CN112285092A

CN112285092A - Safflower dyeing rapid detection method based on surface enhanced Raman spectroscopy

Info

Publication number: CN112285092A
Application number: CN202011184722.9A
Authority: CN
Inventors: 范骁辉; 程翼宇; 王毅
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-10-29
Filing date: 2020-10-29
Publication date: 2021-01-29
Anticipated expiration: 2040-10-29
Also published as: CN112285092B

Abstract

The invention discloses a safflower dyeing rapid detection method based on a surface enhanced Raman spectroscopy technology, and belongs to the field of traditional Chinese medicine quality detection. The method comprises the following steps: (1) soaking a sample to be detected in an ethanol solution, and collecting an extracting solution; (2) a nylon membrane is used as an extraction membrane to adsorb dye substances in the extraction solution; (3) eluting the dye substance adsorbed on the nylon membrane by using an alkaline aqueous solution to prepare a solution to be detected; (4) adding mercaptoethylamine into the gold nanorod sol, mixing, adding the solution to be detected, mixing to obtain a detection solution, performing Raman spectrum collection to obtain a Raman spectrogram of a sample to be detected, and identifying the dye types or performing quantitative detection by contrasting with a standard solution spectrogram. The invention adopts the nylon membrane to separate the coloring agent from the ethanol extract of the safflower; and further, the AuNRs have good Raman enhancement effect on the acid dye, and the handheld Raman spectrometer is adopted, so that the detection and analysis of the dye can be realized.

Description

Safflower dyeing rapid detection method based on surface enhanced Raman spectroscopy

Technical Field

The invention belongs to the field of traditional Chinese medicine quality detection, and particularly relates to a method for detecting safflower dyeing based on a Surface Enhanced Raman Spectroscopy (SERS) technology.

Background

Safflower, also known as Daphne giraldii Nitsche and Caohuahong, is a traditional Chinese medicinal material in China and has the effects of promoting blood circulation to remove blood stasis, relaxing muscles and tendons, activating collaterals, preventing and treating cardiovascular and cerebrovascular diseases and the like. In recent years, a large amount of safflower is applied to clinical treatment, the price is increased, dyed safflower is found in the market to be better, and great potential safety hazards are brought to a user. The drug inspection supplement inspection method and inspection project approval parts of the State food and drug administration published by the State food and drug administration (CFDA) No. 2013006 and No. 2014016 respectively relate to 6 coloring agents such as golden orange II and the like to monitor the quality of safflower medicinal materials.

At present, the safflower identification is mainly artificial identification. The Yihong soldier introduces a method for quickly identifying dyed safflower, which identifies the dyed safflower from the aspects of shape, color, smell and water test, and carefully inspects to find that the flower column and the flower crown barrel are cracked although the color of the extracted and dyed safflower is similar to that of the certified safflower. The shape of each part of the anther is curled, not stretched and the color is fuzzy; the water extract of the certified safflower is golden yellow, and the water extract of the dyed sample is orange red and the flower fades quickly (a fast identification of dyed safflower, china pharmaceutical industry, 2012, vol. 21, No. 4). The manual identification has high professional requirements, results are easily influenced by subjective factors and objective environments, the manual identification is difficult to popularize in daily life, and the specific type of the dyeing agent cannot be identified.

Compared with the traditional sense organ, the intelligent detection instrument can realize quick, accurate and objective detection. The traditional detection method for evaluating the quality of the traditional Chinese medicine mainly applies the technologies of chromatography, mass spectrum, atomic absorption spectrum and the like, the pretreatment time of a sample is long, most of the traditional detection methods depend on large-scale instruments, and the traditional detection methods are not suitable for on-site rapid detection. The Surface Enhanced Raman Spectroscopy (SERS) technology absorbs molecules to be detected to the surface of gold and silver nanoparticles to enable Raman signals of the gold and silver nanoparticles to be exponentially enhanced, has the advantages of high sensitivity, strong specificity, no fluorescence bleaching, no need of complex pretreatment and the like, is widely applied to the fields of chemistry, material analysis, environment and food safety, biological medicine and the like, and has good potential of rapid detection of the quality of traditional Chinese medicines.

Because the colors of the dyes are mostly similar, especially the trace dyes are nearly colorless, and the added dyes are difficult to identify by adopting a visual inspection method, how to realize the field dye identification and quantitative detection by utilizing the SERS technology is a problem to be solved by the technical personnel in the field.

Disclosure of Invention

The invention aims to solve the problem of safflower dyeing adulteration and provides a detection method capable of quickly and accurately identifying safflower dyeing so as to monitor the quality of a safflower medicinal material.

In order to achieve the purpose, the invention adopts the following technical scheme:

a safflower dyeing rapid detection method based on a surface enhanced Raman spectroscopy technology comprises the following steps:

(1) soaking a sample to be detected in an ethanol solution, standing and extracting, and collecting an extracting solution;

(2) a nylon membrane is taken as an extraction membrane, and a micro-extraction technology is utilized to adsorb dye substances in the extraction solution;

(3) eluting the dye substance adsorbed on the nylon membrane filter head by using an alkaline aqueous solution to obtain an eluent, and adding an acid solution with the same concentration into the eluent to adjust back the pH value to prepare a solution to be detected;

(4) adding mercaptoethylamine into the gold nanorod sol, mixing, adding the solution to be detected, mixing to obtain a detection solution, performing Raman spectrum collection to obtain a Raman spectrogram of a sample to be detected, and identifying the dye types or performing quantitative detection by contrasting with a standard solution spectrogram.

The safflower dye of the invention is an acid dye, and specifically comprises golden orange II, lemon yellow, azorubine, sunset yellow, acid red 73 and carmine.

The invention combines the film micro-extraction technology to separate the coloring agent from the safflower extract. Research shows that compared with a mixed cellulose Membrane (MCE) filter head and a polyether sulfone membrane (PES) filter head, the Nylon membrane (Nylon) filter head adopted by the invention has a better adsorption effect on the dye, the Nylon membrane is polyamide, the acid dye added in safflower can be fully adsorbed, and natural pigment and other molecules in safflower extract basically cannot be adsorbed and trapped by the membrane, so that convenient extraction and separation are realized.

In the step (1), ethanol solution with the volume percentage of 70% is used as leaching liquor, and the material ratio is 1 g: 20mL, 5min extraction time.

In the step (2), the micro-extraction comprises: absorbing the extract with a syringe, and extruding the waste liquid through a nylon membrane filter head to make the dye adsorbed on the filter membrane. The nylon membrane filter head can adopt a disposable needle filter, and the aperture of the filter membrane is 0.22 mu m. Specifically, the specification of the nylon membrane filter head is 25mm × 0.22 μm.

Further, in the step (3), before elution by using the alkaline aqueous solution, a formic acid solution with the volume percentage of 10-40% is filtered through a nylon membrane for washing, and then the nylon membrane is washed by water. The formic acid solution is used for eluting a small amount of adsorbed colored substances such as natural pigments on the clean film, and then the residual formic acid on the film is eluted by clear water through the nylon film. More preferably, the concentration of the formic acid solution is 20%.

