CN116500014A - Method for simultaneously and quantitatively detecting concentration of uric acid and creatinine in complex matrix based on paper chromatography and surface-enhanced Raman scattering technology - Google Patents

Abstract

A method for simultaneously and quantitatively detecting uric acid and creatinine concentration in a complex matrix based on paper chromatography and surface enhanced Raman scattering technology relates to the field of detection methods. The invention aims to solve the problems that the existing creatinine and uric acid detection method is easy to be interfered, needs a large-scale instrument, is complex in operation and difficult to detect on site, and simple surface enhanced Raman scattering is limited by competitive adsorption and is difficult to accurately detect target substances in a complex matrix. 1. Preparing a paper chromatograph-surface enhanced Raman scattering substrate based on an aggregation-promoting agent induced assembly technology; 2. separating and enriching uric acid and creatinine in a complex solution at positions with specific shift values of 0 and 0.64 by using a paper chromatography-surface enhanced Raman scattering substrate; 3. in-situ detection of uric acid and muscle using portable raman spectrometerThe surface enhanced raman scattering signal of the anhydride was analyzed quantitatively. The detection limit of the invention on uric acid and creatinine in urine is respectively as low as 10 ^‑5 M and 10 ^‑4 M, satisfy actual detection demand.

Description

A technique based on paper chromatography and surface-enhanced Raman scattering in complex matrices Method for Quantitative Detection of Uric Acid and Creatinine Concentration

technical field

The invention relates to the field of detection methods, in particular to a method for simultaneously quantitatively detecting the concentrations of uric acid and creatinine in complex matrices based on paper chromatography and surface-enhanced Raman scattering technology.

Background technique

Uric acid is the final product of purine metabolism in the body. It is slightly soluble in water. Uric acid is usually produced by kidney metabolism, and almost all of it is excreted through urine. The normal value of serum uric acid in healthy adults is 0.15~0.4mM, and the normal value in urine is 1.1~ 4.4mM, purine metabolism disorder and kidney disease can lead to abnormal uric acid metabolism. Currently, uric acid determination is the best indicator for diagnosing hyperuricemia caused by abnormal purine metabolism. Uric acid can also be used for the diagnosis and monitoring of kidney disease. Therefore, uric acid Detection is important.

Creatinine is the metabolite of creatine in the body, which is filtered out by the glomerulus and excreted. The normal value of urine creatinine for women is 7.9-14.1mmol/24h, and that for men is 9.7-24.7mmol/24h. Urinary creatinine and serum creatinine are important indicators for detecting kidney disease and evaluating its development, and are closely related to the health of the kidneys. In addition, urinary creatinine is often used as an internal standard to correct the content of other substances in urine, such as drugs and toxins, so the development of A method for rapid detection of creatinine is of great significance.

The current methods for detecting uric acid are mainly phosphotungstic acid colorimetric method and enzymatic method, but the operation is cumbersome and the detection cost is high. Commonly used methods for urinary creatinine are the Jaffe reaction method and enzymatic method, but due to the interference of other substances in body fluids, the detection specificity is low. Other detection methods such as HPLC and chromatography-mass spectrometry have extremely low detection limits, but the equipment is expensive. Unable to detect on site.

Surface-enhanced Raman scattering (SERS) technology is a fast and highly sensitive detection technology that can provide molecular fingerprint information of the analyte. Therefore, SERS technology has been widely studied in recent years, but it is limited by the competitive adsorption effect. SERS It is difficult for technology to complete detection tasks in complex matrices, and the signal of analyte substances with small Raman cross-sections will be completely submerged and cannot be accurately detected. Paper chromatography (PC) is an effective liquid-liquid partition chromatography. Due to the different distribution coefficients of the components in the sample in the stationary phase and the mobile phase, they move at different speeds on the filter paper, and finally each substance stays on the filter paper differently. The location achieves separation. The ratio shift value (Rf) is often used to represent the distribution of each substance after separation. Nanoparticles were assembled on the surface of chromatographic paper to make a PC-SERS substrate, and the two technologies were combined to quickly realize the simultaneous separation and detection of uric acid and creatinine in complex matrices. However, although simple SERS detection has high sensitivity, it is difficult to detect target substances in complex media due to competitive adsorption.

