CN114998621B - SERS quantitative analysis method based on coffee ring concentration effect and image processing technology - Google Patents

Abstract

The invention discloses a SERS quantitative analysis method based on coffee ring concentration effect and image processing technology, comprising the following steps: S1, SERS substrate preparation; S2, SERS substrate pretreatment; S3, uniformly spraying probe molecules on the SERS substrate; S4, performing two-dimensional SERS imaging with the highest scattering peak of the probe molecules to obtain an imaging image; S5, obtaining intensity data [x, y, s] at different positions according to the imaging image, and obtaining a corresponding two-dimensional pseudo-color image. In the invention, a large amount of data is collected through two-dimensional SERS imaging detection, and at least 24 intensity curves of different ring structures of the SERS substrate are obtained through image processing technology, and the maximum intensity value of each curve is recorded, and 3 maximum values and 3 minimum values are removed from the maximum intensity value, and the remaining intensity values are averaged, and the average value is the concentration intensity value of the probe molecule solution on the SERS substrate, thereby improving the accuracy of measuring the concentration.

Description

SERS quantitative analysis method based on coffee ring concentration effect and image processing technology

Technical Field

The invention relates to the technical field of surface enhanced Raman scattering, and in particular to a SERS quantitative analysis method based on coffee ring concentration effect and image processing technology.

Background technique

As a new type of spectral analysis method, surface enhanced Raman scattering (SERS) technology has been gradually widely used in the fields of chemistry, catalysis, environmental science, biomedicine, and analytical detection. Compared with some traditional spectral techniques, SERS technology has the advantages of fingerprint information recognition, single-molecule level detection, and real-time in-situ detection, and is not affected by photobleaching, spectral overlap, and the background of water molecules in the body, which makes this technology have greater potential in analytical detection.

However, the SERS signal is not evenly distributed on the substrate surface. In actual application, since Raman spectroscopy is randomly sampled, and the number of samples is very single when the probe molecules are pipetted and dripped on the substrate by a pipette gun or a micro-injector, the randomness, inability to quantify and uncertainty in the experimental process are greatly increased, and the effect of the substrate in actual analysis and detection is poor. In order to improve the problems of randomness, inability to quantify and uncertainty in the research, the present invention provides a SERS quantitative analysis method based on the coffee ring concentration effect and image processing technology. First, the "coffee ring" effect is applied to separation analysis. Secondly, the sensitivity of SERS detection is improved by two-dimensional image processing technology, and the detection limit of the electrodeposited silver substrate is further reduced, while the accuracy of concentration measurement is improved.

Summary of the invention

In order to solve the technical problems mentioned in the above background technology, a SERS quantitative analysis method based on coffee ring concentration effect and image processing technology is proposed.

In order to achieve the above object, the present invention adopts the following technical solutions:

The SERS quantitative analysis method based on the coffee ring concentration effect and image processing technology includes the following steps:

S1. Preparation of SERS substrate: At room temperature, a SERS substrate was prepared on a copper sheet by pulse electrodeposition. The working solution concentration was V _{mother solution} : V _{deionized water} = 1:1, wherein the mother solution was a cyanide-free silver plating solution, the pH of the working solution was 9.5-10.5, the cation and cathode area ratio was 1:10, the peak current density was 1-2 A·dm ^-2 , the duty cycle was 40%-50%, the pulse period was 0.05-0.1s, and the pulse plating time was 600-750s;

S2. SERS substrate pretreatment:

S21, washing the SERS substrate with deionized water and ethanol;

S22, immersing the cleaned SERS substrate in a surface treatment solution for 3 to 5 hours, wherein the surface treatment solution comprises 0.5 mmol·L ^-1 alkylthiol and 0.15 mmol·L ^-1 4-mercapto-1-butanol, and the concentration ratio of alkylthiol to 4-mercapto-1-butanol is 1:2.5 to 1:3, wherein the chemical expression of alkylthiol is: HS(CH ₂ ) _n CH ₃ , n=5 to 7;

S23, after soaking for 3 to 5 hours, take out the SERS substrate, and wash the SERS substrate with ethanol and deionized water in turn;

S3, spraying the probe molecules uniformly on the SERS substrate, controlling the droplet size to be 100-200 μm, and the number of droplets sprayed at a single time to be 15-20/mm ² , and the probe molecules evaporate and concentrate to form particles, and a ring structure appears on the surface of the SERS substrate, wherein the probe molecules include rhodamine 6G molecules, methyl orange molecules, 2,2'-bipyridine molecules, 4-mercaptopyridine molecules and 1,4-benzenedithiol;

S4, select the ring structure formed by the deposition of the probe molecule, and perform two-dimensional SERS imaging with its highest scattering peak to obtain an imaging map. The imaging range is 100μm×100μm. Combined with the droplet size, the Raman laser intensity is selected to be 3.5-4.0mW. The imaging map is used to express the strength distribution of the SERS signal in a certain area on the SERS substrate. The red area indicates a strong SERS signal in the Raman spectrum, and the blue area indicates a weak SERS signal in the Raman spectrum.