The elution dye adopts NaOH aqueous solution with the concentration of 1-100 mM. The concentration of the NaOH aqueous solution is preferably 10 to 100mM, and more preferably 10 mM.

The preparation of the solution to be tested can be as follows: sucking 0.5ml of dyed safflower extract by using a 1ml syringe, passing through a membrane filter head and extruding waste liquid to make dye adsorbed on a filter membrane; then, 1ml of 20% formic acid solution is absorbed by a 1ml syringe, a rubber plug is used for blocking the water outlet of the filter head, the syringe is pumped and beaten for 10 times back and forth for eluting a small amount of adsorbed colored substances such as natural pigments on the clean film, and 5ml of water is absorbed by a 10ml syringe for eluting residual formic acid in the filter head; and finally, sucking 0.5ml of 10mM NaOH aqueous solution by using a 1ml syringe to completely elute the dye adsorbed on the membrane, and adding 0.5ml of hydrochloric acid solution with the same concentration into the collected solution to adjust the pH value to obtain the solution to be detected.

In the step (4), a detection sensor based on a surface enhanced Raman spectroscopy technology is constructed, and the rapid inspection and analysis of the quality of the safflower medicinal material are realized. The preparation method of the gold nanorod sol comprises the following steps:

a. preparation of gold seed solution: adding a chloroauric acid solution into a hexadecyl trimethyl ammonium bromide aqueous solution, adding a freshly prepared and ice-bath precooled sodium borohydride solution, uniformly mixing, and reacting to prepare a CTAB solution containing the gold seed solution;

b. preparing a growth solution: adding a chloroauric acid solution and a silver nitrate solution into a hexadecyl trimethyl ammonium bromide aqueous solution, shaking and uniformly mixing, sequentially adding HCl and freshly prepared ascorbic acid, and enabling the solution to become colorless and transparent to prepare a growth solution;

c. and (c) adding the gold seed solution prepared in the step (a) into the growth solution prepared in the step (b), standing overnight, washing, and concentrating to obtain the gold nanorod sol.

And c, adding the gold seed solution in the step c into the growth solution, standing overnight at 27 ℃, washing with ultrapure water, and concentrating by 25 times to obtain the gold nanorod sol. The relative consistency of the concentration of each batch is ensured by preparing gold nanorod solution with the same concentration factor.

In the step (4), the gold nanorod sol and the concentration are 10^-5The mercaptoethylamine is mixed in an equal volume of M to 1M, and the concentration of the mercaptoethylamine is preferably 0.001M to 0.1M, more preferably 0.01M. Research shows that good Raman enhancement effect requires that molecules of a substance to be detected are adsorbed on the surface of the nanoparticles or the distance is close enough, and the gold and silver nanospheres prepared by the traditional method are difficult to fully contact with acid dye molecules added into safflower due to negative charges on the surface, so that good Raman enhancement effect cannot be generated. According to the invention, micromolecular positive electricity modified gold nanorod (AuNRs) sol with optimized concentration is used as an SERS detection substrate, so that the gold nanorod is closely adsorbed with acid dye with negative electricity groups, and the SERS detection substrate has good SERS detection performance.

And scanning the Raman spectrum by adopting a handheld Raman instrument, specifically, dripping detection liquid on the surface of a blank silicon wafer, standing and then carrying out Raman spectrum collection. Raman spectrum parameter conditions are as follows: the power is 300mW, the integration time is 2s, and the integration times are 1.

And taking the SERS spectrum of the dye standard solution as a comparison spectrum, comparing the similarity of the sample spectrum and the comparison spectrum by adopting a correlation coefficient method, and identifying the safflower dye. Specifically, a Pearson correlation coefficient method is adopted to calculate the similarity, the similarity of 0.9 is taken as a judgment standard, and when the similarity is greater than 0.9, the corresponding dye is judged; meanwhile, the types of the safflower dyes are identified by combining the characteristic peaks.

The invention has the beneficial effects that:

the invention adopts the nylon membrane filter head to separate the coloring agent from the ethanol extract of the safflower, and can directly judge whether the safflower is dyed according to the color of the dye extract; and then the AuNRs modified by the micromolecule positive electricity has a good Raman enhancement effect on the acid dye, and a hand-held Raman spectrometer is adopted to quickly acquire a Raman spectrogram of the dye extracting solution of the dyed safflower medicinal material, so that the detection and analysis of the dye can be realized.

Drawings

FIG. 1 is a flow chart of a specific experiment of the present invention.

FIG. 2 shows the results of optimizing the micro-extraction conditions of lemon yellow dye; wherein (a) is the adsorption rate of different membrane materials to the lemon yellow dye; (b) the adsorption rate of NaOH solutions with different concentrations on the lemon yellow dye is shown.

FIG. 3 is a visual inspection of the eluates of lemon yellow dye at different concentrations; wherein, the lemon yellow dyeing concentration is 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1mg/g in sequence from left to right.

FIG. 4 is a representation of an AuNRs substrate; wherein, (a) is a TEM image of an AuNRs substrate; (b) is a UV-Vis spectrum diagram of an AuNRs substrate.

FIG. 5 shows the result of the repeatability test of AuNRs substrates; wherein, (a) is the repeatability of random point picking investigation of the AuNRs substrate surface; (b) repeatability was investigated for AuNRs substrate batches.

Fig. 6 shows the stability of AuNRs substrates.

Fig. 7 is a SERS spectrum of a lemon yellow dye standard solution, wherein (a) is a 50ppm concentration lemon yellow standard solution SERS spectrum; (b) the SERS spectra of the lemon yellow standard solution are different in concentration gradient.

FIG. 8 shows the molecular structural formulas of six dyes; wherein, (a) lemon yellow; (b) sunset yellow; (c) golden orange II; (d) acid red 73; (e) carmine; (f) and (3) azo jade red.

FIG. 9 is a comparison graph of lemon yellow SERS spectra and a Raman spectrum calculated by DFT theory.

Fig. 10 is a graph of differential SERS from lemon yellow staining at different concentrations.

FIG. 11 shows the result of optimizing the dye micro-extraction conditions of aurantium II; wherein (a) is the adsorption rate of different membrane materials to the golden orange II dye; (b) the adsorption rate of NaOH solutions with different concentrations on the golden orange II dye is shown.

FIG. 12 is a visual inspection of different concentrations of dye eluate of aurantium II; wherein, the dyeing concentration of the golden orange II is 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1mg/g in sequence from left to right.

FIG. 13 is a SERS spectrum of a standard solution of a dye, Citrus aurantium II, wherein (a) is a SERS spectrum of a standard solution of Citrus aurantium II at a concentration of 50 ppm; (b) SERS spectra of different concentration gradient golden orange II standard solutions.