Contents of the invention

The purpose of the present invention is to solve the problem that the existing creatinine and uric acid detection methods are susceptible to interference, require large-scale instruments, are cumbersome to operate, and are difficult to detect on-site. Simple surface-enhanced Raman scattering is limited by competitive adsorption, and it is difficult to accurately detect target substances in complex matrices. To provide a method for the simultaneous quantitative detection of uric acid and creatinine in complex matrices based on paper chromatography and surface-enhanced Raman scattering.

The invention designs a PC-SERS substrate, which effectively overcomes the interference caused by competitive adsorption on SERS detection, and provides a method that does not require pretreatment, low cost, high sensitivity, and can be detected on-site with a portable Raman spectrometer. Simultaneous detection of uric acid and creatinine in complex matrices.

A method for simultaneous quantitative detection of uric acid and creatinine concentrations in complex matrices based on paper chromatography and surface-enhanced Raman scattering technology, specifically completed by the following steps:

1. Preparation of PC-SERS substrate:

①. Immerse the substrate material in the aggregation accelerator solution for a period of time, take it out and air-dry it to obtain the substrate material treated with the aggregation accelerator;

②. First, immerse the substrate material treated with the aggregation accelerator into the concentrated solution of silver nanoparticles for a period of time, then take it out, rinse it with deionized water, and finally air-dry it to obtain the PC-SERS substrate;

2. Separation of uric acid and creatinine in complex matrix:

Spot a small amount of the solution to be tested at a distance of 15 mm from the lower end of the PC-SERS substrate, air-dry it, then immerse the lower end of the substrate in the developer in a closed container and spread it upwards, take it out when it develops to the end of the chromatography, and dry it naturally to obtain a paper PC-SERS substrate after chromatographic separation;

3. SERS detection of uric acid and creatinine:

The PC-SERS substrate separated by paper chromatography was pasted on a glass slide for SERS detection. The strongest signals of uric acid and creatinine were detected at the positions of ratio shift value 0 and 0.64, respectively. After noise suppression and baseline subtraction were performed on the spectrum, Quantitative analysis based on characteristic peak intensities.

The present ^invention is based on wavelet threshold noise reduction, iterative penalty partial least squares method, multivariate scattering correction and ^other algorithms. The peak intensity is substituted into the concentration-characteristic peak intensity curve to obtain the concentration of uric acid and creatinine in the tested solution.

Beneficial effects of the present invention:

1. The PC-SERS substrate prepared by the present invention can achieve high stability, high accuracy, and high sensitivity while reducing the cost of detection. With the help of a portable Raman spectrometer, it can be used for large-scale screening of related diseases in medically backward areas. Provides a new solution for on-site rapid detection of uric acid and creatinine;

2. The present invention is based on PC-SERS technology, which overcomes the difficulty of SERS technology in the detection of complex matrix to a certain extent, and realizes the simultaneous detection of uric acid and creatinine. The sample does not need pretreatment, and can directly detect the substrate in the complex matrix. test substance;

3. The PC-SERS substrate prepared by the present invention, according to the detection method described in the present invention, has a detection limit of ^10-4 M for creatinine in artificial urine, and a detection limit of ^10-5 M for uric acid. ;

4. The present invention has the advantages of fast detection speed, no need for sample pretreatment, low cost, high sensitivity, simultaneous detection of multiple targets, and portability, and can overcome the shortcomings of SERS technology that is difficult to complete detection in complex matrices. Rapid detection provides a new solution.

