of the characteristic peak of 4-MBA molecules at 1074 cm ^-1 ;

6. Extract the characteristic peak intensities of 1074cm ^-1 and 1002cm ^-1 from the SERS spectrum after background subtraction in step 4, and combine them with the relative SERS scattering factor obtained from the SERS file card in step 5, and substitute them into equations (4-1-2) and (4-1-3) to directly calculate the content value of 2-MPY molecules.

The measured SERS spectrum is shown in Figure 8(a). The spectral lines of different 2-MPY molecular contents change sequentially. The 2-MPY molecular content value calculated in step 6 according to the SERS file card and the above formula group is compared with the content value of the actual solution in step 2. The result is shown in Figure 8(b). It can be seen from the figure that the two are in good agreement.

Example 2

2. Prepare a mixed solution of 4-MBA and 2-MPY molecules, take 4-MBA as the added reference substance, the ratio of the number of 4-MBA and 2-MPY molecules in the mixed solution is 1:4, 2:3, 3:2, 4:1, and the corresponding content of 2-MPY molecules is 80%, 60%, 40%, 20%, and take 2-MPY in the mixed solution as the selected molecule, and the concentration of the added reference molecule 4-MBA is 2×10 ^-6 mol/L(M), 4×10 ^-6 M, 6×10 ^-6 M, 8×10 ^-6 M, respectively;

3. Use the SERS substrate selected in step 1 to measure the different mixed solutions prepared in step 2. Select the laser wavelength to be 785nm, the spot to be 80μm, the power to be 30mW, and drop 5μL of the mixed solution. Add it to the SERS substrate with a size not exceeding 5 mm × 5 mm in step 1, wait for it to dry naturally and then test the SERS spectrum;

4. Subtract the background from the SERS spectra of the different mixed solutions tested in step 3, find the SERS file cards between the 2-MPY molecule and the 4-MBA molecule according to the substrate material in step 1 and the laser wavelength in step 3, and calculate their relative SERS scattering factors by transferring molecular profiles;

5. According to the SERS file cards of 2-MPY molecules and 4-MBA molecules, the relative SERS scattering factor of 2-MPY molecules relative to 4-MBA molecules is indirectly calculated, and the normalized SERS spectra of 2-MPY molecules and 4-MBA molecules are obtained;

6. According to the SERS spectra of 2-MPY and 4-MBA molecules, their characteristic peaks are most concentrated between 900-1250 cm ^-1 . Therefore, the SERS spectrum after deducting the background in step 4 is selected to have a characteristic peak range of 988 cm ^-1 to 1202 cm ^-1 , and combined with the relative SERS scattering factor and normalized SERS spectrum obtained from the SERS file card in step 5, they are brought into equations (4-2-11) and (4-2-12), and the content value of the 2-MPY molecule can be directly calculated. Then, the concentration of the 2-MPY molecule can be calculated by substituting it together with the concentration of the reference molecule 4-MBA into equation (4-1-4);

The measured SERS spectrum is shown in Figure 9(a). The spectral lines of different 2-MPY molecular contents change sequentially. The spectrum of the selected interval is normalized by integral area, and the calculated first principal component is shown in Figure 9(b). Finally, the 2-MPY molecular content value calculated according to the SERS file card and the formula group is shown in Figure 9(c). The result is consistent with the content value of the actual solution in step 2. The 2-MPY molecular concentration calculated according to the 4-MBA molecule added as the reference molecule in step 2 is shown in Figure 9(d), which is consistent with the actual molecular concentration.

Example 3

2. Prepare a mixed solution of 4-MBA and 2-MPY molecules. The ratio of the number of 4-MBA and 2-MPY molecules in the mixed solution is 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and the corresponding content of 2-MPY molecules is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, with 4-MBA molecules as the added reference molecules, the concentrations of 4-MBA molecules were 1×10 ^-6 M, 2×10 ^-6 M, 3×10 ^-6 M, 4×10 ^-6 M, 5×10 ^-6 M, 6×10 ^-6 M, 7×10 ^-6 M, 8×10 ^-6 M, 9×10 ^-6 M;

3. Use the SERS substrate selected in step 1 to measure the different mixed solutions prepared in step 2, select a laser wavelength of 785nm, a spot of 80μm, and a power of 15mW, and drop 20μL of the mixed solution onto the SERS substrate in step 1 with a size not exceeding 10mm×10mm, and test the SERS spectrum after it is naturally dried;

6. The full spectrum range of the SERS spectrum selector after deducting the background in step 4, combined with the relative SERS scattering factor indirectly calculated from the SERS file card in step 5, is substituted into equations (4-3-3) and (4-3-4) to directly calculate the content value of the 2-MPY molecule, and then substituted into equation (4-1-4) together with the concentration of the added reference molecule 4-MBA to calculate the concentration of the 2-MPY molecule.

The calculated 2-MPY molecular content value is shown in Figure 10(a), which is consistent with the content value of the actual solution in step 2. The 2-MPY molecular concentration calculated according to formula (4-1-4) and the 4-MBA molecules added as a reference in step 2 is shown in Figure 10(b), which is consistent with the actual 2-MPY molecular concentration.