S5. Obtain intensity data [x, y, s] at different positions according to the imaging image, wherein (x, y) represents the position coordinates, s represents the SERS signal intensity at different (x, y) positions, and obtain a corresponding two-dimensional pseudo-color image through the position coordinates (x, y) and the intensity value s;

S6. Quantitative or semi-quantitative detection of SERS substrates by image processing technology:

S61, extracting the target area in the two-dimensional pseudo-color image, that is, the irregular ring structure representing the concentration, converting the image coordinate system [x, y, s] data of the target area into the corresponding pixel coordinate system [X, Y, s], normalizing the pixel coordinates (X, Y) based on the origin, and keeping the intensity value s of each point unchanged;

S62, determining the center point of the target area: converting the two-dimensional pseudo-color image from the RGB color space to the HSV space, by setting the value ranges of the three channels of the HSV space, the three channels are: hue, saturation and brightness. In the RGB color space, the RGB value of blue is [0,0,255]. In the HSV space, the value range of the blue hue is: H _min =100, H _max =124, the saturation value range is: S _min =43, S _max =255, and the brightness value range is: V _min =46, V _max =255. Extract the blue area as the target area;

Traverse all pixel coordinates (X, Y) of the target area, search for n points within the value range of the three channels, and calculate the average value of the coordinates of the n points as the center point (x ₀ , y ₀ ). The calculation formula is as follows:

x ₀ =(x ₁ +x ₂ +···+x _n )/n;

y ₀ =(y ₁ +y ₂ +···+ _yn )/n;

Wherein, x represents the abscissa of a point within the value range of the three channels, y represents the ordinate of a point within the value range of the three channels, and the subscripts of x and y represent the 1st, 2nd, ..., nth points within the value range of the three channels;

S63, normalize the distance from the center point ( _x0 , _y0 ) to the ring boundary: according to the pixel coordinate system [X, Y, s], from the center point ( _x0 , _y0 ) to the ring boundary, diverge 360° in all directions at intervals of 15°, obtain pixel coordinates (X, Y) in sequence, normalize the pixel coordinates (X, Y) based on the center point ( _x0 , _y0 ), and the intensity value s remains unchanged. The pixel coordinates (X', Y') are traversed to obtain the corresponding intensity value s, and an intensity curve from the center point ( _x0 , _y0 ) to the ring boundary is drawn;

S64. Repeat the above steps to obtain at least 24 intensity curves of different ring structures of the SERS substrate, record the maximum intensity value of each curve, remove the 3 maximum values and 3 minimum values from the maximum intensity values, and calculate the average value of the remaining intensity values. The average value is the concentration intensity value of the probe molecule solution on the SERS substrate.

As a further description of the above technical solution:

In step S1, the cyanide-free silver plating solution includes 40-50 g·L ^-1 silver ion complexing agent, 5-10 g·L ^-1 copper ion complexing agent, 0.01-0.05 g·L ^-1 organic brightener, 15-25 g·L ^-1 silver ion source and pH adjuster, the silver ion complexing agent includes at least one of aminonicotinic acid, 6-aminonicotinic acid methyl ester, and 5-methylnicotinic acid, the copper ion complexing agent includes at least one of cysteine, Z-proline- _NH2 or 1,2-cyclohexanediaminetetraacetic acid, the organic brightener includes at least one of 3-methacryloyl dopamine, dopamine 4-0-sulfate or ractopamine methyl ether, the silver ion source includes one of silver oxide or silver carbonate, and the pH adjuster includes at least one of potassium hydroxide or sodium hydroxide.

As a further description of the above technical solution:

In step S1, the copper sheet is also pre-processed, and the specific processing steps are as follows:

S11, ultrasonic cleaning with acetone and deionized water at room temperature for 10 to 20 minutes respectively;

S12, under the condition of 35-45°C water bath heating, the copper sheet after ultrasonic cleaning was subjected to cathodic electrolytic degreasing for 5 minutes, with a current density of 3-5A·dm ^-2 ;

S13, washing the degreased copper sheet with deionized water, and performing anodic electrochemical polishing at room temperature for 40 to 60 seconds until the surface of the copper sheet is smooth and bright, with a current density of 5 to 7 A·dm ^-2 ;

S14, washing the smooth and bright copper sheet with deionized water, and electro-depositing acid copper at a constant current for 5 min at room temperature with stirring, and the current density is 2A·dm ^-2 ;

S15, washing the electrodeposited copper sheet with deionized water, and soaking it in a dilute hydrochloric acid solution with a volume fraction of 5-7% for 5 minutes at room temperature, taking it out and washing it with deionized water, thereby completing the pretreatment of the copper sheet.

As a further description of the above technical solution:

In step S22, the contact angle between the probe molecule and the SERS substrate is between 110° and 130°.