FIG. 14 is a comparison chart of Raman peak assignment of orange II dye molecules.

FIG. 15 is a graph of SERS by staining with different concentrations of aurantium II.

FIG. 16 shows the results of the micro-extraction conditions for azo rubine dyes; wherein (a) is the adsorption rate of different membrane materials to azorubine dye; (b) the adsorption rate of the NaOH solution with different concentrations on the azo rubine dye.

FIG. 17 is a visual inspection of azorubine dye eluents of different concentrations; wherein, the azo rubine dyeing concentration is 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1mg/g in sequence from left to right.

Fig. 18 is a SERS spectrum of an azorubine dye standard solution, wherein (a) is a 50ppm concentration azorubine standard solution SERS spectrum; (b) the SERS spectra of the azorubine standard solution with different concentration gradients.

FIG. 19 is a comparison chart of Raman peak assignment of molecules of azorubine dye.

Fig. 20 is a graph of differential SERS from different concentrations of azorubine staining.

FIG. 21 shows the results of optimizing the sunset yellow dye microextraction conditions; wherein (a) is the adsorption rate of different membrane materials to the sunset yellow dye; (b) the adsorption rates of NaOH solutions with different concentrations on the sunset yellow dye are shown.

FIG. 22 is a visual inspection of sunset yellow dye eluates at different concentrations; wherein, the sunset yellow dyeing concentration is 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1mg/g in sequence from left to right.

Fig. 23 is a SERS spectrum of a sunset yellow dye standard solution, wherein (a) is a 50ppm concentration SERS spectrum of the sunset yellow standard solution; (b) SERS spectra of sunset yellow standard solutions with different concentration gradients.

FIG. 24 is a comparison chart of molecular Raman peak assignments of sunset yellow dyes.

Fig. 25 is a graph of differential SERS from sunset yellow staining at different concentrations.

FIG. 26 shows the results of the micro-extraction conditions for acid Red 73 dye; wherein (a) is the adsorption rate of different membrane materials to acid red 73 dye; (b) the adsorption rate of NaOH solution with different concentrations to acid red 73 dye.

FIG. 27 is a visual inspection of different concentrations of acid Red 73 dye eluate; wherein the dyeing concentration of acid red 73 is 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1mg/g in sequence from left to right.

Fig. 28 is a SERS spectrum of an acid red 73 dye standard solution, wherein (a) is a 50ppm concentration acid red 73 standard solution SERS spectrum; (b) the SERS spectra of the acidic red 73 standard solution are obtained in different concentration gradients.

FIG. 29 is a comparison chart of Raman peak assignment of acid red 73 dye molecules.

Fig. 30 is a graph of differential SERS from different concentrations of acid red 73 staining.

FIG. 31 shows the results of optimizing the condition of the carmine dye microextraction; wherein (a) is the adsorption rate of different membrane materials to carmine dye; (b) the adsorption rate of NaOH solution with different concentrations on carmine dye.

FIG. 32 is a visual inspection of different concentrations of carmine dye eluates; wherein the dyeing concentration of the carmine from left to right is 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1mg/g in sequence.

Fig. 33 is a SERS spectrum of a carmine dye standard solution, wherein (a) is a 50ppm concentration carmine standard solution SERS spectrum; (b) SERS spectra of carmine standard solutions with different concentration gradients.

FIG. 34 is a comparison chart of Raman peak assignments of carmine dye molecules.

FIG. 35 is a graph of differential SERS staining for cochineal at different concentrations.

Detailed description of the preferred embodiment

The present invention will be further described with reference to the following specific examples.

Example 1 lemon yellow stained safflower assay

1. Preparation of lemon yellow dyeing safflower extract

Taking 1g of undyed safflower (natural safflower medicinal material is not added with dye through high performance liquid chromatography detection), adding 1mg/ml of lemon yellow dye into 1ml of undyed safflower, stirring and kneading for 1min to fully and uniformly dye the dye solution on the surface of the safflower, repeating the above operations to required experimental amount, transferring and flatly spreading the mixture in a tray, drying the mixture in a 60 ℃ oven for 1h, and taking out the dried mixture to obtain the safflower added with the 1mg/g of lemon yellow dye.

Dyeing at other concentrations by using lemon yellow dye solution with corresponding concentration, and obtaining safflower dyed by the lemon yellow dye with series concentrations of 0.1, 0.2, 0.4, 0.6, 0.8 and 1 mg/g.

Referring to the dye extraction method recorded in the safflower medicinal material (decoction pieces) supplement inspection method and inspection items promulgated by the national food and drug administration, 1g of lemon yellow-dyed safflower medicinal material is taken and placed in a 50ml centrifuge tube, 20ml of 70% ethanol solution is added, the mixture is kept stand for 5min, the mixture is shaken and uniformly mixed, and the extracting solution is poured into another centrifuge tube for treatment.

2. Micro-extraction condition optimization

Dye standard curve preparation: preparing lemon yellow dye stock solution with the concentration of 5mg/ml (5000ppm), diluting the lemon yellow dye stock solution to 100, 50, 25, 12.5, 6.25 and 3.125ppm step by step, taking 100 mu l of lemon yellow dye solution with the concentration of 50ppm to a 96-well plate, scanning an absorbance value in a wavelength range of 700nm by using an enzyme reader, determining the maximum absorption wavelength of the lemon yellow dye, detecting the absorbance value of the lemon yellow dye solution with each concentration at the wavelength, and making a standard curve about the concentration and the absorbance value of the lemon yellow dye for the dye concentration calculation in the processes of membrane material selection and eluent optimization.

Selecting a membrane material: 0.5ml of 50ppm of lemon yellow dye solution is sucked by a syringe each time, and is filtered by a Nylon (Nylon) membrane, the dye concentration of the filtrate is detected, and the adsorption rate of the filter head to various dyes is calculated. The experimental operation was repeated by replacing the filter with a mixed cellulose Membrane (MCE) filter and a polyethersulfone membrane (PES) filter to obtain the respective adsorption rates. The 3 filter heads with the specification of 25mm × 0.22 μm are all purchased from Shanghai' an spectral scientific instruments, Inc.

As shown in fig. 2(a), the nylon membrane has the highest adsorption rate to the lemon yellow dye, so that the nylon membrane is selected as the micro-extraction membrane material in the subsequent experiments.

And (3) optimizing the concentration of the NaOH eluent: the filter membrane on which the dye was adsorbed was eluted with 0.5ml of NaOH solutions having different concentrations (1, 10, 100mM), and the dye concentration of the eluate was measured to calculate the dye recovery rate. As shown in fig. 2(b), the adsorption rates of the 10mM and 100mM NaOH solutions to the lemon yellow dye were high, but the adsorption rate was not significantly improved at 100mM concentration, so the 10mM NaOH solution was selected for elution in the subsequent experiments.

