CN116500014B - 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 - Google Patents

Abstract

A method for simultaneously and quantitatively detecting the concentrations of uric acid and creatinine in a complex matrix based on paper chromatography and surface enhanced Raman scattering technology, which relates to the field of detection methods. The purpose of the present invention is to solve the problems that the existing creatinine and uric acid detection methods are susceptible to interference, require large instruments, are cumbersome to operate, are difficult to detect on-site, and simple surface enhanced Raman scattering is limited by competitive adsorption, and it is difficult to accurately detect the target substance in a complex matrix. 1. Prepare paper chromatography-surface enhanced Raman scattering substrate based on aggregation promoter induced assembly technology; 2. Use paper chromatography-surface enhanced Raman scattering substrate to separate and enrich uric acid and creatinine in complex solutions at positions with ratio shift values of 0 and 0.64; 3. Use a portable Raman spectrometer to detect the surface enhanced Raman scattering signals of uric acid and creatinine on-site and quantitatively analyze them. The detection limits of uric acid and creatinine in urine of the present invention are as low as 10 ^‑5 M and 10 ^‑4 M, respectively, which meet the actual detection needs.

Description

A method for simultaneous quantitative detection of uric acid and creatinine concentrations in complex matrices based on paper chromatography and surface-enhanced Raman scattering

Technical Field

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

Background technique

Uric acid is the final product of purine metabolism in the body and is slightly soluble in water. Uric acid is usually produced by kidney metabolism and is almost entirely excreted from the body 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 disorders and kidney diseases can lead to abnormal uric acid metabolism. At present, uric acid determination is the best indicator for diagnosing hyperuricemia caused by abnormal purine metabolism. At the same time, uric acid can also be used for the diagnosis and monitoring of kidney disease. Therefore, it is of great significance to carry out uric acid testing.

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

At present, the main methods for detecting uric acid are phosphotungstic acid colorimetry and enzyme method, but the operation is cumbersome and the detection cost is high. The commonly used methods for urine creatinine are Jaffe reaction method and enzyme 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 and cannot be detected on site.

Surface enhanced Raman scattering (SERS) technology is a fast and highly sensitive detection technology that can provide molecular fingerprint information of the object being measured. Therefore, SERS technology has been widely studied in recent years. However, it is limited by the competitive adsorption effect. SERS technology is difficult to complete the detection task in complex matrices. The signal of the substance being measured with a small Raman cross section 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 at different positions on the filter paper to achieve separation. The relative shift value (Rf) is often used to represent the distribution of each substance after separation. Nanoparticles are assembled on the surface of chromatography paper to make PC-SERS substrates. The two technologies are combined to quickly achieve the simultaneous separation and detection of uric acid and creatinine in complex matrices. However, although the simple SERS detection has high sensitivity, it is difficult to detect target substances in complex media due to competitive adsorption.

Summary of the invention

The purpose of the present invention is to solve the problems that the existing creatinine and uric acid detection methods are susceptible to interference, require large instruments, are cumbersome to operate, are 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 complex matrices. A method for simultaneously quantitatively detecting uric acid and creatinine concentrations in complex matrices based on paper chromatography and surface enhanced Raman scattering technology is provided.

The present invention designs a PC-SERS substrate, which effectively overcomes the interference of competitive adsorption on SERS detection. In combination with a portable Raman spectrometer, a method is provided that does not require pretreatment, is low-cost, highly sensitive, and can be detected on-site for the simultaneous detection of uric acid and creatinine in complex matrices.

A method for simultaneously and quantitatively detecting the concentrations of uric acid and creatinine in a complex matrix based on paper chromatography and surface enhanced Raman scattering technology is specifically completed in the following steps:

1. Preparation of PC-SERS substrate:

①, immerse the base material in the aggregation promoter solution for a period of time, take it out and air-dry it to obtain the base material treated with the aggregation promoter;

② First, immerse the substrate material treated with the aggregation promoter into the silver nanoparticle concentrate 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 matrices:

A trace amount of the solution to be tested is spotted at a distance of 15 mm from the lower end of the PC-SERS substrate. After air drying, the lower end of the substrate is immersed in a developing agent in a sealed container and developed upward. When the development reaches the end point of the chromatography, the substrate is taken out and naturally air dried to obtain a PC-SERS substrate after paper chromatography separation.

3. SERS detection of uric acid and creatinine:

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

The present invention performs noise suppression and baseline subtraction on the spectrum based on wavelet threshold noise reduction, iterative penalty partial least squares method, multivariate scattering correction and other algorithms, and then substitutes the measured 1135cm ^-1 characteristic peak intensity of uric acid and 1426cm ^-1 characteristic peak intensity of creatinine into the concentration-characteristic peak intensity curve to obtain the concentrations of uric acid and creatinine in the measured solution.

