CN102288592B - Method for quantitative detection of uric acid based on surface enhanced Raman spectroscopy (SERS) technology - Google Patents
Method for quantitative detection of uric acid based on surface enhanced Raman spectroscopy (SERS) technology Download PDFInfo
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- CN102288592B CN102288592B CN2011101291640A CN201110129164A CN102288592B CN 102288592 B CN102288592 B CN 102288592B CN 2011101291640 A CN2011101291640 A CN 2011101291640A CN 201110129164 A CN201110129164 A CN 201110129164A CN 102288592 B CN102288592 B CN 102288592B
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Abstract
The invention relates to a method for the quantitative detection of uric acid based on surface enhanced Raman spectroscopy (SERS). The method comprises the following steps of: adding NaOH and hydroxylamine hydrochloride into an AgNO3 solution to obtain silver colloid; centrifuging the silver colloid to obtain high-concentration silver colloid; mixing uric acid solutions of different concentrations with the high-concentration silver colloid and adding a K2SO4 solution to perform a Raman spectroscopy test to obtain a uric acid SERS spectrum; testing the high-concentration silver colloid to obtain a silver colloid background SERS spectrogram; performing intensity normalization by using a silver colloid background Raman signal as internal standard, establishing a relative intensity-concentration standard working curve diagram of uric acid SERS spectral line; and deducing uric acid concentration by comparing the SERS spectrogram of the uric acid with unknown concentration with the relative intensity-concentration standard working curve diagram of uric acid SERS spectrum. The method provided by the invention can be used for solving the problems that low-concentration uric acid solution has weak Raman signal and can be interfered with other impurities easily, obtaining high-quality uric acid SERS spectrum and realizing the quantitative detection of uric acid by means of the SERS spectrum.
Description
Technical field
The present invention relates to the quantitative detecting method of uric acid, specifically be meant the method for utilizing SERS (being called for short SERS) detection by quantitative uric acid concentration, belong to medicine, drugs detection technique field.
Background technology
SERS is that people such as Fleischmann carried out electrochemistry experiment in 1974, finds first when research is adsorbed on the pyridine Raman scattering of molecule experiment on the coarse silver electrode surface.Surface enhanced Raman scattering effect is meant that the more conventional raman spectral signal of its Raman scattering signal obtains the phenomenon of huge enhancing when target molecule is adsorbed in some coarse noble metal good conductor surfaces or the colloidal sol.SERS (SERS) but technology because its many advantages such as resolution characteristic with high detection sensitivity quench fluorescence, non-destructive finger-print type are widely used in interface orientation and configuration, conformational analysis, trace and the trace detection of study of surfaces, biomacromolecule, even can carry out Single Molecule Detection.Uric acid is the final product of human body exogenous (diet) and endogenous purine metabolism.When uric acid in the blood surpasses 7mg/dl, uric acid just begin with crystal form separate out and in some tissues deposition can cause kidney stone and gout.When blood uric acid can be diagnosed as hyperuricemia during more than or equal to 7mg/dl.Correlative study shows had the disease incidence of substantial connection obviously in rising trend with hyperuricemia in the last few years, and age of onset more and more receives people's concern in advance.Therefore, seek a kind of harmless, highly sensitive detection method uric acid in the human body is monitored, for vast scientist, clinical medicine worker and extensive patients, have important application.In patent of the present invention, we have announced a kind of novelty, the method for SERS technology detection by quantitative uric acid easily.
Utilize SERS technology detection by quantitative uric acid, do not see relevant report both at home and abroad as yet, this field still is blank at present.
Summary of the invention
The purpose of this invention is to provide a kind of is the method that strengthens the SERS detection by quantitative uric acid of substrate with the Nano silver grain.At first set up the SERS intensity-concentration standard contrast working curve diagram of known uric acid concentration; With the dielectric sample that contains uric acid to be detected; Detect with SERS, obtain containing the sample SERS spectrogram to be checked of uric acid, this spectrogram and standard working curve figure are contrasted; Can directly calculate the concentration of uric acid from the spectral line relative intensity; Thereby realize the detection by quantitative of uric acid, and detection limit reaches 1mg/L, for the uric acid detection by quantitative provide a kind of directly, new method fast and accurately.