Description of drawings

Fig. 1 is a schematic diagram of PC-SERS substrate preparation and on-site detection method for simultaneous quantitative detection of uric acid and creatinine in a complex matrix of the present invention;

Fig. 2 is the scanning electron micrograph of the PC-SERS substrate that embodiment 1 prepares;

Fig. 3 is the detection effect of utilizing the PC-SERS substrate prepared in embodiment 1 to rhodamine 6G;

Fig. 4 directly detects the SERS signal and the uric acid standard solution of the mixed solution to be tested (comprising fetal bovine serum 1mg/mL, glucose 4mM, creatinine 0.5mM, uric acid 0.5mM), the SERS signal normalization contrast figure of creatinine standard solution, Fig. Among them, creatinine is creatinine, mix is the mixed solution to be tested, and uric acid is uric acid;

Figure 5 is a waterfall diagram of the SERS signal and the sampling position drawn at a sampling interval of 5mm on the PC-SERS substrate;

Figure 6 is the ratio shift value on the horizontal axis, and the 1135cm ^-1 characteristic peak intensity of uric acid and the 1426cm ^-1 characteristic peak intensity of creatinine are respectively used as the vertical axis to perform B-spline fitting to obtain the substances to be tested on the PC-SERS substrate Distribution;

Fig. 7 is the uric acid concentration-characteristic peak intensity curve obtained by using the B-spline fitting method to obtain the uric acid concentration and the uric acid 1135cm ^-1 characteristic peak intensity detected by PC-SERS substrate, ^R2 is 0.9945;

Figure 8 is the creatinine concentration-characteristic peak intensity curve obtained by using the B-spline fitting method to obtain the creatinine concentration and the creatinine 1426cm ^-1 characteristic peak intensity detected by using the PC-SERS substrate, and ^R2 is 0.9940;

Fig. 9 is the result figure of quantitative analysis of uric acid concentration in the test set sample;

Figure 10 is a graph showing the results of quantitative analysis of creatinine concentration in the test set samples.

Detailed ways

Embodiment 1: In this embodiment, a method for simultaneously quantitatively detecting the concentration of uric acid and creatinine in a complex matrix based on paper chromatography and surface-enhanced Raman scattering technology is specifically completed according to the following steps:

1. Preparation of PC-SERS substrate:

①. Immerse the substrate material in the aggregation accelerator solution for a period of time, take it out and air-dry it to obtain the substrate material treated with the aggregation accelerator;

②. First, immerse the substrate material treated with the aggregation accelerator into the concentrated solution of silver nanoparticles for a period of time, then take it out, rinse it with deionized water, and finally air-dry it to obtain the PC-SERS substrate;

2. Separation of uric acid and creatinine in complex matrix:

Spot a small amount of the solution to be tested at a distance of 15 mm from the lower end of the PC-SERS substrate, air-dry it, then immerse the lower end of the substrate in the developer in a closed container and spread it upwards, take it out when it develops to the end of the chromatography, and dry it naturally to obtain a paper PC-SERS substrate after chromatographic separation;

3. SERS detection of uric acid and creatinine:

The PC-SERS substrate separated by paper chromatography was pasted on a glass slide for SERS detection. The strongest signals of uric acid and creatinine were detected at the positions of ratio shift value 0 and 0.64, respectively. After noise suppression and baseline subtraction were performed on the spectrum, Quantitative analysis based on characteristic peak intensities.

Specific embodiment two: the difference between this embodiment and specific embodiment one is: the base material described in step one 1. is cellulose chromatography paper; the aggregation promoting agent solution described in step one 1. is NaCl solution or KCl solution , the concentration is 10mmol/L～100mmol/L. Other steps are the same as in the first embodiment.

Embodiment 3: This embodiment differs from Embodiment 1 or Embodiment 2 in that: in step ①, the base material is immersed in the aggregation accelerator solution for 10 minutes to 60 minutes. Other steps are the same as those in Embodiment 1 or 2.

Embodiment 4: This embodiment differs from Embodiment 1 to Embodiment 3 in that: the particle size of the silver nanoparticles in the silver nanoparticle concentrate described in Step 1② is 30nm-120nm. Other steps are the same as those in Embodiments 1 to 3.