Example 4

1. A high-purity gold nanostructured SERS substrate was used as the 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 target analysis system;

3. Use the SERS substrate selected in step 1 to measure the different mixed solutions prepared in step 2, select a laser wavelength of 785nm, a spot of 80μm, and a power of 60mW, and drop 10μL of the mixed solution onto the SERS substrate in step 1 with a size not exceeding 5mm×5mm, and test the SERS spectrum after it is naturally dried;

The calculated 2-MPY molecular content value is shown in Figure 12(a), which is consistent with the content value of the actual solution in step 2. The 2-MPY molecular concentration calculated according to formula (4-1-4) and the 4-MBA molecules added as a reference in step 2 is shown in Figure 12(b), which is consistent with the actual 2-MPY molecular concentration.

By comparing Figure 10 in Example 3 with Figure 12 in Example 4, it can be seen that although the relative SERS scattering factors of 4-MBA molecules and 2-MPY molecules on different materials are different, as long as the SERS file cards of corresponding materials and laser wavelengths are used for quantitative analysis, their correct molecular content and concentration can be obtained, and the quantitative analysis results obtained on SERS substrates of different materials are consistent.

It can be seen from the above specific embodiments that the SERS file card provided by the present invention, the method for establishing the SERS file card and the method for applying the SERS file card in quantitative analysis, by defining and measuring two physical quantities, namely, the intramolecular relative SERS scattering cross section and the intermolecular relative SERS scattering factor, is constructed to be used for quantitative analysis. The SERS file cards, in which all parameters are universal among SERS substrates with the same material properties but different geometric morphologies, can eliminate the adverse effects of factors such as uniformity of SERS substrates, batch differences, fluctuations in test conditions, and changes in geometric morphology on quantitative analysis. They can be used to construct a quantitative analysis database in the SERS field, and can make SERS technology, like X-ray diffraction technology, have a standard powder diffraction file card library, which can facilitate various quantitative analyses. It has broad application prospects and plays an important basic supporting role in the field of quantitative analysis of trace molecules.

The method for quantitative analysis using SERS file cards according to the present invention is described above by way of example with reference to the accompanying drawings. However, it should be understood by those skilled in the art that various improvements can be made to the method for quantitative analysis using SERS file cards according to the present invention without departing from the content of the present invention, and all of them fall within the scope of protection of the present invention.

Claims

A surface enhanced Raman scattering file card, the file card comprising:

Relative scattering cross sections of “surface enhanced Raman scattering” of selected molecules;

Relative scattering factors of Surface Enhanced Raman Scattering of a selected molecule compared to a reference molecule.
The surface enhanced Raman scattering file card according to claim 1, characterized in that the file card includes the material and test wavelength of the surface enhanced Raman scattering substrate.
The surface enhanced Raman scattering file card according to claim 1 or 2, characterized in that the file card includes selected reference peaks of selected molecules and reference molecules.
The surface enhanced Raman scattering file card according to claim 1 or 2, characterized in that the file card includes a normalized surface enhanced Raman scattering spectrum of a selected molecule.
A method for making a surface enhanced Raman scattering file card according to any one of claims 1 to 4, characterized in that it comprises the following steps:

Determine the "surface enhanced Raman scattering" spectrum of the selected molecule, select the characteristic peak with the strongest peak intensity in the spectrum as the reference peak, calculate the relative value of the peak intensity of other characteristic peaks and the peak intensity of the reference peak, and obtain the relative scattering cross section of the "surface enhanced Raman scattering";

The "surface enhanced Raman scattering" spectra of the selected molecule and the reference molecule are measured respectively, and the characteristic peaks with the strongest peak intensity 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 "surface enhanced Raman scattering" spectrum of the mixture of the selected molecule and the reference molecule is measured, and the relative value of the peak intensity of the reference peak of the selected molecule and the reference molecule is calculated to obtain the relative scattering factor of the "surface enhanced Raman scattering".
The production method according to claim 5 is characterized in that after selecting the characteristic peak with the strongest peak intensity in the spectrum as the reference peak, the intensity value of the reference peak is set to 100, and the peak intensities of other characteristic peaks are normalized. The peak intensities of other characteristic peaks after normalization are the relative scattering cross sections of "surface enhanced Raman scattering".
The preparation method according to claim 5 or 6 is characterized in that the selected molecule and the reference molecule are mixed in different molar ratios, and the "surface enhanced Raman scattering" spectra of different molar ratios are measured respectively, and the characteristic peaks with the strongest peak intensity 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, and the intensities of the reference peaks of the selected molecule and the reference molecule are calculated. The linear regression coefficient between the intensity ratio and the molar ratio is calculated by the least square regression method, which is the relative scattering factor of "surface enhanced Raman scattering" between molecules.
The manufacturing method according to claim 7 is characterized in that the intensity ratio is in the range of 0.1 to 10.
The preparation method according to any one of claims 5 to 8 is characterized in that, when measuring the "surface enhanced Raman scattering" spectrum, the molecule to be tested is dripped onto the surface of the substrate in the form of a solution, and after the solvent is naturally dried, its spectrum is tested, and the fluorescence background signal is subtracted to obtain the "surface enhanced Raman scattering" spectrum.
The preparation method according to claim 9, characterized in that the total concentration of the solution is 10 -8 to 10 -5 mol/L, and the average intra-plane volume range of the dropwise addition amount is 0.1 to 5 μL/mm 2 .
A method for quantitative analysis using a SERS file card, characterized in that it comprises the following steps:

