CN116500014B - 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
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 Download PDFInfo
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- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 title claims abstract description 105
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 title claims abstract description 91
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 title claims abstract description 91
- 229940116269 uric acid Drugs 0.000 title claims abstract description 91
- 229940109239 creatinine Drugs 0.000 title claims abstract description 86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
- G01N30/94—Development
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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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. and detecting surface enhanced Raman scattering signals of uric acid and creatinine on site by adopting a portable Raman spectrometer and quantitatively analyzing. The detection limits of uric acid and creatinine in urine are respectively as low as 10 ‑5 M and 10 ‑4 M, so that the actual detection requirements are met.
Description
Technical Field
The invention relates to the field of detection methods, in particular to a method for simultaneously and quantitatively detecting uric acid and creatinine concentration in a complex matrix based on paper chromatography and a surface enhanced Raman scattering technology.
Background
Uric acid is a final product of purine metabolism in vivo, is slightly soluble in water, is usually produced by kidney metabolism, is almost completely discharged outside a human body through urine, has a normal value of 0.15-0.4 mM of serum uric acid of healthy adult human, has a normal value of 1.1-4.4 mM in urine, causes abnormal uric acid metabolism due to purine metabolic disorder and kidney diseases, is an optimal index for diagnosing hyperuricemia caused by abnormal purine metabolism, and can be used for diagnosing and monitoring kidney diseases, so that uric acid detection has important significance.
Creatinine is a metabolic product of creatine in vivo, and is discharged from the body after being filtered by glomeruli, the normal value of urine creatinine of women is 7.9-14.1 mmol/24h, and the normal value of creatinine of men is 9.7-24.7 mmol/24h. Urinary creatinine and blood creatinine are important indexes for detecting kidney diseases and evaluating the development of the diseases, are closely related to the health condition of the kidney, and are often used as internal standard substances for correcting the content of other substances in urine, such as medicines and toxins, so that the development of a method for rapidly detecting creatinine has important significance.
At present, the method for detecting uric acid mainly comprises a phosphotungstic acid colorimetric method and an enzymatic method, but the operation is complex, and the detection cost is high. Common methods of urinary creatinine are the Jaffe reaction method and the enzyme method, but due to interference of other substances in body fluid, the detection specificity is low, and other detection methods such as HPLC and chromatography-mass spectrometry have extremely low detection limit, but equipment is expensive and cannot be detected on site.
The Surface Enhanced Raman Scattering (SERS) technology is a fast and high-sensitivity detection technology, and can provide molecular fingerprint information of a detected object, so that the SERS technology has been widely studied in recent years, but is limited by competitive adsorption effect, the SERS technology is difficult to complete detection tasks in a complex matrix, and a detected substance signal with a small raman cross section can be completely submerged and cannot be accurately detected. Paper Chromatography (PC) is an effective liquid-liquid partition chromatography, and as the partition coefficients of the constituent substances in the sample in the stationary phase and the mobile phase are different, the moving speeds of the constituent substances on the filter paper are different, and finally, the respective substances stay at different positions on the filter paper to realize separation. The specific shift value (Rf) is often used to represent the distribution of each substance after separation. And assembling nano particles on the surface of the chromatographic paper to prepare a PC-SERS substrate, and combining two technologies to quickly realize simultaneous separation and detection of uric acid and creatinine in a complex matrix. However, simple SERS detection has high sensitivity, but detection of a target substance in a complex medium is difficult to achieve due to competitive adsorption.
Disclosure of Invention
The invention aims to solve the problems that the existing creatinine and uric acid detection method is easy to be interfered, a large-scale instrument is needed, the operation is complicated, the field detection is difficult, the simple surface enhanced Raman scattering is limited by competitive adsorption, and the target substance is difficult to accurately detect in a complex matrix, and provides a method for simultaneously and quantitatively detecting the concentration of uric acid and creatinine in the complex matrix based on paper chromatography and the surface enhanced Raman scattering technology.
The invention designs a PC-SERS substrate, effectively overcomes interference caused by competitive adsorption to SERS detection, and provides a method which does not need pretreatment, has low cost and high sensitivity and can be used for detecting uric acid and creatinine in complex matrixes simultaneously by matching with a portable Raman spectrometer.
The method for simultaneously and 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 by 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;
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.
The invention is based on wavelet threshold noise reduction, iterative punishment partial least square method, multivariate scattering correction and other algorithms, and after noise suppression, baseline subtraction and other treatments are carried out on the spectrum, the measured 1135cm -1 characteristic peak intensity and the measured 1426cm -1 characteristic peak intensity of the uric acid are substituted into a concentration-characteristic peak intensity curve, so that the uric acid and creatinine concentration in the measured solution is obtained.