Embodiment five: the difference between this embodiment and embodiment one to four is: the preparation method of the silver nanoparticle concentrate described in step one 2. is: 0.036g silver nitrate is dissolved in 200mL deionized water, stir And heat until the solution boils, then add 2mL ~ 8mL sodium citrate solution with a mass fraction of 1%, keep boiling for 0.5h ~ 1h, stop heating, cool to room temperature, after 1 ~ 3 times of centrifugation, take the precipitate and redissolve in Dilute to 1/10-1/4 of the volume before centrifugation with deionized water. Other steps are the same as those in Embodiments 1 to 4.

Specific embodiment six: the difference between this embodiment and one of specific embodiments one to five is: in step 1 ②, the base material treated with the aggregation promoting agent is immersed in the silver nanoparticle concentrated solution for 2h to 24h; in step 1 ②, use Rinse with deionized water for 2 to 5 times, each time for 3s to 6s. Other steps are the same as those in Embodiments 1 to 5.

Embodiment 7: This embodiment differs from Embodiment 1 to Embodiment 6 in that: the sample volume of the trace solution to be tested described in step 2 is 1 μL to 10 μL; the developing agent described in step 2 is water and isocyanate. A mixture of propanol, in which the volume ratio of water and isopropanol is 3:2. Other steps are the same as those in Embodiments 1 to 6.

Embodiment 8: This embodiment differs from Embodiments 1 to 7 in that: in step 2, the lower end of the PC-SERS substrate is immersed in the developing agent for 3 mm to 7 mm, and the span is 30 mm to 140 mm when reaching the end point of the chromatography. Other steps are the same as those in Embodiments 1 to 7.

Embodiment 9: The difference between this embodiment and Embodiment 1 to Embodiment 8 is that the solution to be tested in step 2 is urine, serum or tears. Other steps are the same as those in Embodiments 1 to 8.

Embodiment 10: The difference between this embodiment and Embodiments 1 to 9 is that the range of SERS detection in step 3 is 500 cm ^-1 to 1800 cm ^-1 , and after collecting the SERS signal, according to the 1135 cm ^-1 characteristic of uric acid Quantitative analysis of peak intensity and 1426cm ^-1 characteristic peak intensity of creatinine; in step 3, the laser line scanning method is used to determine the distribution of uric acid and creatinine according to the change of characteristic peak intensity, and determine the detection position. Other steps are the same as those in Embodiments 1 to 9.

Adopt the following examples to verify the beneficial effects of the present invention:

Embodiment 1: a kind of preparation method of PC-SERS substrate is specifically finished according to the following steps:

1. Preparation of PC-SERS substrate:

①. Immerse the substrate material in the aggregation accelerator solution for 20 minutes, take it out and air-dry it to obtain the substrate material treated with the aggregation accelerator;

The base material described in step 1.1 is cellulose chromatography paper with a size of 1cm×10cm;

The aggregation promoting agent solution described in step 1. is NaCl solution, and the concentration is 20mmol/L;

②. First, immerse the base material treated with the aggregation accelerator into the concentrated solution of silver nanoparticles for 4 hours, then take it out, and rinse it with deionized water for 3 times, each time for 3 seconds, so as to completely remove the particles that are not tightly combined with the chromatography paper. The silver nanoparticles are finally air-dried to obtain the PC-SERS substrate;

The particle diameter of the silver nanoparticles in the silver nanoparticle concentrate described in step 1.2 is 30nm～120nm, and it is prepared by the sodium citrate reduction method. The specific preparation method is: 0.036g of silver nitrate is dissolved in 200mL of deionized water, and stirred And heat until the solution boils, then add 5mL of sodium citrate solution with a mass fraction of 1%, keep boiling for 0.8h, stop heating, cool to room temperature, centrifuge at 8000r/min for 10min, discard the supernatant, take the precipitate and redissolve Dilute to 1/8 of the volume before centrifugation in deionized water.

Fig. 2 is the scanning electron micrograph of the PC-SERS substrate that embodiment 1 prepares;

It can be seen from Figure 2 that silver nanoparticles were successfully assembled on the substrate surface, and the particle size and density were relatively uniform.