3. Lemon yellow staining safflower eluate acquisition

Sucking 0.5ml of lemon yellow dyed safflower extract by using a 1ml syringe, passing through a nylon membrane filter head and extruding waste liquid to make the dye adsorbed on the filter membrane; then, 1ml of formic acid solution with the concentration of 20% is absorbed by a 1ml syringe, a self-made rubber plug is used for plugging the water outlet of the filter head, the syringe is pumped back and forth for 10 times for eluting a small amount of adsorbed colored substances such as natural pigments on the nylon membrane, and 5ml of water is absorbed by a 10ml syringe for eluting residual formic acid in the filter head; and finally, sucking 0.5ml of 10mM NaOH aqueous solution by using a 1ml syringe to completely elute the lemon yellow dye adsorbed on the membrane, and adding 0.5ml of hydrochloric acid solution with the same concentration into the collected solution to adjust the pH value to obtain the lemon yellow dyed safflower eluate.

4. Quick visual judgment of lemon yellow dyed safflower eluate

And directly observing whether the color of the lemon yellow dyeing safflower eluate is colorless and transparent by naked eyes, and judging whether the safflower is dyed. As shown in figure 3, the concentration of lemon yellow dye in the centrifuge tubes arranged in the figure is 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1mg/g from left to right in sequence, and it is observed that the undyed safflower eluate is almost colorless and transparent after being extracted by a nylon membrane, and the color of the lemon yellow dyed safflower extract deepens along with the increase of the concentration of the dye.

5. Preparation and characterization of AuNRs

5.1 Synthesis of AuNRs:

(1) preparation of gold seed solution: 50mL of 0.1M aqueous cetyltrimethylammonium bromide (CTAB) solution was prepared and placed in a 27 ℃ water bath for use. 9.75mL was taken and 0.25mL of 10mM chloroauric acid (HAuCl) was added₄·3H₂O) solution, then adding 0.6mL of 10mM sodium borohydride which is freshly prepared and precooled by an ice bath, whirling for 2min, and carrying out water bath at 27 ℃ for 2 h. (2) Preparing a growth solution: 40mL of 0.1M CTAB solution was taken and HAuCl was added₄·3H₂O (10mM,2mL) and AgNO₃(10mM,0.4mL), shaken well and added HCl (1M,0.8mL) followed by freshly prepared ascorbic acid (0.1M,0.32mL) the solution became colorless and transparent indicating that the yellow Au (III) in the solution was reduced to colorless Au (I). (3) Mu.l of a 0.1M CTAB solution containing 10% gold seed solution was added to the growth solution and allowed to stand at 27 ℃ overnight. The AuNRs solution obtained is centrifugally washed for 2 times with ultra-pure water at 4000rpm for 10min, and is resuspended to obtain an AuNRs concentrated solution which is stored at 4 ℃.

5.2 characterization of gold nanorods:

TEM representation: and dropping the gold nanorod sol on a copper mesh of an electron microscope, and observing the appearance of the sample in a transmission electron microscope after the solution is dried. The electron microscope model is JEM-1200EX transmission electron microscope, and the magnification is 150 k. As shown in fig. 4 (a). The prepared nanoparticles have good dispersibility and uniform shape.

UV-Vis spectral characterization: and (3) taking 100 mu l of the gold nanorod sol into a 96-well plate, and scanning the absorbance value within the wavelength range of 300-900nm by using an enzyme-linked immunosorbent assay to obtain the UV-Vis spectrogram of the sample. As shown in fig. 4 (b).

5.3AuNRs substrate repeatability inspection

Get 10^-6M R6 solution 6G and gold nanorod sol are mixed to be dripped on the surface of a blank silicon wafer after being mixed, a self-made 3D printing Raman detection groove is placed, a portable Raman spectrometer is used for carrying out Raman spectrum collection, and the repeatability of randomly taking points of the substrate is inspected by randomly taking 10 points of scanning spectrum from the mixed liquid drops; 5 mixed detection samples were prepared in parallel according to the above procedure, and the Raman light was scanned separatelySpectra, the substrate was examined for multiple sampling repeatability. Raman spectrum parameter conditions are as follows: the power is 300mW, the integration time is 2s, and the integration times are 1. The raman spectrum acquisitions performed thereafter all use this parameter.

The results of the examination are shown in FIG. 5. As can be seen from fig. 5(a), the AuNRs substrate has good random dot-picking repeatability; as can be seen from fig. 5(b), the AuNRs substrate lot-to-lot reproducibility was good.

5.4AuNRs substrate stability Studies

Get 10^-6M R6 and 5 mul of each of the gold nanorod sol and the solution 6G are mixed, dropped on the surface of a blank silicon wafer, a Raman spectrum is scanned, the parallel is performed for 3 times and recorded as Day 0, then, the substrate Raman spectrum collection is performed every 4 days according to the operation, and the Raman enhancement performance stability of the gold nanorod sol substrate when the gold nanorod sol substrate is placed for 0, 4, 8, 12 and 16 days is respectively inspected.

The results of the examination are shown in FIG. 6. As can be seen from fig. 6, the AuNRs substrates were stable for more than half a month.

6. AuNRs detection lemon yellow dye standard solution

1mg/ml (namely 1000ppm) of lemon yellow dye mother liquor is prepared and is diluted into different concentration gradients step by step until the concentration gradient reaches 100ng/ml (namely 0.1ppm) to be used as a lemon yellow dye standard solution. And (3) taking 5 mul of each gold nanorod sol and 0.01M mercaptoethylamine, blowing and beating the gold nanorod sol and the mercaptoethylamine for 5 times by using a pipette, mixing the gold nanorod sol and the mercaptoethylamine by using the same method, adding 5 mul of lemon yellow dye standard solutions with different concentrations, mixing the mixture, sucking 10 mul of the mixture, dripping the mixture on the surface of a blank silicon wafer, standing for 8min, and then performing Raman spectrum collection.

The results are shown in FIG. 7. As can be seen from fig. 7(a), since the aurrs substrate modified electropositive small molecule mercaptoethylamine can enhance the raman signal of the acid dye, the raman signal of the lemon yellow dye is stronger, and there is substantially no interference of background signals. As can be seen from FIG. 7(b), the intensity of the Raman signal of the lemon yellow dye increases with increasing concentration, with a lower detection limit of 0.1 ppm.

7. Gold orange II dye Raman characteristic peak attribution based on DFT calculation

The DFT is adopted to carry out geometric configuration optimization and vibration frequency calculation on the lemon yellow dye molecules, so that attribution of Raman spectrum characteristic peaks can be realized, and the DFT is compared with an experimental detection value to serve as a theoretical basis for distinguishing and detecting the dye molecules by the SERS technology. The DFT calculation adopts Gaussian 09 software, and B3LYP mixed exchange functional and 6-31G + (d, p) base group are selected as functions and base groups, namely B3LYP/6-31G + (d, p) to carry out structure optimization and frequency calculation on the lemon yellow dye molecules.