In summary, due to the adoption of the above technical scheme, the beneficial effects of the present invention are as follows: in the present invention, firstly, a simple and easy-to-operate pulse electrodeposition method is selected to prepare an excellent SERS substrate. During the pulse electrodeposition process, the continuous growth of crystals is hindered due to the existence of pulse intervals, so that fine nanoparticles are more easily obtained. Secondly, in order to improve the hydrophilicity of the substrate surface, the substrate is surface treated with a surface treatment liquid, and in order to eliminate the influence of a single number of added samples or strong randomness during the immersion process, the method of spraying droplets is combined to improve sampling and averaging. The above operation is combined with the statistical average method to eliminate the influence of randomness. Finally, the two-dimensional SE RS imaging detection collects a large amount of data to display the distribution of Raman signals on the substrate and facilitate image processing. Using image processing technology, at least 24 intensity curves of different ring structures of the SERS substrate are obtained, and the maximum intensity value of each curve is recorded. The 3 maximum values and 3 minimum values are removed from the maximum intensity value, and the remaining intensity values are averaged. The average value is the intensity value of the concentration of the probe molecule Rhodamine 6G on the SERS substrate. This averaging method is used to remove some intensity values that are too concentrated or have little to do with the concentration, and extract the intensity value representing the concentration, thereby reducing the detection limit of the SERS substrate and improving the accuracy of concentration measurement.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows a schematic flow chart of a SERS quantitative analysis method based on the coffee ring concentration effect and image processing technology according to an embodiment of the present invention;

FIG2 is a schematic diagram showing a flow chart of an image processing technique of a SERS quantitative analysis method based on a coffee ring concentration effect and an image processing technique provided in an embodiment of the present invention;

FIG3 shows a schematic diagram of a local enhancement effect of a SERS substrate according to a SERS quantitative analysis method based on a coffee ring concentration effect and an image processing technique provided in an embodiment of the present invention;

FIG4 shows a schematic diagram of the hydrophobicity of a Rhodamine 6G molecule according to a SERS quantitative analysis method based on the coffee ring concentration effect and image processing technology provided in an embodiment of the present invention;

FIG5 shows a schematic diagram of the hydrophilicity of Rhodamine 6G molecules according to a SERS quantitative analysis method based on the coffee ring concentration effect and image processing technology provided in an embodiment of the present invention;

FIG6 shows a first two-dimensional SERS imaging diagram of a SERS quantitative analysis method based on a coffee ring concentration effect and an image processing technique provided in an embodiment of the present invention;

FIG. 7 shows a second two-dimensional SERS imaging of the SERS quantitative analysis method based on the coffee ring concentration effect and image processing technology provided in an embodiment of the present invention;

FIG8 shows a third two-dimensional SERS imaging schematic diagram of a SERS quantitative analysis method based on the coffee ring concentration effect and image processing technology provided in an embodiment of the present invention;

FIG9 shows a schematic diagram of the linear range of the probe molecule Rhodamine 6G molecule of the SERS quantitative analysis method based on the coffee ring concentration effect and image processing technology provided in an embodiment of the present invention;

FIG. 10 shows SEM images of a fast current pulse electrodeposited SERS substrate at different pulse periods according to a SERS quantitative analysis method based on the coffee ring concentration effect and image processing technology provided by an embodiment of the present invention.

Detailed ways

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Embodiment 1

Please refer to Figures 1-10. The present invention provides a technical solution: a SERS quantitative analysis method based on coffee ring concentration effect and image processing technology, comprising the following steps:

S1. Preparation of SERS substrate: at room temperature, a SERS substrate is prepared by pulse electrodeposition on a copper sheet, and the working solution concentration used is V _{mother solution} : V _{deionized water} = 1:1, wherein the mother solution is a cyanide-free silver plating solution, and the cyanide-free silver plating solution includes 40-50 g·L ^-1 silver ion complexing agent, 5-10 g·L ^-1 copper ion complexing agent, 0.01-0.05 g·L ^-1 organic brightener, 15-25 g·L ^-1 silver ion source and pH regulator, the silver ion complexing agent includes at least one of aminonicotinic acid, 6-aminonicotinic acid methyl ester, and 5-methylnicotinic acid, the copper ion complexing agent includes at least one of cysteine, Z-proline- _NH2 or 1,2-cyclohexanediaminetetraacetic acid, the organic brightener includes at least one of 3-methacryloyl dopamine, dopamine 4-0-sulfate or ractopamine methyl ether, the silver ion source includes one of silver oxide or silver carbonate, and the pH regulator includes at least one of potassium hydroxide or sodium hydroxide;

The pH of the working solution is 9.5-10.5, the ratio of the positive and negative areas is 1:10, the peak current density is 1-2A·dm ^-2 , the duty cycle is 40%-50%, the pulse period is 0.05-0.1s, and the pulse plating time is 600-750s;

Specifically, the composition of the working fluid selected in this embodiment is shown in Table 1 below:

Table 1

The preparation of active substrate is the premise of obtaining high-quality SERS signals. Excellent SERS substrate should have the following characteristics: first, the substrate has high SERS activity, i.e., sensitivity, and controlled metal nanoparticle size (less than 50nm) and particle gap (less than 10nm); second, the substrate should have good uniformity and reproducibility, and the nanoparticles on the surface should be arranged in an orderly manner to ensure that the deviation of the entire surface enhancement effect is less than 20%;