Using the PC-SERS substrate prepared in Example 1 to detect rhodamine 6G solutions with different concentrations, the detection effect diagram is shown in Figure 3;

Fig. 3 is the detection effect of utilizing the PC-SERS substrate prepared in embodiment 1 to rhodamine 6G;

It can be seen from FIG. 3 that the detection limit of the PC-SERS substrate prepared in Example 1 to rhodamine 6G is 100 pM, and it can be seen that the prepared substrate has excellent SERS performance.

Embodiment 2: A kind of method that facilitates the PC-SERS substrate prepared in embodiment 1 to quantitatively detect uric acid and creatinine concentration simultaneously in a complex matrix based on paper chromatography and surface-enhanced Raman scattering technology is specifically completed in the following steps:

1. Separation of uric acid and creatinine in complex matrix:

Spot 3 μL of the mixed solution to be tested at a distance of 15mm from the lower end of the PC-SERS substrate, air-dry, then immerse the lower end of the substrate in the developing agent in a closed container and spread upward, and take it out when it develops to the end of chromatography, and air-dry naturally , to obtain the PC-SERS substrate after paper chromatographic separation;

The concentration of fetal bovine serum in the trace mixed solution to be tested described in step 1 is 1mg/mL, the concentration of glucose is 4mM, the concentration of creatinine is 0.5mM, and the concentration of uric acid is 0.5mM;

The developing agent described in step 1 is a mixed solution of water and Virahol, wherein the volume ratio of water and Virahol is 3:2;

In step 1, the lower end of the PC-SERS substrate is immersed in the developing agent for 5mm, and the spread distance is 70mm when reaching the end point of chromatography;

2. SERS detection of uric acid and creatinine:

Paste the PC-SERS substrate separated by paper chromatography on the glass slide for SERS detection, use 785nm laser as excitation light source, integrate time 15s, average 2 times, detection range is 500～1800cm ^-1 , and collect once every 2.5mm The SERS signal, as shown in Figure 5, is a waterfall diagram of the SERS signal and the sampling position drawn at a sampling interval of 5mm; it can be seen from Figure 5 that the signal of the uric acid molecule is the strongest at the Rf value of 0 (0mm), which is due to Uric acid has very low solubility in developing solvent. The creatinine signal was the strongest at the Rf value of 0.64 (45mm), indicating that creatinine moved here with the developing agent. Due to the volatility of isopropanol, it does not interfere with the measurement. Taking the ratio shift as the horizontal axis, and taking the 1135cm ^-1 characteristic peak intensity of uric acid and the 1426cm ^-1 characteristic peak intensity of creatinine as the vertical axis respectively, B-spline fitting was carried out to obtain the material distribution on the PC-SERS substrate as shown in Figure 6 Show. The above results prove that the simultaneous detection of uric acid and creatinine in complex matrices cannot be detected solely based on SERS technology, but the PC-SERS substrate described in the present invention can realize simultaneous detection of uric acid and creatinine in complex matrices.

Detect the SERS signal of creatinine standard solution (creatinine 0.5mM), detect the SERS signal of uric acid standard solution (uric acid 0.5mM), detect the mixed solution to be tested (the concentration of fetal calf serum in the mixed solution to be tested is 1mg/mL, The concentration of glucose is 4mM, the concentration of creatinine is 0.5mM, and the concentration of uric acid is 0.5mM;) the SERS signal is drawn in Figure 4 after normalizing the obtained SERS signal;

It can be seen from Figure 4 that the signal of the mixed solution to be tested is almost identical to the signal of the uric acid standard solution, and several main characteristic peaks of creatinine are almost invisible, because the Raman cross section of uric acid is much higher than that of creatinine and several other interfering substances. The signal enhancement hotspot will be preempted by uric acid, and it is impossible to detect uric acid and creatinine simultaneously in complex matrices directly based on SERS technology.