Collecting a lemon yellow dye standard product by Raman spectrum: and (3) spreading a proper amount of dye solid powder on the surface of the blank silicon wafer, putting the blank silicon wafer into a Raman detection tank, and performing Raman spectrum collection.

FIG. 8 is a molecular structural formula of a dye, and the molecular structure of the dye is divided into R for convenience of describing chemical bonds when molecular vibration mode attribution is carried out on Raman shift peaks₁、R₂And so forth. And comparing the SERS spectrum of the lemon yellow standard solution with a spectrum obtained by the theoretical calculation of density functional and matching characteristic peaks, and referring to fig. 9. And performing total attribution on the matched Raman characteristic peak according to a density functional theory calculation result and related documents, providing a theoretical basis for subsequent dye molecule identification based on the characteristic peak, wherein attribution information is shown in table 1.

TABLE 1 molecular Raman characteristic peak assignment of lemon yellow dyes

Note: s is strong; m is medium; w is weak; upsilon is telescopic vibration; upsilon is_sSymmetric telescopic vibration is adopted; upsilon is_asAsymmetric stretching vibration is adopted; delta is in-plane shear mode vibration; rho is in-plane pendulum vibration; omega is out-of-plane pendulum vibration; τ is out-of-plane torsional vibration.

8. AuNRs detection of lemon yellow dyeing safflower eluate

Raman spectrum collection is carried out on lemon yellow dyeing safflower eluent with different concentrations (10, 50, 250 and 1000 mu g/g). And taking the SERS spectrum of the standard solution of the lemon yellow II dye as a comparison spectrum, comparing the similarity of the sample spectrum and the comparison spectrum by adopting a correlation coefficient method, and identifying the lemon yellow dye dyeing under the conditions of different dye concentrations.

SERS spectra are shown in FIG. 10, the limit of detection of the lemon yellow dye safflower staining is within the range10 mu g/g, and the sample solution is colorless and transparent at the concentration, which can fully meet the actual detection requirement. And (3) calculating and comparing the similarity of the SERS spectrum of the actual sample and the control spectrum of the standard solution, thereby realizing the identification of the dye. The main characteristic peak of the dye molecule is concentrated at 1000-1800cm^-1Therefore, the map data points of the shift band are selected, and similarity calculation is performed by using a Pearson correlation coefficient method, and the result is shown in Table 2.

TABLE 2 similarity between the spectrum of the lemon yellow stained safflower sample and the 6 dye control spectra (1000 + 1800 cm)^-1)

The similarity judgment standard of whether dye is present or not is set to be 0.9, and according to a calculation result, the lemon yellow dye in the safflower sample can be accurately identified at the concentration of 10 mu g/g or more.

Example 2 gold orange II staining for safflower detection

1. Preparation of golden orange II dyed safflower extract

The dye used was gold orange II and the other treatments were the same as in example 1.

2. Micro-extraction condition optimization

The procedure is as in example 1. As shown in fig. 11(a), the nylon membrane has the highest adsorption rate to the aurantii ii dye, so the nylon membrane was selected as the micro-extraction membrane material in the subsequent experiments. As shown in FIG. 11(b), the adsorption rate of the 10mM NaOH solution to the gold orange II dye is high, so that the 10mM NaOH solution is selected for elution in the subsequent experiment.

3. Nylon membrane micro-extraction gold orange II dyeing safflower dyeing eluent

The procedure is as in example 1.

4. Quick visual judgment of gold orange II dyed safflower eluent

And directly observing whether the color of the safflower eluent of the golden orange II dyeing is colorless and transparent by naked eyes, and judging whether the safflower is dyed. As shown in FIG. 12, the staining concentrations of gold orange II in the centrifuge tubes arranged in the figure from left to right are 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1mg/g in sequence, and it is observed that the undyed safflower eluate is almost colorless and transparent after being extracted by a nylon membrane, and the color of the gold orange II-stained safflower extract deepens along with the increase of the staining concentration.

5. Preparation and characterization of AuNRs

The same as in example 1.

6. AuNRs detection golden orange II dye standard solution

Preparing 1mg/ml (1000 ppm) of golden orange II dye mother liquor, and gradually diluting the golden orange II dye mother liquor to different concentration gradients until the concentration gradient reaches 100ng/ml (0.1 ppm) to be used as a golden orange II dye standard solution. Taking 5 mul of each gold nanorod sol and 0.01M mercaptoethylamine, blowing and beating the gold nanorod sol and the mercaptoethylamine for 5 times by using a pipette, mixing the gold nanorod sol and the mercaptoethylamine for 5 times, adding 5 mul of standard solution of the gold orange II dye with different concentrations, mixing the mixture by the same method, sucking 10 mul of mixed solution, dripping the mixed solution on the surface of a blank silicon wafer, standing for 8min, and then performing Raman spectrum collection.

The results are shown in FIG. 13. As can be seen from fig. 13(a), since the aurrs substrate modified electropositive small molecule mercaptoethylamine can enhance the raman signal of the acid dye, the raman signal of the aurantii ii dye is stronger, and there is substantially no interference of background signal. As can be seen from FIG. 13(b), the Raman signal intensity of the gold orange II dye increased with increasing concentration, with a lower detection limit of 0.5 ppm.

The procedure is as in example 1. The results are shown in FIG. 14. And performing total attribution on the matched Raman characteristic peaks according to the calculation result of the density functional theory and related documents, wherein attribution information is shown in a table 3.

TABLE 3 molecular Raman characteristic peak assignment of gold orange II dye

8. AuNRs detection of gold orange II staining safflower eluate

Raman spectrum collection is carried out on the safflower eluent with different concentrations (50, 250 and 1000 mu g/g) of golden orange II staining. And taking the SERS spectrum of the golden orange II dye standard solution as a comparison spectrum, comparing the similarity of the sample spectrum and the comparison spectrum by adopting a correlation coefficient method, and identifying the golden orange II dye dyeing under the conditions of different dye concentrations.

The SERS spectrum is shown in FIG. 15, the detection limit of the golden orange II dye safflower dyeing can reach 50 mug/g, and the sample solution is colorless and transparent under the concentration, so that the actual detection requirement can be fully met. And (3) calculating and comparing the similarity of the SERS spectrum of the actual sample and the control spectrum of the standard solution, thereby realizing the identification of the dye. The main characteristic peak of the dye molecule is concentrated at 1000-1800cm^-1Therefore, the map data points of the shift band are selected, and similarity calculation is performed by using the Pearson correlation coefficient method, and the result is shown in Table 4.