There are many methods for preparing SERS substrates in current research, such as metal nanosol method (poor repeatability and potential sampling challenges), self-assembly method (susceptible to the influence of experimental environment), nano-etching method (time-consuming process, low output) and template method (many experimental influencing factors, poor repeatability), etc. Some SERS substrates can obtain low detection limits and wide linear ranges, but the preparation methods are complicated to operate, time-consuming and difficult to mass produce. The substrates prepared by some methods are easily affected by the environment, have poor repeatability, and cannot obtain low detection limits. Therefore, the present invention selects a simple, easy-to-operate, mass-producible pulse electrodeposition method to prepare the substrate, wherein pulse electrodeposition hinders the continuous growth of crystals due to the existence of pulse intervals, thereby making it easier to obtain fine nanoparticles;

Secondly, the temperature is controlled at room temperature or below room temperature, which is easy to control and has a better substrate enhancement effect. The concentration of silver ions in the working solution is reduced, which can save experimental costs.

The preparation process is also affected by a variety of experimental factors, such as current density, temperature, working solution concentration, anode and cathode area ratio, working solution pH, silver ion concentration in the working solution, and electrodeposition method. By adjusting these influencing factors, the particle size of the prepared substrate can meet the conditions of an ideal SERS substrate, and at the same time, there are narrow particle gaps on the substrate that can generate SERS hotspots. In the present invention, the preparation process of the substrate is simple, the production is environmentally friendly, and the substrate can be prepared in batches, thereby improving the efficiency of analysis and detection.

Specifically, in step S1, the copper sheet is also pre-processed, and the specific processing steps are as follows:

S11, ultrasonic cleaning with acetone and deionized water at room temperature for 10 to 20 minutes respectively;

S12, under the condition of 35-45°C water bath heating, the copper sheet after ultrasonic cleaning was subjected to cathodic electrolytic degreasing for 5 minutes, with a current density of 3-5A·dm ^-2 ;

S13, washing the degreased copper sheet with deionized water, and performing anodic electrochemical polishing at room temperature for 40 to 60 seconds until the surface of the copper sheet is smooth and bright, with a current density of 5 to 7 A·dm ^-2 ;

S14, washing the smooth and bright copper sheet with deionized water, and electro-depositing acid copper at a constant current for 5 min at room temperature with stirring, and the current density is 2A·dm ^-2 ;

S15, washing the electrodeposited copper sheet with deionized water, and soaking it in a dilute hydrochloric acid solution with a volume fraction of 5-7% for 5 minutes at room temperature, taking it out and washing it with deionized water, thereby completing the pretreatment of the copper sheet;

Before the preparation of the pulse electrodeposition silver substrate, ultrasonic cleaning, cathode degreasing, anode electrolytic polishing, dilute acid activation and other methods are used to make the surface of the copper plated piece smoother, brighter and more bonded;

Specifically, during the pulse electrodeposition process, when the current is turned on, the electrochemical cathode polarization increases, the metal ions near the cathode area are fully deposited, and the deposited layer is finely crystalline and bright. When the current is turned off, the discharge ions near the cathode area return to the initial concentration, the concentration polarization is eliminated, and the peak current density is often higher, the higher the overpotential is, which also makes it more difficult for the grains to grow;

Furthermore, it is still generally believed that the SERS effect is related to the electromagnetic enhancement generated by localized surface plasmon resonance (LSPR) and chemical enhancement based on charge transfer. Electromagnetic field enhancement is usually considered to be the main contributor to SERS enhancement, and localized surface plasmon resonance contributes the most to electromagnetic field enhancement. The shape and size of the particles in the SERS substrate have a particularly significant effect on the LSPR effect. Figure 10a-d shows SEM images of pulsed electrodeposited silver substrates with pulse periods of 5, 0.5, 0.1 and 0.01 s, respectively. The surfaces of the silver substrates prepared by pulsed electrodeposition with different pulse periods are very uniform and flat, and their appearance is smooth and bright. This is because pulsed electrodeposition increases the activation polarization of the cathode and reduces the concentration polarization of the cathode, which greatly improves the physical and chemical properties of the deposited silver layer, making the deposited layer high in purity, dense in structure and low in porosity. When the pulse period is 0.01 to 2.5 s, the appearance of the silver substrate also presents a dark blue or slightly blue color. The silver substrate is silvery white at 5-10s, and the silver nanoparticles in its SEM image are relatively larger. However, overall, the size of the silver nanoparticles in the substrate prepared by pulse electrodeposition does not exceed 50nm, and the appearance of the prepared silver substrate is blue. The size of the silver nanoparticles is smaller and more delicate. When used for Raman spectroscopy detection, the substrate may show a more significant enhancement effect. Direct observation of the SEM image shows that the size of the silver nanoparticles under the 0.1s pulse period is significantly smaller than that of the silver substrate prepared under other pulse periods. From the results of the SEM image, the deposited silver layers prepared under different pulse periods are composed of delicate spherical grains with relatively regular shapes and relatively uniform particle sizes. The particles are arranged very closely and there are narrow gaps between some adjacent particles. The sizes range from 10nm to tens of nanometers. These characteristics indicate that the performance of the substrate deposition layer prepared by pulse electrodeposition is very excellent.