For realizing that the technical scheme that the object of the invention adopts is:
1, at the bottom of the SERS active group-preparation of elargol
Join 6 * 10 to the NaOH solution of an amount of 0.1M/L
-2In the oxammonium hydrochloride solution of M/L, add potpourri to 1.11 * 10 fast then
-3The AgNO of M/L
3In the solution, stir fast and obtained uniform newborn grey fulmargin in 10 minutes.
An amount of fulmargin is placed in the centrifuge tube with 10000r/min centrifugal 10 minutes, and it is subsequent use to take off floor height concentration elargol.
In the above-mentioned steps, NaOH: oxammonium hydrochloride: AgNO
3Volume ratio be 1:1-1.3:18-25.
2, set up uric acid SERS optic spectrum line relative intensity-concentration standard contrast working curve diagram
Accurately take by weighing a certain amount of uric acid, with the Na of 11.6mmol/L
2CO
3Solution is 70
0In the C water-bath dissolving and use the ultrapure water constant volume, again with this solution dilution, being mixed with concentration respectively is the uric acid standard solution sample of 5～6 gradient concentrations.The setting of gradient concentration can be explained the increase along with uric acid concentration, and its SERS spectral signal strengthens gradually.In 5～6 gradient concentrations, its graded value is between 20mg/L～140mg/L.
The uric acid standard solution of 5～6 gradient concentrations is evenly mixed the back dropping on highly purified aluminium flake with the high concentration silver colloid of isopyknic step (1) preparation respectively, and drip 10 μ L K above that respectively
2SO
4Solution leaves standstill after 1 minute and carries out the SERS spectrum test.
Get the high concentration silver colloid of step (1) preparation and carry out the Raman spectrum test separately, obtain elargol background Raman spectrum spectrogram.
With elargol background Raman spectrum 240cm
-1The spectrum peak at place carries out spectral strength normalization as interior mark to detecting the uric acid standard solution SERS spectrum that obtains variable concentrations, utilizes uric acid SERS spectrum at 636cm
-1The SERS of wave number place spectrum peak relative intensity and uric acid concentration are linear, set up uric acid SERS optic spectrum line relative intensity-concentration standard working curve diagram.
3, infer unknown uric acid concentration
The uric acid solution of unknown concentration is evenly mixed with the high concentration silver colloid of step (1) preparation and adds an amount of K
2SO
4Solution leaves standstill the uric acid solution S ERS spectrum test that carries out unknown concentration after 1 minute, and the uric acid SERS spectrum that detects the unknown concentration that obtains is carried out spectral strength normalization handle.
Obtain detection and contrast, can directly infer the concentration of uric acid from the spectral line relative intensity through unknown concentration uric acid solution S ERS spectrogram and uric acid SERS spectrum relative intensity-concentration standard working curve that intensity normalization is handled.
Advantage of the present invention is: the time weak point that needs measurement and analyze, and amount of samples is few, and can realize the detection by quantitative of uric acid directly, quickly and accurately, and detectability reaches 1mg/L.
Description of drawings
Fig. 1 is the technical scheme block scheme that the present invention adopts.
Fig. 2 is uric acid solution SERS spectral signal under different pH values of the same concentrations that records of the embodiment of the invention 1.
Fig. 3 is that the embodiment of the invention 1 records the SERS spectrum of (A) uric acid and elargol mixed solution and (B) uric acid and elargol mixed solution and add K
2SO
4SERS spectrum behind the solution.
Fig. 4 is the embodiment of the invention 1 uric acid, elargol, uric acid and the elargol mixed solution that record and uric acid, elargol, K
2SO
4The absorption spectrum of three's mixed solution.
Fig. 5 is the SERS figure of 5 gradient concentration uric acid recording of the embodiment of the invention 1.
Fig. 6 is that the uric acid SERS spectrum that calculates of the embodiment of the invention 1 is at 636cm
-1Spectrum peak, wave number place relative intensity is with the standard working curve of change in concentration.
Among Fig. 2, the pH value of spectral line A, B, C correspondence is respectively 5.8,7.5,10.7.