TABLE 4 similarity between the spectrum of the golden orange II stained safflower sample and the 6 dye control spectra (1000 + 1800 cm)^-1)

According to the calculation result, the golden orange II dyeing in the safflower sample can be obviously distinguished from other acid dye spectrums of 4 types of lemon yellow, acid red 73, carmine and azorubine at the concentration of 10 mu g/g and above, for sunset yellow, the similarity difference is smaller because the molecular structures of the golden orange II dyeing safflower sample and the golden orange II control spectrum are highly similar, but the similarity of the golden orange II dyeing safflower sample and the golden orange II control spectrum is still higher than that of the sunset yellow, and meanwhile, the comparison is combined with a local characteristic peak: such as 1229cm yellow sunset compared with golden orange II^-1The intensity of Raman peak is more than 1389cm^-1And at 1335cm^-1With a stronger signal peak. Thereby realizing the accurate identification of the dyeing of the safflower sample golden orange II.

Example 3 Azolorubine-stained safflower assay

1. Preparation of azorubine dyed safflower extract

The dye used was azorubine, the other treatments being the same as in example 1.

2. Micro-extraction condition optimization

The procedure is as in example 1. As shown in fig. 16(a), the nylon membrane has the highest adsorption rate to azo rubine dye, so the nylon membrane was selected as the micro-extraction membrane material in the subsequent experiments. As shown in FIG. 16(b), the adsorption rate of the azo rubine dye by the 10mM NaOH solution is high, so that the 10mM NaOH solution is selected for elution in the subsequent experiment.

3. Nylon membrane micro-extraction azorubine dyeing safflower dyeing eluent

The procedure is as in example 1.

4. Quick visual judgment of azorubine dyed safflower eluent

And directly observing whether the color of the azorubine dyeing safflower eluent is colorless and transparent by naked eyes, and judging whether the safflower is dyed. As shown in FIG. 17, the concentration of azo rubine dye in the centrifuge tubes arranged in the figure is 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1mg/g from left to right in sequence, and it is observed that the undyed safflower eluate is almost colorless and transparent after being extracted by a nylon membrane, and the color of the azo rubine dyed safflower extract deepens along with the increase of the dye concentration.

5. Preparation and characterization of AuNRs

The preparation is as in example 1.

6. AuNRs detection azo rubine dye standard solution

Preparing 1mg/ml (namely 1000ppm) of azorubine dye mother liquor, and gradually diluting the azorubine dye mother liquor to different concentration gradients until the concentration is 100ng/ml (namely 0.1ppm) to be used as an azorubine dye standard solution. Taking 5 mul of each gold nanorod sol and 0.01M mercaptoethylamine, blowing and beating the gold nanorod sol and the mercaptoethylamine for 5 times by using a pipette, mixing the gold nanorod sol and the mercaptoethylamine for 5 times, adding 5 mul of azorubine dye standard solution with different concentrations, mixing the mixture by the same method, sucking 10 mul of the mixture, dripping the mixture on the surface of a blank silicon wafer, standing for 8min, and then performing Raman spectrum collection.

The results are shown in FIG. 18. As can be seen from fig. 18(a), since the aurrs substrate modified electropositive small molecule mercaptoethylamine can enhance the raman signal of the acid dye, the raman signal of the azorubine dye is stronger, and there is substantially no interference of background signals. As can be seen from fig. 18(b), the raman signal intensity of the azorubine dye increases with increasing concentration, with a lower detection limit of 0.1 ppm.

7. Azoic rubine dye Raman characteristic peak attribution based on DFT calculation

The procedure is as in example 1. The results are shown in FIG. 19. And performing total attribution on the matched Raman characteristic peaks according to the calculation result of the density functional theory and related documents, wherein attribution information is shown in a table 5.

TABLE 5 molecular Raman characteristic peak assignment of azorubine dyes

8. AuNRs detection of azorubine dyeing safflower eluate

Raman spectrum collection is carried out on azorubine dyeing safflower eluent with different concentrations (10, 50, 250 and 1000 mu g/g). And taking the SERS spectrum of the azo rubine dye standard solution as a comparison spectrum, comparing the similarity of the sample spectrum and the comparison spectrum by adopting a correlation coefficient method, and identifying the azo rubine dye dyeing under the conditions of different dye concentrations.

The SERS spectrum is shown in figure 20, the detection limit of the azo rubine dye safflower dyeing can reach 10 mug/g, and the sample solution is colorless and transparent under the concentration, so that the actual detection requirement can be fully met. And (3) calculating and comparing the similarity of the SERS spectrum of the actual sample and the control spectrum of the standard solution, thereby realizing the identification of the dye. The main characteristic peak of the dye molecule is concentrated at 1000-1800cm^-1Therefore, the map data points of the shift band are selected, and similarity calculation is performed by using the Pearson correlation coefficient method, and the result is shown in Table 6.

TABLE 6 similarity between the spectrum of the azo rubine-dyed safflower sample and the 6 dye control spectra (1000 + 1800 cm)^-1)

The similarity judgment standard of whether dye dyeing exists is set to be 0.9, and according to a calculation result, the azo rubine dyeing in the safflower sample can be accurately identified at the concentration of 10 mu g/g or more.

Example 4 sunset yellow-stained safflower assay

1. Preparation of sunset yellow-dyed safflower extract

The dye used was sunset yellow and the other treatments were the same as in example 1.

2. Micro-extraction condition optimization

The procedure is as in example 1. As shown in fig. 21(a), the nylon membrane has the highest adsorption rate to the sunset yellow dye, so that the nylon membrane was selected as the micro-extraction membrane material in the subsequent experiments. As shown in fig. 21(b), 10mM and 100mM NaOH solutions have high adsorption rates to the sunset yellow dye, but the adsorption rate is not significantly improved at 100mM concentration, so that the 10mM NaOH solution is selected for elution in the subsequent experiment.

3. Nylon membrane micro-extraction sunset yellow dyeing safflower dyeing eluent

The procedure is as in example 1.

4. Fast visual judgment of sunset yellow dyeing safflower eluate

Directly and visually observing whether the color of the sunset yellow dyeing safflower eluate is colorless and transparent, and judging whether the safflower is dyed. As shown in FIG. 22, the concentration of the sunset yellow dye in the centrifuge tubes arranged in the figure is 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1mg/g from left to right in sequence, and it is observed that the undyed safflower eluate is almost colorless and transparent after being extracted by a nylon membrane, and the color of the sunset yellow-dyed safflower extract deepens along with the increase of the dye concentration.

5. Preparation and characterization of AuNRs

The same as in example 1.

6. AuNRs detection sunset yellow dye standard solution

Preparing 1mg/ml (namely 1000ppm) sunset yellow dye mother liquor, and gradually diluting the sunset yellow dye mother liquor to different concentration gradients until the concentration is 100ng/ml (namely 0.1ppm) to be used as a sunset yellow dye standard solution. And (3) taking 5 mul of each gold nanorod sol and 0.01M mercaptoethylamine, blowing and beating the gold nanorod sol and the mercaptoethylamine for 5 times by using a pipette, mixing the gold nanorod sol and the mercaptoethylamine for 5 times, adding 5 mul of sunset yellow dye standard solutions with different concentrations, mixing the solutions by the same method, sucking 10 mul of the mixed solution, dripping the mixed solution on the surface of a blank silicon wafer, standing for 8min, and then performing Raman spectrum collection.