S2. SERS substrate pretreatment:

S21, washing the SERS substrate with deionized water and ethanol;

S22, immersing the cleaned SERS substrate in a surface treatment solution for 3 to 5 hours, wherein the surface treatment solution includes 0.5 mmol·L ^-1 alkylthiol and 0.15 mmol·L ^-1 4-mercapto-1-butanol, and the concentration ratio of alkylthiol to 4-mercapto-1-butanol is 1:2.5 to 1:3, wherein the chemical expression of alkylthiol is: HS(CH ₂ ) _n CH ₃ , n=5 to 7;

S23, after soaking for 3 to 5 hours, take out the SERS substrate, and wash the SERS substrate with ethanol and deionized water in turn;

S3, spraying Rhodamine 6G molecules uniformly on the SERS substrate, controlling the droplet size to be 100-200 μm, and the number of droplets sprayed at a single time to be 15-20/mm ² , and the Rhodamine 6G molecules evaporate and concentrate to form particles, and a ring structure appears on the surface of the SERS substrate, and the contact angle between the Rhodamine 6G molecules and the SERS substrate is between 110° and 130°;

The prepared substrate surface has a certain hydrophilicity. The addition of rhodamine 6G molecules or the immersion of the substrate in rhodamine 6G molecules leads to a process of naturally drying the substrate. The experimental results are easily affected by the operation and there are uncontrollable factors in evaporation and concentration. Therefore, in the present invention, first, in order to improve the hydrophilicity of the substrate surface, the substrate is surface treated with a surface treatment liquid. 4-mercapto-1-butanol can adjust the contact angle between the probe molecule and the substrate to between 110° and 130°, and self-assemble with alkylthiols with a chain length of C6 to C8. After 3 to 5 hours, the alkylthiols are orderly self-assembled on the substrate. There is a certain hydrophobicity on the substrate. After drying, a ring structure will appear on the substrate due to the action of surface tension;

First, if only alkylthiols are selected to modify the active substrate, the contact angle between the probe molecule and the substrate surface is 175° to 177°. After drying, the solution will only form a shallow small-scale "water stain", which is not conducive to image processing. Secondly, alkylthiols with a chain length lower than C6 have a very strong odor that is difficult to eliminate and are not recommended. Alkylthiols with a chain length higher than C8 have a poor self-assembly effect, which affects the experimental efficiency. Similarly, in order to eliminate the influence of the single number of measured samples during dropwise addition or the randomness generated during the immersion process, the droplets are sprayed to improve sampling and averaging. The above operation is combined with statistical averaging to eliminate the influence of randomness.

S4. Select the ring structure formed by the deposition of probe molecules, and perform two-dimensional SERS imaging with the highest scattering peak of rhodamine 6G molecules at 611 cm ^-1 to obtain an imaging image. The imaging range is 100 μm × 100 μm. Combined with the droplet size, the Raman laser intensity is selected to be 3.5-4.0 mW. This allows the substrate to just enter a dry state when the SERS imaging image is tested, and the droplet will not evaporate too quickly to affect the enhancement effect. The imaging image is used to express the strength distribution of the SERS signal in a certain area on the SERS substrate. The red area indicates the position where the SERS signal is stronger in the Raman spectrum, and the blue area indicates the position where the SERS signal is weaker in the Raman spectrum. The two-dimensional SERS imaging method can collect a large amount of data, display the distribution of signals on the substrate, and facilitate image recognition processing;

After the sprayed droplets were subjected to two-dimensional SERS imaging, due to the effect of surface tension, the obtained image was generally a "coffee ring" distribution, with a weaker Raman signal in the middle and a stronger Raman signal on the ring.

S5. Obtain intensity data [x, y, s] at different positions according to the imaging image, wherein (x, y) represents the position coordinates, s represents the SERS signal intensity at different (x, y) positions, and obtain a corresponding two-dimensional pseudo-color image through the position coordinates (x, y) and the intensity value s;

S6. Quantitative or semi-quantitative detection of SERS substrates by image processing technology:

S61, extracting the target area in the two-dimensional pseudo-color image, that is, the irregular ring structure representing the concentration, converting the image coordinate system [x, y, s] data of the target area into the corresponding pixel coordinate system [X, Y, s], normalizing the pixel coordinates (X, Y) based on the origin, and keeping the intensity value s of each point unchanged;

S62, determining the center point of the target area: converting the two-dimensional pseudo-color image from the RGB color space to the HSV space, by setting the value ranges of the three channels of the HSV space, the three channels are: hue, saturation and brightness. In the RGB color space, the RGB value of red is [255,0,0]. In the HSV space, the value range of the red hue is: H _min = 0, H _max = 10 or H _min = 156, H _max = 180, the value range of the saturation is: S _min = 43, S _max = 255, and the value range of the brightness is: V _min = 46, V _max = 255;