Beneficial effects of the present invention:

1. The PC-SERS substrate prepared by the present invention reduces the detection cost while achieving high stability, high accuracy and high sensitivity. It can be used for large-scale screening of related diseases in medically backward areas with a portable Raman spectrometer, and provides a new solution for on-site rapid detection of uric acid and creatinine.

Second, the present invention is based on PC-SERS technology, which overcomes the problem of SERS technology being difficult to detect in complex matrices to a certain extent, and realizes the simultaneous detection of uric acid and creatinine. The sample does not need to be pre-treated, and the substance to be tested in the complex matrix can be directly detected;

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

Fourth, 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 easy portability. It can overcome the deficiency that SERS technology is difficult to complete detection in complex matrices, and provides a new solution for on-site rapid detection of creatinine and uric acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of a PC-SERS substrate preparation and on-site detection method for simultaneous quantitative detection of uric acid and creatinine in a complex matrix according to the present invention;

FIG2 is a scanning electron microscope image of the PC-SERS substrate prepared in Example 1;

FIG3 shows the detection effect of Rhodamine 6G using the PC-SERS substrate prepared in Example 1;

FIG4 is a normalized comparison diagram of the SERS signal of the direct detection of the mixed solution to be tested (including fetal bovine serum 1 mg/mL, glucose 4 mM, creatinine 0.5 mM, uric acid 0.5 mM) and the uric acid standard solution, and the SERS signal of the creatinine standard solution, in which creatinine is creatinine, mix is the mixed solution to be tested, and uric acid is uric acid;

FIG5 is a waterfall diagram of SERS signals and sampling positions plotted on a PC-SERS substrate with a sampling interval of 5 mm;

FIG6 is a B-spline fitting with the ratio shift as the horizontal axis and the characteristic peak intensity of 1135 cm ^-1 of uric acid and the characteristic peak intensity of 1426 cm ^-1 of creatinine as the vertical axis, to obtain the distribution diagram of the substance to be tested on the PC-SERS substrate;

FIG7 is a 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 1135 cm ^-1 characteristic peak intensity detected using the PC-SERS substrate, and R ² is 0.9945;

FIG8 is a curve of creatinine concentration-characteristic peak intensity obtained by using the B-spline fitting method to fit the creatinine concentration to the characteristic peak intensity of 1426 cm ^-1 of creatinine detected using the PC-SERS substrate, and R ² is 0.9940;

FIG9 is a diagram showing the quantitative analysis results of uric acid concentration in the test set samples;

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

Detailed ways

Specific implementation method 1: This implementation method is 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, which is specifically completed in the following steps:

1. Preparation of PC-SERS substrate:

①, immerse the base material in the aggregation promoter solution for a period of time, take it out and air-dry it to obtain the base material treated with the aggregation promoter;

② First, immerse the substrate material treated with the aggregation promoter into the silver nanoparticle concentrate 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 matrices:

A trace amount of the solution to be tested is spotted at a distance of 15 mm from the lower end of the PC-SERS substrate. After air drying, the lower end of the substrate is immersed in a developing agent in a sealed container and developed upward. When the development reaches the end point of the chromatography, the substrate is taken out and naturally air dried to obtain a PC-SERS substrate after paper chromatography separation.

3. SERS detection of uric acid and creatinine:

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

Specific embodiment 2: This embodiment differs from specific embodiment 1 in that: the base material in step 1① is cellulose chromatography paper; the aggregation promoter solution in step 1① is NaCl solution or KCl solution, with a concentration of 10mmol/L to 100mmol/L. The other steps are the same as those in specific embodiment 1.

Specific implementation method 3: This implementation method is different from specific implementation method 1 or 2 in that: in step 1①, the substrate material is immersed in the aggregation promoter solution for 10 min to 60 min. The other steps are the same as those of specific implementation method 1 or 2.

Specific embodiment 4: This embodiment differs from specific embodiments 1 to 3 in that the particle size of the silver nanoparticles in the silver nanoparticle concentrate described in step 1② is 30nm to 120nm. The other steps are the same as those of specific embodiments 1 to 3.

Specific embodiment 5: This embodiment differs from specific embodiments 1 to 4 in that: the preparation method of the silver nanoparticle concentrate described in step 1 ② is: dissolve 0.036g of silver nitrate in 200mL of deionized water, stir and heat until the solution boils, then add 2mL to 8mL of a 1% sodium citrate solution by mass, keep boiling for 0.5h to 1h, then stop heating, cool to room temperature, centrifuge 1 to 3 times, take the precipitate and redissolve it in deionized water, and make up to 1/10 to 1/4 of the volume before centrifugation. The other steps are the same as those of specific embodiments 1 to 4.