1) measuring a surface enhanced Raman scattering spectrum (SERS spectrum) to be analyzed based on the test 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, select 1 to n characteristic peaks for molecule i, and select 1 to m characteristic peaks for molecule j, and use the following formula group I to solve:

in,

I i,p : peak intensity of the pth SERS characteristic peak of the i-th molecule,

I j,q : peak intensity of the qth SERS characteristic peak of the jth molecule,

RSF i,j : Relative SERS scattering factor of the i-th molecule relative to the j-th molecule,

For k molecules, any i, j belongs to the interval [1, k], and i is not equal to j;

Xi : the content of the i-th molecule,

Ci : the concentration of the i-th molecule,

X j : the content of the jth molecule,

C j : concentration of the jth molecule,

RSC i,p : relative SERS scattering cross section of the pth SERS characteristic peak selected by the i-th molecule,

RSC j,q : relative SERS scattering cross section of the qth SERS characteristic peak selected by the jth molecule,

l and m are the numbers of selected SERS characteristic peaks, respectively.
A method for quantitative analysis using a SERS file card, characterized in that it comprises the following steps:

1) measuring a surface enhanced Raman scattering spectrum (SERS spectrum) to be analyzed based on the 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 n molecules, select spectral intervals 1 to p of the SERS spectrum and use the following formula group II to solve:

in,

Xi : the content of the i-th molecule,

Ci : the concentration of the i-th molecule,

X j : the content of the jth molecule,

C j : concentration of the jth molecule,

RSF i,j : relative SERS scattering factor of molecule i relative to molecule j,

Anorm i,1-p : The integrated area of the SERS spectrum intervals 1 to p selected in the i-th molecule SERS file card,

Anorm j,1-p : The integrated area of the SERS spectrum intervals 1 to p selected in the j-th molecule SERS file card,

PC A,i,1-p : The principal component value corresponding to the integrated area normalized SERS spectrum of the i-th molecule in the spectral interval 1 to p,

PC A,range,1-p : The principal component value corresponding to the integrated area normalized SERS spectrum of the spectrum to be analyzed in the spectral range 1 to p.
A method for quantitative analysis using a SERS file card, characterized in that it comprises the following steps:

1) measuring a surface enhanced Raman scattering spectrum (SERS spectrum) to be analyzed based on the 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 n molecules, the complete spectral interval of the SERS spectrum or a partial spectral interval of the SERS spectrum is selected as the complete spectral interval, and the following formula group III is used for solution:

in,

Xi : the content of the i-th molecule,

Ci : the concentration of the i-th molecule,

X j : the content of the jth molecule,

C j : concentration of the jth molecule,

RSF i,j : relative SERS scattering factor of molecule i relative to molecule j,

α: the common proportional coefficient introduced,

Spec i : the normalized SERS spectrum of the i-th molecule in the SERS file card,

Xi,M : When i ranges from 1 to n, the vector consisting of α×RSF i,j ×Xi,

Spec i,M : The matrix formed by Spec i when i ranges from 1 to n.

Spec mix : SERS spectrum to be analyzed.
The method according to any one of claims 11 to 13 is characterized in that the laser wavelength and substrate material of the SERS file card are the same as the SERS spectrum, and the SERS spectrum is subtracted from the background.
The method according to any one of claims 11 to 13, characterized in that the relative SERS scattering factor of a molecule is obtained through a SERS file card of the molecule, or is indirectly transferred and calculated through SERS file cards of other molecules, and the relative SERS scattering factor of the molecule is calculated through the SERS file cards of other molecules by the following chain transfer formula IV:
RSF i,j =RSF i,p ×RSF p,q ×RSF q,r ×RSF r,s ×RSF r,t ×RSF t,j

Wherein, RSF i,j is the relative SERS scattering factor of molecule i relative to molecule j, RSF i,p is the relative SERS scattering factor of molecule i relative to molecule p, RSF p,q is the relative SERS scattering factor of molecule p relative to molecule q, RSF q,r is the relative SERS scattering factor of molecule q relative to molecule r, RSF r,s is the relative SERS scattering factor of molecule r relative to molecule s, RSF s,t is the relative SERS scattering factor of molecule s relative to molecule t, and RSF t,j is the relative SERS scattering factor of molecule t relative to molecule j.
The method according to claim 15 is characterized in that the number of transmissions of the chain transmission formula IV is ≤ 6.