In the RGB color space, the RGB value of blue is [0, 0, 255]. In the HSV space, the blue hue value range is: H _min = 100, H _max = 124, the saturation value range is: S _min = 43, S _max = 255, and the lightness value range is: V _min = 46, V _max = 255. In the present invention, the blue area is extracted as the target area;

Traverse all pixel coordinates (X, Y) of the target area, search for n points within the value range of the three channels, and calculate the average value of the coordinates of the n points as the center point (x ₀ , y ₀ ). The calculation formula is as follows:

x ₀ =(x ₁ +x ₂ +···+x _n )/n;

y ₀ =(y ₁ +y ₂ +···+ _yn )/n;

Wherein, x represents the abscissa of a point within the value range of the three channels, y represents the ordinate of a point within the value range of the three channels, and the subscripts of x and y represent the 1st, 2nd, ..., nth points within the value range of the three channels;

S63, normalize the distance from the center point ( _x0 , _y0 ) to the ring boundary: according to the pixel coordinate system [X, Y, s], from the center point ( _x0 , _y0 ) to the ring boundary, diverge 360° in all directions at intervals of 15°, obtain pixel coordinates (X, Y) in sequence, normalize the pixel coordinates (X, Y) based on the center point ( _x0 , _y0 ), and the intensity value s remains unchanged. The pixel coordinates (X', Y') are traversed to obtain the corresponding intensity value s, and an intensity curve from the center point ( _x0 , _y0 ) to the ring boundary is drawn;

S64. Repeat the above steps to obtain at least 24 intensity curves of different ring structures of the SERS substrate, record the maximum intensity value of each curve, remove the 3 maximum values and the 3 minimum values from the maximum intensity value, and average the remaining intensity values. The average value is the intensity value of the concentration of Rhodamine 6G molecules on the SERS substrate. By using this averaging method, some intensity values that are too concentrated or have little to do with the concentration are removed, and the intensity values that truly represent the concentration are extracted, thereby improving the accuracy of measuring the concentration.

Through the above method, it was obtained that the linear range of Rhodamine 6G molecule was 1.0×10 ^-9 ～1.0×10 ^-13 mol·L ^-1 (correlation coefficient R ² =0.995), and the detection limit obtained in the experiment was 1.0×10 ^-13 mol·L ^-1 . The lowest concentration detection value currently obtained through experiments is 1.0×10 ^-13 mol·L ^-1 , which is the lowest concentration value that can be obtained within the linear range. The lowest detection limit is a theoretical value, which is the ideal concentration that can be achieved by calculating the standard curve made by standard samples. Combined with the lowest concentration value obtained, the signal-to-noise ratio (S/N) is greater than 3. Therefore, it can be shown that the ideal detection limit may be slightly lower than 1.0×10 ^-13 mol·L ^-1 .

Further, first, in the step of preparing the SERS substrate, there are different preparation methods, such as chemical synthesis, self-assembly, nanolithography, template method and pulse electrodeposition (the preparation method in the present invention);

Among them, chemical synthesis method: SERS substrates of various sizes and shapes can be prepared through some techniques such as chemical reduction, chemical replacement, photochemistry or thermal decomposition. Chang et al. reported a simple replacement reduction method in the literature [A facile method to directly deposit the large-scale Ag nanoparticles on a silicon substrate for sensitive, uniform, reproducible and stable SERS substrate], which can directly deposit large-scale Ag nanoparticles on a silicon substrate at room temperature and a short reaction time (3min). The reaction process is a direct wet chemical method, using silver nitrate and hydrogen fluoride as the silver source and reducing agent respectively;

Self-assembly method: two or more components spontaneously form larger aggregates through covalent or non-covalent bonds, and assemble into a two-dimensional or three-dimensional ordered structure, namely the SERS substrate. In the literature [Self-assembly Ag nanoparticle monolayer film as SERS Substrate for pesticide detection], Zhang prepared a SERS active substrate by chemically self-assembling silver nanoparticles on the surface of ITO functionalized with 3-aminopropyltrimethoxysilane (APTMS). First, synthesize silver nanoparticles: 200mL of AgNO ₃ aqueous solution (2.91×10 ^-4 mol·L ^-1 ) was heated to boiling under vigorous stirring, and 1.3mL of 1% trisodium citrate aqueous solution was added. Finally, a gray solution was obtained, which was ready for chemical assembly, and a 50nm silver nanoparticle film SERS substrate was obtained on the glass surface;

Nanolithography: Nanopores can be obtained on continuous metal ultra-thin films, or nanoparticles can be obtained on solid substrates with controllable optical properties, mainly through focused ion beam (FIB) and electron beam lithography (EBL). In the literature [Nafion stabilized Ag nanopillar arrays as a flexible SERS substrate fortrace chemical detection], Guo et al. first produced nanostructure arrays on the surface of Nafion membranes through colloidal lithography and oxygen plasma etching, and then uniformly metallized the nanostructure arrays through ion exchange and in-situ reduction to generate micro/nanostructured SERS active substrates.