The invention has the beneficial effects that:
1. The PC-SERS substrate prepared by the invention reduces the detection cost while realizing high stability, high accuracy and high sensitivity, can be matched with a portable Raman spectrometer, can be used for large-scale screening of related diseases in a medical laggard area, and provides a new solution for on-site rapid detection of uric acid and creatinine;
2. the invention is based on PC-SERS technology, overcomes the problem of difficult detection of SERS technology in complex matrix to a certain extent, realizes simultaneous detection of uric acid and creatinine, and can directly detect detected substances in complex matrix without pretreatment of samples;
3. According to the PC-SERS substrate prepared by the method, the detection lower limit of creatinine in artificial urine can reach 10 -4 M, and the detection lower limit of uric acid can reach 10 -5 M;
4. The invention has the advantages of high detection speed, no pretreatment of samples, low cost, high sensitivity, simultaneous detection of multiple targets and convenient carrying, can overcome the defect that the SERS technology is difficult to complete detection in complex matrixes, and provides a new solution for on-site rapid detection of creatinine and uric acid.
Drawings
FIG. 1 is a schematic diagram of a PC-SERS substrate preparation and in-situ detection method for simultaneous quantitative detection of uric acid and creatinine in a complex matrix according to the present invention;
FIG. 2 is a scanning electron microscope image of a PC-SERS substrate prepared in example 1;
FIG. 3 shows the detection effect of rhodamine 6G on a PC-SERS substrate prepared by using example 1;
FIG. 4 is a diagram showing normalized comparison of SERS signals of a mixed solution to be tested (including 1mg/mL of fetal bovine serum, 4mM of glucose, 0.5mM of creatinine and 0.5mM of uric acid) and a standard solution of uric acid, wherein creatinine is creatinine, mix is the mixed solution to be tested, and uric acid is uric acid;
FIG. 5 is a waterfall plot of SERS signal plotted at 5mm sampling intervals versus sampling position on a PC-SERS substrate;
FIG. 6 is a graph of distribution of a substance to be tested on a PC-SERS substrate, wherein the graph is obtained by performing B-spline fitting with the specific shift value as the horizontal axis and the 1135cm -1 characteristic peak intensity of uric acid and the 1426cm -1 characteristic peak intensity of creatinine as the vertical axis respectively;
FIG. 7 is a graph of uric acid concentration versus characteristic peak intensity obtained by B-spline fitting of uric acid concentration to 1135cm -1 characteristic peak intensity of uric acid detected using a PC-SERS substrate, R 2 being 0.9945;
FIG. 8 is a graph of creatinine concentration versus characteristic peak intensity obtained by B-spline fitting of creatinine concentration to the characteristic peak intensity of creatinine 1426cm -1 detected using a PC-SERS substrate, R 2 being 0.9940;
FIG. 9 is a graph showing the results of quantitative analysis of uric acid concentration in a sample of a test set;
FIG. 10 is a graph showing the results of the quantitative analysis of creatinine concentration in test set samples.
Detailed Description
The first embodiment is as follows: the 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 in the embodiment is specifically completed by 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;
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.
The second embodiment is as follows: the present embodiment differs from the specific embodiment in that: the substrate material in the first ① is cellulose chromatographic 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. The other steps are the same as in the first embodiment.
And a third specific embodiment: this embodiment differs from the first or second embodiment in that: in the first ① step, the base material is immersed in the aggregation-promoting agent solution for 10min to 60min. The other steps are the same as those of the first or second embodiment.
The specific embodiment IV is as follows: one difference between this embodiment and the first to third embodiments is that: the particle size of the silver nano particles in the silver nano particle concentrated solution in the step one ② is 30 nm-120 nm. The other steps are the same as those of the first to third embodiments.
Fifth embodiment: one to four differences between the present embodiment and the specific embodiment are: 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 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. Other steps are the same as those of the first to fourth embodiments.
Specific embodiment six: the present embodiment differs from the first to fifth embodiments in that: in the first ② step, immersing the substrate material treated by the aggregation-promoting agent into the silver nanoparticle concentrated solution for 2-24 hours; 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. Other steps are the same as those of the first to fifth embodiments.
Seventh embodiment: one difference between the present embodiment and the first to sixth embodiments is that: the sample application amount of the trace solution to be measured in the second step is 1 mu L-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. Other steps are the same as those of embodiments one to six.