Among Fig. 4, A is the absorption spectrum curve of uric acid solution; B is the absorption spectrum curve of fulmargin; C is the absorption spectra of elargol and uric acid solution mixed solution, and D is for to add K in the mixed system of elargol and uric acid solution
2SO
4Absorption spectra behind the solution, visible by figure, add K
2SO
4New broad absorption band appears in mixed system behind the solution at the 840nm place.
Among Fig. 5, the uric acid concentration of A, B, C, D, E uric acid SERS spectral line correspondence is respectively 20mg/L, 50mg/L, 80mg/L, 110mg/L, 140mg/L, from figure, can significantly observe uric acid SERS spectral intensity and do linear change with concentration.
Embodiment
Below with reference to embodiment and accompanying drawing the present invention is further specified.
1, at the bottom of the SERS active group-and the preparation of elargol: is total amount that the NaOH solution (0.1M/L) of 4.5mL joins 4.5mL oxammonium hydrochloride (6 * 10
-2M/L) in, add potpourri to 81mL AgNO fast then
3(1.11 * 10
-3M/L) in, vibration is to obtaining uniform newborn grey fulmargin always.
2, with the centrifugal elargol of 10000r/min 10 minutes, it was subsequent use to take off floor height concentration elargol.
3, accurately take by weighing the 0.0250g uric acid, dissolving is transferred in the 250mL volumetric flask, and constant volume is made into the storing solution of 100mg/L.Pipette the 80.00mL storing solution respectively in the 100mL volumetric flask, add hydrochloric acid solution and regulate pH and be respectively 5.8,7.5,10.7, constant volume is made into the uric acid solution of 80mg/L.The solution that curve A, B, C are followed successively by same concentrations among Fig. 2 is respectively at pH=5.8,7.5,10.7 SERS spectrogram.Can find out that therefrom its SERS spectral signal is stronger in alkaline environment.This also is that the pH value of uric acid standard solution among the present invention is set in the important evidence about 10.
4, the preparation of the accurate solution of alkaline urine acidity scale: accurately take by weighing the 0.020g uric acid, use the Na of concentration as 11.6mmol/L
2CO
3Solution is 70
0Dissolving and constant volume dilute this solution in the 100mL volumetric flask successively in the C water-bath, and compound concentration is the uric acid standard solution sample of 5 variable concentrations of 20mg/L, 50mg/L, 80mg/L, 110mg/L, 140mg/L.
5, uric acid standard solution and the isopyknic high concentration silver colloid with the variable concentrations of 20 μ l steps 4 preparations is to mix on 99.9% aluminium flake in purity, and at each part uric acid and the further interpolation of elargol mixed solution 10 μ l K
2SO
4Solution also mixes with the micropipette rifle, leaves standstill and carries out the SERS spectrum test in 1 minute.
6, in uric acid and elargol mixed solution, further add 10 μ l K
2SO
4Solution can significantly strengthen uric acid SERS signal.As shown in Figure 3, spectral line A is dissolved in Na
2CO
3Concentration is that the uric acid standard solution of 20mg/L mixes and add 10 μ l K with elargol in the solution
2SO
4Uric acid SERS spectral signal behind the solution; Spectral line B is dissolved in Na
2CO
3Concentration is the direct mixed uric acid SERS spectral signal of 20mg/L uric acid standard solution and elargol in the solution.Contrast A, two spectral lines of B, we are very easy to find and add K
2SO
4Solution can measure very desirable uric acid SERS spectral signal, and can observe obviously that mixed solution turns black rapidly, clustering phenomena.
7, detect dropping K
2SO
4The absorption spectrum of uric acid and elargol mixed solution before and after the solution.As shown in Figure 4, uric acid solution and elargol mixed solution drip K
2SO
4Can see in the mixed system significantly blackout, clustering phenomena behind the solution, wide new resonance absorbing peak appears in mixed system about long wave direction 840 nm.Explanation is at K
2SO
4Under the solution effects, the strong interaction takes place in uric acid molecule and Nano silver grain, makes the SERS signal of uric acid obtain further to strengthen.
8, among Fig. 5,5 gradient concentration uric acid standard solution that A, B, C, D, E are respectively preparation in the step 4 mix the back and drip 10 μ l K with elargol
2SO
4Solution leaves standstill the SERS spectrogram after 1 minute.Can find out that by figure along with the increase of uric acid concentration, its SERS spectral signal strengthens gradually.