Template method: It is a method of depositing metal nanoparticles of controllable geometry on a template, which usually includes soft template method and hard template method. In the literature [AAO Template-Assisted Fabrication of Ordered Ag Nanoparticles-Decorated Au Nanotubes Array for Surface-Enhanced Raman Scattering Detection], Sun et al. further oxidized the conical hole AAO template by multi-step anodization and etching to prepare a funnel hole AAO template. Then, the prepared funnel hole AAO was used as a sacrificial template, and physical sputtering was used to assemble Au-NTs and Ag-NPs to obtain a SERS active substrate;

The detection limit of the rhodamine 6G molecule was obtained by the above method, specifically, as shown in the following Table 2:

Table 2

It can be seen from Table 2 that the detection limit of Rhodamine 6G molecules obtained by the SERS substrate prepared by pulse electrodeposition combined with two-dimensional SERS imaging and image processing technology is significantly lower than or close to the detection limit obtained by substrates prepared by other commonly used methods, which proves that the present invention has good sensitivity and strong practicality in analytical detection.

Secondly, in step S1, the formula of the cyanide-free silver plating solution is changed to Formula 1, and the detection limit of the rhodamine 6G molecule is obtained, specifically, as shown in the following Table 3:

Formula 1: Zhang et al. gave a silver plating solution formula in the literature [Preparation of Ag nanosheet hierarchicalclubbed micro/nanostructured arrays and their application based on the SERS effect], which contained 0.5g·L ^-1 silver nitrate, 3.75g·L ^-1 polyvinyl pyrrolidone (PVP) and 0.75g·L ^-1 sodium citrate. The hierarchical rod-shaped micro/nanostructured array of silver nanosheets was prepared by constant current device at room temperature and low current density (-10μA·cm ^-2 ) for 3h. Among them, graphite sheet was used as anode and gold-plated zinc oxide nanorod array was used as cathode;

table 3

Masterbatch formula Detection limit of rhodamine 6G molecule Recipe 1 1.0×10 ^-12 mol·L ^-1 Mother liquor formula (the present invention) as given in Table 1 1.0×10 ^-13 mol·L ^-1

It can be seen from Table 3 that the SERS substrates prepared by the cyanide-free silver plating solution formula used in the present invention have higher SERS activity and sensitivity than other silver plating solution formulas reported in the literature.

Qualitative analysis is generally performed by detecting the characteristics of the corresponding substance through means such as SERS and substrates, while the present invention realizes the preparation of a substrate that is simple to operate, large-scale, reproducible and stable, solves the problems of uniformity, reliability and sensitivity in the test process, improves the possibility of quantitative analysis, and realizes accurate quantitative or semi-quantitative analysis. On the basis of qualitative analysis, the detection means are optimized, and the minimum value of the corresponding substance, such as concentration, is quantitatively obtained to confirm the detection limit, so as to be used for detecting certain prohibited dyes, pigments, etc. that exist in trace amounts in aqueous solutions.

The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims (4)