Specific embodiment 6: This embodiment differs from specific embodiments 1 to 5 in that: in step 1 ②, the substrate material treated with the aggregation promoter is immersed in the silver nanoparticle concentrate for 2 hours to 24 hours; in step 1 ②, deionized water is used for rinsing 2 to 5 times, and each rinsing time is 3 seconds to 6 seconds. The other steps are the same as those of specific embodiments 1 to 5.

Specific embodiment 7: This embodiment differs from specific embodiments 1 to 6 in that: the amount of the sample of the micro-amount of the solution to be tested in step 2 is 1 μL to 10 μL; the developing agent in step 2 is a mixture of water and isopropanol, wherein the volume ratio of water to isopropanol is 3:2. The other steps are the same as those in specific embodiments 1 to 6.

Specific embodiment 8: This embodiment differs from specific 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 spreading distance is 30 mm to 140 mm when the chromatography end point is reached. The other steps are the same as specific embodiments 1 to 7.

Specific embodiment 9: This embodiment differs from specific embodiments 1 to 8 in that the solution to be tested in step 2 is urine, serum or tears. The other steps are the same as those of specific embodiments 1 to 8.

Specific embodiment 10: This embodiment differs from specific embodiments 1 to 9 in that: the range of SERS detection described in step 3 is 500 cm ^-1 to 1800 cm ^-1 , and after collecting SERS signals, quantitative analysis is performed based on the characteristic peak intensity of 1135 cm ^-1 of uric acid and the characteristic peak intensity of 1426 cm ^-1 of creatinine; in step 3, laser line scanning is used to determine the distribution of uric acid and creatinine and the detection position based on the change in the characteristic peak intensity. The other steps are the same as specific embodiments 1 to 9.

The following examples are used to verify the beneficial effects of the present invention:

Example 1: A method for preparing a PC-SERS substrate is specifically completed by the following steps:

1. Preparation of PC-SERS substrate:

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

The base material described in step 1① is cellulose chromatography paper with a size of 1 cm×10 cm;

The aggregation promoter solution described in step 1① is a NaCl solution with a concentration of 20mmol/L;

② First, immerse the substrate material treated with the aggregation promoter into the silver nanoparticle concentrate for 4 hours, then take it out and rinse it with deionized water for 3 times, each rinsing time is 3 seconds, so as to completely remove the silver nanoparticles that are not tightly bound to the chromatography paper, and finally air-dry it to obtain the PC-SERS substrate;

The silver nanoparticles in the silver nanoparticle concentrate described in step 1② have a particle size of 30nm to 120nm and are prepared by sodium citrate reduction method. The specific preparation method is: dissolve 0.036g of silver nitrate in 200mL of deionized water, stir and heat the solution until it boils, then add 5mL of 1% sodium citrate solution by mass, keep boiling for 0.8h, then stop heating, cool to room temperature, centrifuge at 8000r/min for 10min, discard the supernatant, take the precipitate and redissolve it in deionized water, and make up to 1/8 of the volume before centrifugation.

FIG2 is a scanning electron microscope image of the PC-SERS substrate prepared in Example 1;

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

The PC-SERS substrate prepared in Example 1 was used to detect Rhodamine 6G solutions of different concentrations, and the detection effect diagram is shown in FIG3 ;

FIG3 shows the detection effect of Rhodamine 6G using the PC-SERS substrate prepared in Example 1;

As shown in FIG. 3 , the detection limit of the PC-SERS substrate prepared in Example 1 for Rhodamine 6G is 100 pM, which shows that the prepared substrate has excellent SERS performance.

Example 2: A method for simultaneously and quantitatively detecting the concentrations of uric acid and creatinine in a complex matrix based on paper chromatography and surface enhanced Raman scattering technology using the PC-SERS substrate prepared in Example 1 is specifically accomplished by the following steps:

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

3 μL of the mixed solution to be tested is spotted at a distance of 15 mm from the lower end of the PC-SERS substrate. After air drying, the lower end of the substrate is immersed in the developing agent in a sealed container and developed upward. When the development reaches the end point of the chromatography, it is taken out and naturally air-dried to obtain the PC-SERS substrate after paper chromatography separation.

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

The developing solvent described in step 1 is a mixture of water and isopropanol, wherein the volume ratio of water to isopropanol is 3:2;

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

2. SERS detection of uric acid and creatinine:

The PC-SERS substrate separated by paper chromatography was pasted on a glass slide for SERS detection. A 785nm laser was used as the excitation light source. The integration time was 15s, and the average was 2 times. The detection range was 500-1800cm ^-1 . The SERS signal was collected every 2.5mm. As shown in Figure 5, the waterfall diagram of the SERS signal and the sampling position drawn with a sampling interval of 5mm is shown; it can be seen from Figure 5 that the uric acid molecule has the strongest signal at the Rf value of 0 (0mm), which is due to the extremely low solubility of uric acid in the developing agent. The creatinine signal is the strongest at the Rf value of 0.64 (45mm), indicating that creatinine moves to this place with the developing agent. Since isopropanol is volatile, it will not interfere with the measurement. With the ratio shift value as the horizontal axis, the characteristic peak intensity of 1135cm ^-1 of uric acid and the characteristic peak intensity of 1426cm ^-1 of creatinine as the vertical axis, B-spline fitting is performed, and the material distribution on the PC-SERS substrate is obtained as shown in Figure 6. The above results prove that it is impossible to simultaneously detect uric acid and creatinine in a complex matrix based solely on SERS technology, but the PC-SERS substrate described in the present invention can realize the simultaneous detection of uric acid and creatinine in a complex matrix.