Embodiment 3: A kind of method that is beneficial to the PC-SERS substrate prepared in embodiment 1 is based on paper chromatography and surface-enhanced Raman scattering technology to quantitatively detect the method for uric acid and creatinine concentration simultaneously in complex matrix, specifically is finished according to the following steps:

1. Preparation of artificial urine with different concentrations of creatinine and uric acid:

Accurately weigh the pure substance powder of uric acid and creatinine, use artificial urine as the solvent to prepare the mother liquor, add 100mM NaOH solution dropwise to the uric acid mother liquor until the uric acid precipitate is completely dissolved, ultrasonically treat the solution for 5 minutes to disperse the solution evenly, and use a pipette to draw an appropriate amount of The two mother liquors are mixed and diluted with artificial urine to obtain artificial urine with different concentrations of creatinine and uric acid (the concentration of uric acid in the artificial urine is 1 μM to 4 mM, and the concentration of creatinine is 1 μM to 30 mM);

2. Spot 3uL of artificial urine with different concentrations of uric acid and creatinine on the PC-SERS substrate at a distance of 15mm from the bottom and wait for it to air-dry. Put the developing agent configured with water:isopropanol=3:2 in an airtight container , shake to speed up the rate at which the developer vapor reaches saturation in the container;

3. Hang the PC-SERS substrate in the container, immerse the bottom of the substrate in the developing agent for 5mm, spread it upwards, take it out when the spreading distance is 70mm, and air dry it in the fume hood;

4. Paste the separated and air-dried PC-SERS substrate on the glass slide for SERS detection, use 785nm laser as the excitation light source, integrate time 15s, average 2 times, the detection range is 500～1800cm ^-1 , respectively in the ratio shift value The SERS signals of uric acid and creatinine were detected for the 0 and 0.64 positions, and 20 spectra were collected for each gradient concentration to take the average spectrum for subsequent curve fitting.

Based on wavelet threshold noise reduction, iterative penalty partial least squares method, multivariate scattering correction and other algorithms, after noise suppression and baseline subtraction are processed on the spectrum, the 1135cm ^-1 characteristic peak intensity of uric acid and the 1426cm ^-1 characteristic peak intensity of creatinine will be measured Calculate the average value, and use the B-spline fitting method to obtain the concentration-characteristic peak intensity curves of uric acid and creatinine, as shown in Figure 7 and Figure 8 respectively, and the ^R of the fitting curves are 0.9945 and 0.9940 respectively.

Use the above method to detect artificial urine mixed with different concentrations of uric acid and creatinine, and substitute the uric acid and creatinine characteristic peak intensities detected at Rf=0 and Rf=0.64 into the fitting curve to obtain the concentration of uric acid and creatinine in the solution, thereby The simultaneous quantitative detection of uric acid and creatinine is realized, and the test sets of uric acid and creatinine each contain 30 spectra. Figure 9 and Figure 10 show the detection results of uric acid and creatinine in the test set samples respectively. It can be seen that the predicted value and the real value show good consistency, and the relative deviation between most of the predicted results and the real value is less than 10%. Examples illustrate that the present invention can simultaneously achieve good detection effects on uric acid and creatinine in complex matrices.

Claims (10)

1. A method for simultaneously and quantitatively detecting uric acid and creatinine concentration in a complex matrix based on paper chromatography and surface-enhanced Raman scattering technology is characterized by comprising the following steps:

1. preparing a PC-SERS substrate:

(1) immersing the substrate material into the aggregation-promoting agent solution for a period of time, taking out and air-drying to obtain the substrate material treated by the aggregation-promoting agent;

(2) immersing a base material treated by an aggregation-promoting agent into silver nanoparticle concentrated solution for a period of time, taking out, washing with deionized water, and finally air-drying to obtain a PC-SERS substrate;

2. separation of uric acid and creatinine in complex matrices:

spotting a trace amount of solution to be detected at a position 15mm away from the lower end of the PC-SERS substrate, immersing the lower end of the substrate into a developing agent in a closed container for developing upwards after air drying, taking out the substrate when developing to a chromatography end point, and naturally air drying to obtain the PC-SERS substrate after paper chromatography separation;

3. SERS detects uric acid and creatinine:

and sticking the PC-SERS substrate subjected to paper chromatographic separation on a glass slide for SERS detection, respectively detecting the strongest signals of uric acid and creatinine at the positions with the specific shift value of 0 and 0.64, and quantitatively analyzing according to the characteristic peak intensity after noise suppression and baseline deduction of the spectrum.