Eighth embodiment: one difference between the present embodiment and the first to seventh embodiments is that: in the second step, the lower end of the PC-SERS substrate is immersed into the developing agent for 3 mm-7 mm, and the developing distance is 30 mm-140 mm when reaching the chromatography end point. The other steps are the same as those of embodiments one to seven.
Detailed description nine: one of the differences between this embodiment and the first to eighth embodiments is: the solution to be detected in the second step is urine, serum or tears. Other steps are the same as those of embodiments one to eight.
Detailed description ten: the present embodiment differs from the first to ninth embodiments in that: the SERS detection range in the third step is 500cm -1～1800cm-1, and quantitative analysis is carried out 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. The other steps are the same as those of embodiments one to nine.
The following examples are used to verify the benefits of the present invention:
Example 1: the preparation method of the PC-SERS substrate comprises the following steps:
1. Preparing a PC-SERS substrate:
① . Immersing the substrate material into the aggregation-promoting agent solution for 20min, taking out and air-drying to obtain the aggregation-promoting agent treated substrate material;
The substrate material in the first ① is cellulose chromatographic paper, and the size is 1cm multiplied by 10cm;
The aggregation-promoting agent solution in the step one ① is NaCl solution with the concentration of 20mmol/L;
② . Immersing a base material treated by an aggregation-promoting agent into silver nanoparticle concentrated solution for 4 hours, taking out, washing 3 times by using deionized water for 3s each time to thoroughly remove silver nanoparticles which are not tightly combined with chromatographic paper, and finally air-drying to obtain a PC-SERS substrate;
the particle diameter of silver nano particles in the silver nano particle concentrated solution in the step one ② is 30 nm-120 nm, and the silver nano particles are prepared by adopting a sodium citrate reduction method, and the specific preparation method comprises the following steps: 0.036g of silver nitrate is dissolved in 200mL of deionized water, stirred and heated until the solution boils, then 5mL of sodium citrate solution with the mass fraction of 1% is added, heating is stopped after the solution is kept boiling for 0.8h, cooling is carried out to room temperature, centrifugation is carried out for 10min at 8000r/min, supernatant is removed, precipitate is taken and redissolved in deionized water, and the volume is fixed to 1/8 of the volume before centrifugation.
FIG. 2 is a scanning electron microscope image of a PC-SERS substrate prepared in example 1;
As can be seen from fig. 2, silver nanoparticles were successfully assembled on the substrate surface, and the particle size and density were relatively uniform.
The PC-SERS substrate prepared in the embodiment 1 is used for detecting rhodamine 6G solutions with different concentrations, and the detection effect is shown in the figure 3;
FIG. 3 shows the detection effect of rhodamine 6G on a PC-SERS substrate prepared by using example 1;
As can be seen from fig. 3: the PC-SERS substrate prepared in example 1 has a detection limit of 100pM to rhodamine 6G, and the prepared substrate has excellent SERS performance.
Example 2: the method for simultaneously and quantitatively detecting uric acid and creatinine concentrations in a complex matrix by using the PC-SERS substrate prepared in the embodiment 1 based on paper chromatography and surface-enhanced Raman scattering technology is specifically completed by the following steps:
1. separation of uric acid and creatinine in complex matrices:
Spotting 3 mu L of a trace mixed solution to be tested at a position 15mm away from the lower end of the PC-SERS substrate, airing, immersing the lower end of the substrate into a developing agent in a closed container for developing upwards, taking out the substrate after developing to a chromatography end point, and naturally airing to obtain the PC-SERS substrate after paper chromatography separation;
the concentration of fetal bovine serum in the trace mixed solution to be detected in the first step 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 in the first step is a mixed solution of water and isopropanol, wherein the volume ratio of the water to the isopropanol is 3:2;
step one, immersing the lower end of the PC-SERS substrate into a developing agent for 5mm, wherein the developing distance is 70mm when the lower end reaches a chromatography end point;
2. SERS detects uric acid and creatinine:
Sticking the PC-SERS substrate subjected to paper chromatographic separation on a glass slide for SERS detection, using 785nm laser as an excitation light source, integrating for 15s, averaging 2 times, acquiring SERS signals every 2.5mm, wherein the detection range is 500-1800 cm -1, and a waterfall diagram of the SERS signals and sampling positions, which is drawn at sampling intervals of 5mm, is shown in FIG. 5; from FIG. 5, it can be seen that uric acid molecules have the strongest signal at Rf value of 0 (0 mm) due to extremely low solubility of uric acid in the developing agent. The strongest creatinine signal was found at Rf value 0.64 (45 mm), indicating that creatinine moved there with the developer. Since isopropanol is volatile, it does not interfere with the measurement. B-spline fitting was performed with the specific shift value as the horizontal axis and the 1135cm -1 characteristic peak intensity of uric acid and the 1426cm -1 characteristic peak intensity of creatinine as the vertical axis, respectively, to obtain the material distribution on the PC-SERS substrate as shown in FIG. 6. The above results demonstrate that uric acid and creatinine cannot be detected simultaneously in a complex matrix based solely on SERS technology, but the PC-SERS substrate described in the present invention enables simultaneous detection of uric acid and creatinine in a complex matrix.