The results are shown in FIG. 23. As can be seen from fig. 23(a), since the aurrs substrate modified electropositive small molecule mercaptoethylamine can enhance the raman signal of the acid dye, the raman signal of the sunset yellow dye is stronger, and there is substantially no interference of the background signal. As can be seen from FIG. 23(b), the intensity of the Raman signal of the sunset yellow dye increases with increasing concentration, and the lower limit of detection is 0.1 ppm.

7. Sunset yellow dye Raman characteristic peak attribution based on DFT calculation

The procedure is as in example 1. The results are shown in FIG. 24. And performing total attribution on the matched Raman characteristic peaks according to the calculation result of the density functional theory and related documents, wherein attribution information is shown in a table 7.

TABLE 7 molecular Raman characteristic peak assignment of sunset yellow dye

8. AuNRs detection of sunset yellow-dyeing safflower eluate

Raman spectrum collection is carried out on sunset yellow-dyed safflower eluate with different concentrations (10, 50, 250 and 1000 mu g/g). And taking the SERS spectrum of the standard solution of the sunset yellow dye as a comparison spectrum, comparing the similarity of the sample spectrum and the comparison spectrum by adopting a correlation coefficient method, and identifying the dyeing of the sunset yellow dye under the conditions of different dye concentrations.

The SERS spectrum is shown in figure 25, the detection limit of the sunset yellow dye safflower dyeing can reach 10 mug/g, and the sample solution is colorless and transparent at the concentration, so that the actual detection requirement can be fully met. And (3) calculating and comparing the similarity of the SERS spectrum of the actual sample and the control spectrum of the standard solution, thereby realizing the identification of the dye. The main characteristic peak of the dye molecule is concentrated at 1000-1800cm^-1Therefore, the map data points of the shift band are selected, and similarity calculation is performed by using the Pearson correlation coefficient method, and the result is shown in Table 8.

TABLE 8 similarity between the spectrum of the sunset yellow-stained safflower sample and the 6 dye control spectra (1000 + 1800 cm)^-1)

According to the calculation result, the sunset yellow dyeing in the safflower sample can be obviously distinguished from the spectra of 4 other acid dyes including lemon yellow, acid red 73, carmine and azorubine at the concentration of 10 mu g/g and above, for golden orange II, the similarity difference is smaller because the molecular structures of the two are highly similar, but the similarity of the sunset yellow dyeing safflower sample and the sunset yellow control spectrum is still higher than that of the golden orange II, and the comparison is combined with the local characteristic peak: such as 1229cm yellow sunset compared with golden orange II^-1The intensity of Raman peak is more than 1389cm^-1And at 1335cm^-1With a stronger signal peak. Thereby realizing the accurate identification of the sunset yellow stain of the safflower sample.

Example 5 acid Red 73 staining of safflower assay

1. Preparation of acid red 73 dyed safflower extract

The dye used was acid red 73 and the other treatments were the same as in example 1.

2. Micro-extraction condition optimization

The procedure is as in example 1. As shown in fig. 26(a), the nylon membrane has the highest adsorption rate for acid red 73 dye, so that the nylon membrane was selected as the micro-extraction membrane material in the subsequent experiments. As shown in FIG. 26(b), the adsorption rate of 10mM NaOH solution to acid red 73 dye is high, so that 10mM NaOH solution is selected for elution in the subsequent experiment.

3. Nylon membrane micro-extraction acid red 73 dyeing safflower dyeing eluent

The procedure is as in example 1.

4. Quick visual judgment of acid red 73 dyed safflower eluent

Directly and visually observing whether the color of the acid red 73 dyed safflower eluent is colorless and transparent, and judging whether the safflower is dyed. Referring to fig. 27, the concentration of acid red 73 dye in the centrifuge tubes arranged in the figure is 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1mg/g from left to right, and it is observed that the un-dyed safflower eluate is almost colorless and transparent after being extracted by the nylon membrane, and the color of the acid red 73 dyed safflower extract deepens with the increase of the dye concentration.

5. Preparation and characterization of AuNRs

The preparation is as in example 1.

6. AuNRs detection acid red 73 dye standard solution

Preparing 1mg/ml (namely 1000ppm) acid red 73 dye mother liquor, and diluting the mother liquor to different concentration gradients to 100ng/ml (namely 0.1ppm) as an acid red 73 dye standard solution. And (3) taking 5 mul of each gold nanorod sol and 0.01M mercaptoethylamine, blowing and beating the gold nanorod sol and the mercaptoethylamine for 5 times by using a pipette, mixing the gold nanorod sol and the mercaptoethylamine for 5 times, adding 5 mul of acid red 73 dye standard solutions with different concentrations, mixing the mixture by the same method, sucking 10 mul of the mixture, dripping the mixture on the surface of a blank silicon wafer, standing for 8min, and then performing Raman spectrum collection.

The results are shown in FIG. 28. As can be seen from fig. 28(a), since the aurrs substrate modified electropositive small molecule mercaptoethylamine can enhance the raman signal of the acid dye, the raman signal of the acid red 73 dye is stronger and has substantially no interference from background signals. As can be seen from fig. 28(b), the raman signal intensity of the acid red 73 dye increased with increasing concentration, with a lower detection limit of 0.5 ppm.

7. Acid red 73 dye Raman characteristic peak attribution based on DFT calculation

The procedure is as in example 1. The results are shown in FIG. 29. And performing total attribution on the matched Raman characteristic peaks according to the calculation result of the density functional theory and related documents, wherein attribution information is shown in a table 9.

TABLE 9 molecular Raman characteristic peak assignment of acid Red 73 dye

8. AuNRs detection of acid red 73 staining safflower eluate

Raman spectrum collection is carried out on the eluate of the acid red 73 staining safflower with different concentrations (50, 250 and 1000 mu g/g). And taking the SERS spectrum of the acid red 73 dye standard solution as a comparison spectrum, comparing the similarity of the sample spectrum and the comparison spectrum by adopting a correlation coefficient method, and identifying the acid red 73 dye dyeing under the conditions of different dye concentrations.

As shown in the graph 30, the SERS spectrum shows that the detection limit of the safflower dyeing of the acid red 73 dye can reach 50 mug/g, and the sample solution is colorless and transparent at the concentration, so that the actual detection requirement can be fully met. And (3) calculating and comparing the similarity of the SERS spectrum of the actual sample and the control spectrum of the standard solution, thereby realizing the identification of the dye. The main characteristic peak of the dye molecule is concentrated at 1000-1800cm^-1Therefore, the map data points of the shift band are selected, and similarity calculation is performed by using the Pearson correlation coefficient method, and the result is shown in Table 10.

TABLE 10 similarity between the spectrum of acid Red 73 stained safflower sample and the 6 dye control spectra (1000 + 1800 cm)^-1)

The similarity judgment standard of whether dye is available or not is set to be 0.9, and according to a calculation result, the acid red 73 dye in the safflower sample can be accurately identified at the concentration of 10 mu g/g or more.