1. A SERS quantitative analysis method based on coffee ring concentration effect and image processing technology, characterized in that it comprises the following steps: S1. Preparation of SERS substrate: At room temperature, a SERS substrate was prepared on a copper sheet by pulse electrodeposition. The working solution concentration was V _{mother solution} : V _{deionized water} = 1:1, wherein the mother solution was a cyanide-free silver plating solution, the pH of the working solution was 9.5-10.5, the cation and cathode area ratio was 1:10, the peak current density was 1-2 A·dm ^-2 , the duty cycle was 40%-50%, the pulse period was 0.05-0.1s, and the pulse plating time was 600-750s; S2. SERS substrate pretreatment: S21, washing the SERS substrate with deionized water and ethanol; S22, immersing the cleaned SERS substrate in a surface treatment solution for 3 to 5 hours, wherein the surface treatment solution comprises 0.5 mmol·L ^-1 alkylthiol and 0.15 mmol·L ^-1 4-mercapto-1-butanol, and the concentration ratio of alkylthiol to 4-mercapto-1-butanol is 1:2.5 to 1:3, wherein the chemical expression of alkylthiol is: HS(CH ₂ ) _n CH ₃ , n=5 to 7; S23, after soaking for 3 to 5 hours, take out the SERS substrate, and wash the SERS substrate with ethanol and deionized water in turn; S3, spraying the probe molecules uniformly on the SERS substrate, controlling the droplet size to be 100-200 μm, and the number of droplets sprayed at a single time to be 15-20/mm ² , and the probe molecules evaporate and concentrate to form particles, and a ring structure appears on the surface of the SERS substrate, wherein the probe molecules include rhodamine 6G molecules, methyl orange molecules, 2,2'-bipyridine molecules, 4-mercaptopyridine molecules and 1,4-benzenedithiol; S4, select the ring structure formed by the deposition of the probe molecule, and perform two-dimensional SERS imaging with its highest scattering peak to obtain an imaging map. The imaging range is 100μm×100μm. Combined with the droplet size, the Raman laser intensity is selected to be 3.5-4.0mW. The imaging map is used to express the strength distribution of the SERS signal in a certain area on the SERS substrate. The red area indicates a strong SERS signal in the Raman spectrum, and the blue area indicates a weak SERS signal in the Raman spectrum. S5. Obtain intensity data [x, y, s] at different positions according to the imaging image, wherein (x, y) represents the position coordinates, s represents the SERS signal intensity at different (x, y) positions, and obtain a corresponding two-dimensional pseudo-color image through the position coordinates (x, y) and the intensity value s; S6. Quantitative or semi-quantitative detection of SERS substrates by image processing technology: S61, extracting the target area in the two-dimensional pseudo-color image, that is, the irregular ring structure representing the concentration, converting the image coordinate system [x, y, s] data of the target area into the corresponding pixel coordinate system [X, Y, s], normalizing the pixel coordinates (X, Y) based on the origin, and keeping the intensity value s of each point unchanged; S62, determining the center point of the target area: converting the two-dimensional pseudo-color image from the RGB color space to the HSV space, by setting the value ranges of the three channels of the HSV space, the three channels are: hue, saturation and brightness. In the RGB color space, the RGB value of blue is [0,0,255]. In the HSV space, the value range of the blue hue is: H _min =100, H _max =124, the saturation value range is: S _min =43, S _max =255, and the brightness value range is: V _min =46, V _max =255. Extract the blue area as the target area; Traverse all pixel coordinates (X, Y) of the target area, search for n points within the value range of the three channels, and calculate the average value of the coordinates of the n points as the center point (x ₀ , y ₀ ). The calculation formula is as follows: x ₀ =(x ₁ +x ₂ +···+x _n )/n; y ₀ =(y ₁ +y ₂ +···+ _yn )/n; Wherein, x represents the abscissa of a point within the value range of the three channels, y represents the ordinate of a point within the value range of the three channels, and the subscripts of x and y represent the 1st, 2nd, ..., nth points within the value range of the three channels; S63, normalize the distance from the center point ( _x0 , _y0 ) to the ring boundary: according to the pixel coordinate system [X, Y, s], from the center point ( _x0 , _y0 ) to the ring boundary, diverge 360° in all directions at intervals of 15°, obtain pixel coordinates (X, Y) in sequence, normalize the pixel coordinates (X, Y) based on the center point ( _x0 , _y0 ), and the intensity value s remains unchanged. The pixel coordinates (X', Y') are traversed to obtain the corresponding intensity value s, and an intensity curve from the center point ( _x0 , _y0 ) to the ring boundary is drawn; S64. Repeat the above steps to obtain at least 24 intensity curves of different ring structures of the SERS substrate, record the maximum intensity value of each curve, remove the 3 maximum values and 3 minimum values from the maximum intensity values, and calculate the average value of the remaining intensity values. The average value is the concentration intensity value of the probe molecule solution on the SERS substrate. 2. The SERS quantitative analysis method based on the coffee ring concentration effect and image processing technology according to claim 1 is characterized in that, in step S1, the cyanide-free silver plating solution comprises 40-50 g·L ^-1 silver ion complexing agent, 5-10 g·L ^-1 copper ion complexing agent, 0.01-0.05 g·L ^-1 organic brightener, 15-25 g·L ^-1 silver ion source and pH regulator, the silver ion complexing agent comprises at least one of aminonicotinic acid, 6-aminonicotinic acid methyl ester, and 5-methylnicotinic acid, the copper ion complexing agent comprises at least one of cysteine, Z-proline- _NH2 or 1,2-cyclohexanediaminetetraacetic acid, the organic brightener comprises at least one of 3-methacryloyl dopamine, dopamine 4-0-sulfate or ractopamine methyl ether, the silver ion source comprises one of silver oxide or silver carbonate, and the pH regulator comprises at least one of potassium hydroxide or sodium hydroxide. 3. The SERS quantitative analysis method based on coffee ring concentration effect and image processing technology according to claim 1 is characterized in that, in step S1, it also includes pre-treatment of the copper sheet, and the specific processing steps are as follows: S11, ultrasonic cleaning with acetone and deionized water at room temperature for 10 to 20 minutes respectively; S12, under the condition of heating in a water bath at 35-40°C, the copper sheet after ultrasonic cleaning is subjected to cathodic electrolytic degreasing for 5 minutes at a current density of 3-5A·dm ^-2 ; S13, washing the degreased copper sheet with deionized water, and performing anodic electrochemical polishing at room temperature for 40 to 60 seconds until the surface of the copper sheet is smooth and bright, with a current density of 5 to 7 A·dm ^-2 ; S14, washing the smooth and bright copper sheet with deionized water, and electro-depositing acid copper at a constant current for 5 min at room temperature with stirring, and the current density is 2A·dm ^-2 ; S15, washing the electrodeposited copper sheet with deionized water, and soaking it in a dilute hydrochloric acid solution with a volume fraction of 5-7% for 5 minutes at room temperature, taking it out and washing it with deionized water, thereby completing the pretreatment of the copper sheet. 4. The SERS quantitative analysis method based on coffee ring concentration effect and image processing technology according to claim 1 is characterized in that, in step S22, the contact angle between the probe molecule and the SERS substrate is between 110° and 130°.