Detecting SERS signals of creatinine standard solution (creatinine 0.5 mM) and uric acid standard solution (uric acid 0.5 mM), detecting SERS signals of mixed solution to be detected (concentration of fetal calf serum in the mixed solution to be detected is 1mg/mL, concentration of glucose is 4mM, concentration of creatinine is 0.5mM, concentration of uric acid is 0.5mM; the obtained SERS signals are normalized and then are plotted in FIG. 4;
As can be seen from fig. 4: the signal of the mixed solution to be detected is almost identical with that of the uric acid standard solution, and several main characteristic peaks of creatinine are almost invisible, because the Raman section of uric acid is far higher than that of creatinine and other interfering substances, signal enhancement hot spots can be preempted by uric acid, and uric acid and creatinine can not be detected in a complex matrix at the same time directly based on SERS technology.
Example 3: the method for simultaneously and quantitatively detecting uric acid and creatinine concentrations in a complex matrix by using the PC-SERS substrate prepared in the embodiment 1 based on paper chromatography and surface-enhanced Raman scattering technology is specifically completed by the following steps:
1. Preparing artificial urine with different concentrations of creatinine and uric acid:
Accurately weighing pure substance powder of uric acid and creatinine, preparing mother liquor by taking artificial urine as a solvent, dripping 100mM NaOH solution into the uric acid mother liquor until the uric acid precipitate is completely dissolved, carrying out ultrasonic treatment for 5min to uniformly disperse the solution, extracting a proper amount of the two mother liquors by using a pipette gun, mixing, and diluting the mixed mother liquor by the artificial urine to obtain the artificial urine with different concentrations of creatinine and uric acid (the concentration of the uric acid in the artificial urine is 1 mu M-4 mM, and the concentration of the creatinine is 1 mu M-30 mM);
2. 3uL of artificial urine with uric acid and creatinine with different concentrations is spotted on a PC-SERS substrate 15mm away from the bottom to be air-dried, a closed container is filled with a developing agent prepared by water and isopropanol=3:2, and the container is shaken to accelerate the speed of the developing agent vapor reaching a saturated state in the container;
3. suspending the PC-SERS substrate in a container, immersing the bottom of the substrate in a developing agent for 5mm, developing upwards, taking out when the developing distance is 70mm, and airing in a fume hood;
4. And sticking the separated and air-dried PC-SERS substrate on a glass slide for SERS detection, using 785nm laser as an excitation light source, integrating for 15s, averaging 2 times, detecting SERS signals of uric acid and creatinine at positions with specific shift values of 0 and 0.64 respectively, and acquiring 20 spectrums by each gradient concentration to obtain average spectrums for subsequent curve fitting.
Based on wavelet threshold noise reduction, iterative punishment partial least square method, multi-element scattering correction and other algorithms, noise suppression, baseline subtraction and other treatments are carried out on the spectrum, the measured 1135cm -1 characteristic peak intensity and the measured 1426cm -1 characteristic peak intensity of the uric acid are averaged, the concentration-characteristic peak intensity curves of the uric acid and the creatinine are obtained by using a B-spline fitting method, the curves are respectively shown in fig. 7 and fig. 8, and R 2 of the fitting curves are respectively 0.9945 and 0.9940.
The method is used for detecting artificial urine mixed with uric acid and creatinine with different concentrations, uric acid detected at the positions of Rf=0 and Rf=0.64 is substituted into a fitting curve to obtain the concentration of uric acid and creatinine in the solution, so that simultaneous quantitative detection of uric acid and creatinine is realized, and a test set of uric acid and creatinine respectively comprises 30 spectra. Fig. 9 and fig. 10 show the test results of uric acid and creatinine in the test set sample, respectively, and it can be seen that the predicted value and the actual value show good consistency, and the relative deviation between most of the predicted values and the actual value is less than 10%, which illustrates that the present embodiment can obtain good detection effects on uric acid and creatinine in complex substrates.
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.
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