The SERS signals of the creatinine standard solution (creatinine 0.5 mM), the SERS signals of the uric acid standard solution (uric acid 0.5 mM), and the SERS signals of the mixed solution to be tested (the concentration of fetal bovine serum in the mixed solution to be tested is 1 mg/mL, the concentration of glucose is 4 mM, the concentration of creatinine is 0.5 mM, and the concentration of uric acid is 0.5 mM) are detected, and the obtained SERS signals are normalized and plotted in FIG4 ;

As can be seen from Figure 4, 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. This is 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 occupied by uric acid, and it is impossible to directly detect uric acid and creatinine simultaneously in a complex matrix based on the SERS technology.

Example 3: A method for simultaneously quantitatively detecting the concentrations of uric acid and creatinine in a complex matrix based on paper chromatography and surface enhanced Raman scattering technology using the PC-SERS substrate prepared in Example 1 is specifically accomplished by the following steps:

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

Accurately weigh pure powders of uric acid and creatinine, prepare a mother solution with artificial urine as a solvent, add 100 mM NaOH solution to the uric acid mother solution until the uric acid precipitate is completely dissolved, perform ultrasonic treatment for 5 minutes to make the solution evenly dispersed, use a pipette to extract an appropriate amount of the two mother solutions, mix them, and then dilute them 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);

Second, 3uL of artificial urine with different concentrations of uric acid and creatinine was spotted on the PC-SERS substrate 15mm from the bottom and allowed to air dry. A developing agent with a ratio of water to isopropanol = 3:2 was placed in a sealed container and shaken to speed up the speed at which the developing agent vapor reached saturation in the container.

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

Fourth, the separated and air-dried PC-SERS substrate was pasted on a glass slide for SERS detection. A 785 nm laser was used as the excitation light source, the integration time was 15 s, and the average was 2 times. The detection range was 500-1800 cm ^-1 . The SERS signals of uric acid and creatinine were detected at the positions of ratio shift values of 0 and 0.64, respectively. 20 spectra were collected for each gradient concentration and the average spectrum was used for subsequent curve fitting.

After noise suppression and baseline subtraction of the spectrum based on wavelet threshold denoising, iterative penalty partial least squares, multivariate scattering correction and other algorithms, the average of the characteristic peak intensity of uric acid at 1135 cm ^-1 and the characteristic peak intensity of creatinine at 1426 cm ^-1 was calculated, and the concentration-characteristic peak intensity curves of uric acid and creatinine were obtained using the B-spline fitting method as shown in Figures 7 and 8, respectively. The R ² of the fitting curves were 0.9945 and 0.9940, respectively.

The above method is used to detect artificial urine mixed with different concentrations of uric acid and creatinine. The characteristic peak intensities of uric acid and creatinine detected at Rf=0 and Rf=0.64 are substituted into the fitting curve to obtain the concentrations of uric acid and creatinine in the solution, thereby achieving simultaneous quantitative detection of uric acid and creatinine. The test sets of uric acid and creatinine each contain 30 spectra. Figures 9 and 10 show the test results of uric acid and creatinine in the test set samples, respectively. It can be seen that the predicted value and the true value show good consistency. The relative deviation between the vast majority of the predicted results and the true value is less than 10%. This specific embodiment shows that the present invention can achieve good detection effects on uric acid and creatinine in a complex matrix at the same time.

Claims (9)

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 aggregation-promoting agent treated substrate material;

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

The preparation method of the silver nanoparticle concentrated solution in the step one ② comprises the following steps: 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 0.5-1 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;

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 determination 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 immersing 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 (SERS) technology as defined in 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; in the first ② steps, deionized water is used for washing for 2 to 5 times, and the washing time is 3 to 6 seconds each time.

6. 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.

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 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.

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 (SERS) according to claim 1, wherein the solution to be detected in the second step is urine, serum or tear.

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 technology as defined in claim 1, wherein the SERS detection range in the step three is 500cm ^-1～1800cm^-1, and quantitative analysis is performed according to 1135cm ^-1 characteristic peak intensity of uric acid and 1426cm ^-1 characteristic peak intensity of creatinine after SERS signals are acquired; 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.