9, get high concentration silver colloid and carry out the Raman spectrum test separately, obtain elargol background Raman spectrum.
10, with elargol background Raman spectrum 240cm
-1Spectral strength utilizes the origin data processing software to carry out spectral strength normalization processing to detecting the uric acid SERS spectrum that obtains variable concentrations, with uric acid 636cm as interior mark
-1The SERS that the wave number place is the strongest composes the peak relative intensity as the Y axle, and uric acid concentration is the standard working curve figure of uric acid solution S ERS spectrum spectrum peak relative intensity with change in concentration as the X axle, and is as shown in Figure 6.Each concentration is surveyed five times, averages the drawing standard working curve.Regression equation is y=0.0094x+0.1328, coefficient R
2=0.9892.
11, obtain detection and contrast with uric acid SERS spectrum relative intensity-concentration standard working curve through the unknown concentration uric acid solution S ERS spectrogram that intensity normalization is handled; Can infer directly that from the spectral line relative intensity concentration of uric acid to be measured is 65mg/L; Thereby realize the detection by quantitative of uric acid, detect and be limited to 1mg/L.
Embodiment 2
1, at the bottom of the SERS active group-and the preparation of elargol: is total amount that the NaOH solution (0.1M/L) of 4.5mL joins 5.8mL oxammonium hydrochloride (6 * 10
-2M/L) in, add potpourri to 100mL AgNO fast then
3(1.11 * 10
-3M/L) in, vibration is to obtaining uniform newborn grey fulmargin always.
2, with the centrifugal elargol of 10000r/min 10 minutes, it was subsequent use to take off floor height concentration elargol.
3, the preparation of alkaline urine acid solution: accurately take by weighing the 0.02g uric acid, use the Na of concentration as 11.6mmol/L
2CO
3Solution is 70
0Dissolving and constant volume dilute this solution in the 100mL volumetric flask successively in the C water-bath, and compound concentration is the uric acid standard solution sample of 5 different gradient concentrations of 20mg/L, 40mg/L, 70mg/L, 100mg/L, 140mg/L.
4, uric acid standard solution and the isopyknic high concentration silver colloid with the variable concentrations of 20 μ L steps 3 preparations is to mix on 99.9% aluminium flake in purity, and in each part uric acid and elargol mixed solution further interpolation 10 μ lK
2SO
4Solution also mixes with the micropipette rifle, leaves standstill and carries out the SERS spectrum test in 1 minute.Along with the increase of uric acid concentration, its SERS spectral signal strengthens gradually.
5, get high concentration silver colloid and carry out the Raman spectrum test separately, obtain elargol background Raman spectrum.
6, with elargol background Raman spectrum 240cm
-1Spectral strength is as interior mark, utilizes data processing software such as origin to carry out spectral strength normalization and handle detecting the uric acid SERS spectrum that obtains variable concentrations, with uric acid 636cm
-1The SERS that the wave number place is the strongest composes the peak relative intensity as the Y axle, and uric acid concentration is the standard working curve figure of uric acid solution S ERS spectrum spectral strength with change in concentration as the X axle.Each concentration is surveyed five times, averages the drawing standard working curve.Regression equation is y=0.0092x+0.1325, coefficient R
2=0.9881.
7, obtain detection and contrast with uric acid SERS spectrum relative intensity-concentration standard working curve diagram through the unknown concentration uric acid solution S ERS spectrogram that intensity normalization is handled; Can infer directly that from the spectral line relative intensity concentration of uric acid to be measured is 63.5mg/L; Thereby realize the detection by quantitative of uric acid, detect and be limited to 1mg/L.