Example 6 carmine-stained safflower assay

1. Preparation of carmine-dyed safflower extract

The dye used was carmine and the other treatments were the same as in example 1.

2. Micro-extraction condition optimization

The procedure is as in example 1. As shown in fig. 31(a), the nylon membrane has the highest adsorption rate to the carmine dye, so the nylon membrane was selected as the micro-extraction membrane material in the subsequent experiments. As shown in FIG. 31(b), 10mM and 100mM NaOH solutions have high adsorption rates to carmine dye, but the adsorption rate is not significantly improved at 100mM concentration, so that the 10mM NaOH solution is selected for elution in subsequent experiments.

3. Nylon membrane micro-extraction carmine dyeing safflower dyeing eluent

The procedure is as in example 1.

4. Rapid visual judgment of carmine-dyed safflower eluate

And directly observing whether the color of the carmine dyed safflower eluate is colorless and transparent by naked eyes, and judging whether the safflower is dyed. Referring to FIG. 32, the concentrations of the carmine dye in the centrifuge tubes arranged in the figure are 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1mg/g from left to right, and it is observed that the un-dyed safflower eluate is almost colorless and transparent after being extracted by the nylon membrane, and the color of the carmine-dyed safflower extract deepens with the increase of the dye concentration.

5. Preparation and characterization of AuNRs

The preparation is as in example 1.

6. AuNRs detection carmine dye standard solution

A carmine dye stock solution of 1mg/ml (i.e. 1000ppm) was prepared and diluted stepwise to different concentration gradients up to 100ng/ml (i.e. 0.1ppm) as a carmine dye standard solution. And (3) taking 5 mul of gold nanorod sol and 0.01M mercaptoethylamine, blowing and beating the gold nanorod sol and the mercaptoethylamine for 5 times by using a pipette, mixing the gold nanorod sol and the mercaptoethylamine for 5 times, adding 5 mul of carmine dye standard solutions with different concentrations, mixing the mixture by the same method, sucking 10 mul of the mixture, dripping the mixture on the surface of a blank silicon wafer, standing for 8min, and then performing Raman spectrum collection.

The results are shown in FIG. 33. As can be seen from fig. 33(a), since the aurrs substrate modified electropositive small molecule mercaptoethylamine can enhance the raman signal of the acid dye, the raman signal of the carmine dye is stronger and has substantially no interference from background signals. As can be seen from FIG. 33(b), the intensity of Raman signal of carmine dye increases with increasing concentration, and the lower limit of detection is 0.5 ppm.

7. Carmine dye Raman characteristic peak attribution calculated based on DFT

The procedure is as in example 1. The results are shown in FIG. 34. And performing total attribution on the matched Raman characteristic peaks according to the calculation result of the density functional theory and related documents, wherein attribution information is shown in a table 11.

TABLE 11 molecular Raman characteristic Peak assignment of carmine dyes

8. AuNRs detection of carmine-dyed safflower eluate

Raman spectra were collected from the eluates of cochineal color safflower at different concentrations (50, 250, 1000. mu.g/g). And taking the SERS spectrum of the carmine dye standard solution as a comparison spectrum, comparing the similarity of the sample spectrum and the comparison spectrum by adopting a correlation coefficient method, and identifying the carmine dye dyeing under the conditions of different dye concentrations.

The SERS spectrum is shown in FIG. 35, the limit of detection of the carmine dye safflower dyeing can reach 50 μ g/g, and the sample solution is colorless and transparent at the concentration, which can fully meet the actual detection requirementAnd (4) measuring the requirement. And (3) calculating and comparing the similarity of the SERS spectrum of the actual sample and the control spectrum of the standard solution, thereby realizing the identification of the dye. The main characteristic peak of the dye molecule is concentrated at 1000-1800cm^-1Therefore, the map data points of the shift band are selected, and similarity calculation is performed by using the pearson correlation coefficient method, and the result is shown in table 12.

TABLE 12 similarity between spectrum of carmine-stained safflower sample and 6 dye control spectra (1000 + 1800 cm)^-1)

The similarity judgment standard of whether dye is present or not is set to be 0.9, and according to a calculation result, the carmine dyeing in the safflower sample can be accurately identified at the concentration of 50 mu g/g or more.

Claims

1. A safflower dyeing rapid detection method based on a surface enhanced Raman spectroscopy technology is characterized by comprising the following steps:

(3) eluting the dye substance adsorbed on the nylon membrane by using an alkaline aqueous solution to obtain an eluent, and adding an acid solution with the same concentration into the eluent to adjust back the pH value to prepare a solution to be detected;

2. The method for rapidly detecting safflower dyeing based on the surface-enhanced Raman spectroscopy of claim 1, wherein in the step (1), an ethanol solution with a volume percentage of 70% is used as a leaching solution, and the material ratio is 1 g: 20mL, 5min extraction time.

3. The method for rapidly detecting safflower dyeing based on the surface enhanced Raman spectroscopy of claim 1, wherein in the step (3), before elution with the alkaline aqueous solution, formic acid solution with volume percentage of 10% -40% is filtered through the nylon membrane for washing, and then water is used for washing.

4. The method for rapidly detecting safflower dyeing based on the surface enhanced Raman spectroscopy of claim 1, wherein in the step (3), NaOH aqueous solution with a concentration of 1-100 mM is used as the alkaline aqueous solution, and the acid solution is hydrochloric acid solution.

5. The method for rapidly detecting safflower dyeing based on the surface-enhanced Raman spectroscopy of claim 1, wherein in the step (4), the preparation method of the gold nanorod sol comprises the following steps:

6. The method for rapid detection of safflower dyeing based on surface enhanced Raman spectroscopy of claim 1, wherein in the step (4), the gold nanorod sol and the concentration of the gold nanorod sol are 10%^-5Mixing M-1M mercaptoethylamine in equal volume.

7. The method for rapid detection of safflower staining based on surface enhanced Raman spectroscopy of claim 6, wherein the concentration of mercaptoethylamine is 0.001-0.1M.

8. The method for rapidly detecting safflower dyeing based on surface enhanced Raman spectroscopy according to claim 1, wherein in the step (4), Raman spectrum parameter conditions are as follows: the power is 300mW, the integration time is 2s, and the integration times are 1.

9. The method for rapidly detecting safflower dyeing according to the surface-enhanced raman spectroscopy technique of claim 1, wherein in the step (4), the SERS spectrum of the dye standard solution is used as the control spectrum in the step (4), and the correlation coefficient method is used to compare the similarity between the sample spectrum and the control spectrum, so as to identify the safflower dye.

10. The method for rapidly detecting safflower dyeing based on the surface-enhanced Raman spectroscopy according to claim 1, wherein in the step (4), the dye is golden orange II, lemon yellow, azorubine, sunset yellow, acid red 73 or carmine.