2. The method for simultaneous quantitative detection of uric acid and creatinine concentrations in a complex matrix based on paper chromatography and surface enhanced raman scattering techniques as defined in claim 1, wherein the substrate material in step one (1) is cellulose chromatography paper; the aggregation-promoting agent solution in the step one (1) is NaCl solution or KCl solution, and the concentration is 10 mmol/L-100 mmol/L.

3. The method for simultaneous quantitative detection of uric acid and creatinine concentrations in a complex matrix based on paper chromatography and surface enhanced raman scattering techniques as defined in claim 1, wherein the step one (1) is to immerse the substrate material in the aggregation-promoting agent solution for 10min to 60min.

4. The method for simultaneous quantitative detection of uric acid and creatinine concentrations in a complex matrix based on paper chromatography and surface-enhanced raman scattering (surface-enhanced raman scattering) according to claim 1, wherein the particle size of silver nanoparticles in the silver nanoparticle concentrate in step one (2) is 30nm to 120nm.

5. The method for simultaneous quantitative detection of uric acid and creatinine concentrations in a complex matrix based on paper chromatography and surface enhanced raman scattering (surface enhanced raman scattering) according to claim 1, wherein the preparation method of the silver nanoparticle concentrate in step one (2) is as follows: dissolving 0.036g of silver nitrate into 200mL of deionized water, stirring and heating to boil the solution, adding 2 mL-8 mL of sodium citrate solution with mass fraction of 1%, keeping boiling for 0.5 h-1 h, stopping heating, cooling to room temperature, centrifuging for 1-3 times, taking precipitate, redissolving in deionized water, and fixing the volume to 1/10-1/4 of the volume before centrifuging.

6. The method for simultaneous quantitative detection of uric acid and creatinine concentrations in a complex matrix based on paper chromatography and surface-enhanced raman scattering (surface-enhanced raman scattering) technology according to claim 1, wherein in step one (2), the aggregation-promoting agent-treated base material is immersed in a silver nanoparticle concentrate for 2-24 h; and (3) in the step (2), deionized water is used for washing for 2 to 5 times, and the washing time is 3 to 6 seconds each time.

7. The method for simultaneously and quantitatively detecting uric acid and creatinine concentrations in a complex matrix based on paper chromatography and surface-enhanced Raman scattering technology as defined in claim 1, wherein the sample application amount of the trace amount of the solution to be detected in the second step is 1-10 mu L; the developing agent in the second step is a mixed solution of water and isopropanol, wherein the volume ratio of the water to the isopropanol is 3:2.

8. The method for simultaneously and quantitatively detecting uric acid and creatinine concentrations in a complex matrix based on paper chromatography and surface-enhanced Raman scattering technology as claimed in claim 1, wherein the lower end of the PC-SERS substrate is immersed in a developing agent for 3-7 mm, and the developing distance is 30-140 mm when reaching the chromatography end point.

9. The method for simultaneously and quantitatively detecting uric acid and creatinine concentrations in a complex matrix based on paper chromatography and surface-enhanced raman scattering (SERS) according to claim 1, wherein the solution to be detected in the second step is urine, serum or tear.

10. The method for simultaneous quantitative detection of uric acid and creatinine concentrations in a complex matrix based on paper chromatography and Surface Enhanced Raman Scattering (SERS) techniques as defined in claim 1, wherein the SERS detection in step three is performed in a range of 500cm ^-1 ～1800cm ^-1 1135cm based on uric acid after acquisition of SERS signal ^-1 1426cm of characteristic peak intensity and creatinine ^-1 Quantitative analysis of the intensity of the characteristic peak; and step three, determining the distribution condition of uric acid and creatinine according to the intensity change of the characteristic peak by adopting a laser line scanning mode, and determining the detection position.