Claims (5)
1. uric acid quantitative detecting method based on SERS technology; At first set up the SERS intensity-concentration standard contrast working curve diagram of known uric acid concentration, the dielectric sample that contains uric acid with to be detected detects with SERS; Obtain containing the sample surfaces enhancing Raman spectrum spectrogram to be checked of uric acid; This spectrogram and standard control working curve diagram are contrasted, can directly calculate the concentration of uric acid, it is characterized in that from the spectral line relative intensity:
1) at the bottom of the SERS active group-preparation of elargol
Join NaOH solution in the oxammonium hydrochloride solution, mix, and add AgNO fast to
3In the solution, stir and got fulmargin in 10 minutes, centrifugal, it is subsequent use to take off floor height concentration elargol;
2) set up uric acid SERS spectral line relative intensity-concentration standard contrast working curve diagram
Take by weighing uric acid, with the Na of 11.6mmol/L
2CO
3Solution is 70
0Dissolve and constant volume in the C water-bath, again this solution dilution is mixed with the uric acid standard solution sample of 5-6 gradient concentration respectively;
The uric acid standard solution sample of 5-6 gradient concentration is evenly mixed the back dropping on aluminium flake with the high concentration silver colloid of isopyknic step (1) preparation respectively, and drip 10 respectively above that
K
2SO
4Solution carries out the SERS test after leaving standstill;
Get the high concentration silver colloid of step (1) preparation and carry out the Raman spectrum test separately, obtain elargol background Raman signal;
As interior mark uric acid standard solution SERS is carried out intensity normalization with elargol background Raman signal, set up uric acid SERS spectral line relative intensity-concentration standard contrast working curve diagram;
3) infer unknown uric acid concentration
The uric acid solution of unknown concentration is evenly mixed with the high concentration silver colloid of step 1) preparation and adds K
2SO
4Solution carries out SERS test after leaving standstill, and carry out spectral strength normalization handle the uric acid SERS spectrogram of unknown concentration;
The uric acid SERS spectrogram of unknown concentration and uric acid SERS spectral line relative intensity-concentration standard working curve diagram contrast, directly infer the concentration of uric acid from the spectral line relative intensity.
2. a kind of uric acid quantitative detecting method according to claim 1 based on SERS technology, it is characterized in that the SERS active group at the bottom of-when elargol prepared, described NaOH solution concentration was 0.1M/L; Described oxammonium hydrochloride solution concentration is 6 * 10
-2M/L; Described AgNO
3Solution concentration is 1.11 * 10
-3M/L.
3. a kind of uric acid quantitative detecting method according to claim 1 based on SERS technology, it is characterized in that the SERS active group at the bottom of-when elargol prepares, described NaOH solution: oxammonium hydrochloride solution: AgNO
3The volume ratio of solution is 1:1～1.3:18～25.
4. a kind of uric acid quantitative detecting method based on SERS technology according to claim 1, the change in concentration value of uric acid standard solution sample that it is characterized in that described 5～6 gradient concentrations is between 10mg/L～140mg/L.
5. a kind of uric acid quantitative detecting method according to claim 1 based on the SERS technology; It is characterized in that describedly when setting up uric acid SERS spectral line relative intensity-concentration standard contrast working curve diagram, is elargol background Raman spectrum 240cm as interior target spectrum peak
-1The spectrum peak.
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| CN102590177A (en) * | 2012-03-19 | 2012-07-18 | 中国政法大学 | Method for detecting methamphetamine through surface enhanced Raman spectroscopy |
| CN103357885B (en) * | 2012-04-01 | 2016-06-22 | 深圳市宇驰检测技术有限公司 | A kind of Raman strengthens preparation method and the application thereof of elargol |
| CN105181670B (en) * | 2015-08-26 | 2017-10-10 | 福建师范大学 | One kind utilizes cell secretory protein SERS spectra detection drug cytotoxicity method |
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| CN109668870B (en) * | 2019-01-24 | 2020-07-17 | 清华大学 | Method for detecting concentration of copper ions in solution based on surface enhanced Raman spectroscopy |
| CN110082336A (en) * | 2019-04-25 | 2019-08-02 | 宁波大学 | A method of total purine content in aquatic products is quickly detected based on diazo-reaction and SERS |
| CN113466210B (en) * | 2021-07-29 | 2024-04-02 | 浙江澍源智能技术有限公司 | Apparatus and method for improving water signal intensity in raman spectroscopy |
| CN116183576B (en) * | 2022-12-09 | 2024-01-26 | 中国矿业大学 | Method for measuring content of free silicon dioxide in coal dust based on internal standard method |
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