CN110082336A - A method of total purine content in aquatic products is quickly detected based on diazo-reaction and SERS - Google Patents
A method of total purine content in aquatic products is quickly detected based on diazo-reaction and SERS Download PDFInfo
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- 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
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- 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
Abstract
The invention discloses a kind of method for quickly detecting total purine content in aquatic products based on diazo-reaction and SERS, feature is the following steps are included: perchloric acid solution is added in (1) in aquatic products sample, and heats hydrolysis under alkaline condition and extract purine；(2) NaNO is added in analyte sample fluid2And HCl, heating occur hydrolysis and generate phenol；Then xanthine oxidase is added and generates final product uric acid；(3) Au@AgNPs is synthesized；(4) by the sample to be tested containing uric acid and step Au@AgNPs solution by volume 1:1 ratio mix, survey its SERS signal intensity, according to the quantitative relationship between uric acid Raman peak intensity and uric acid concentration, calculate the concentration for obtaining uric acid in sample to be tested, according to diazo-reaction formula, total purine content is calculated in aquatic products, and advantage is fast detection speed, high sensitivity and high specificity.
Description
Technical field
The present invention relates to a kind of detection methods of purine content total in aquatic products, anti-based on diazotising more particularly, to one kind
The method that total purine content in aquatic products should be quickly detected with SERS.
Background technique
Purine is a kind of natural materials, is present in all cells and almost all of food of human body.Marine product is because of it
Flavor is delicious, protein and unsaturated fatty acid content are high and eaten extensively.However, it is considered as a kind of high purine food
Product, safety receive significant attention.People only obtain about 20% purine from diet, but these purine are seldom by human body
It uses, is almost converted to uric acid.The final product of purine metabolism is uric acid, takes in the food of high purine for a long time, in addition some
Inducement leads to the raising of uric acid level, so as to cause gout and hyperuricemia.Therefore measurement marine product in purine it is total
Content is of great significance.
Total purine content generally uses high performance liquid chromatography in measurement food at present, and it is fast first to measure adenine, bird respectively
Summation is calculated after purine, hypoxanthine and xanthine content, it is not every that the Detection wavelength of selection, which is the compromise wavelength of this 4 kinds of purine,
The most suitable Detection wavelength of a purine, therefore, HPLC method can not accurately reflect the content of total purine；In addition purine in the sample can
It degrades, therefore the sample pre-treatments of HPLC method, the true purine content of sample is had a significant impact.Due to surface enhanced
Raman spectrum (Surface-enhanced Raman spectroscopy, SERS) has very high sensitivity and quickly detection
The advantages of, people are more and more interested in the application of food security aspect in it.SERS technology is by using specially treated
Rough surface metal (such as gold, silver) is used as active substrate, and tested molecule or functional group are adsorbed onto metal surface, so that tested
Molecule or the Raman scattering signal of its functional group are increased to 105~106Times.Currently, open about being based on not yet both at home and abroad
Diazo-reaction and SERS quickly detect the correlative study report of the method for total purine content in aquatic products.
Summary of the invention
Technical problem to be solved by the invention is to provide one kind to have fast detection speed, high sensitivity and high specificity
The method that total purine content in aquatic products is quickly detected based on diazo-reaction and SERS.
The technical scheme of the invention to solve the technical problem is: a kind of quick based on diazo-reaction and SERS
The method for detecting total purine content in aquatic products, comprising the following steps:
(1) purine extracts
0.15-0.25 g aquatic products sample is weighed in centrifuge tube, the perchloric acid solution that 3.0 mL concentration are 10wt% is added, is vortexed
Stir 35-45 s, heating hydrolysis 50-70 min；Sample after hydrolysis sodium hydroxide is adjusted into pH to alkalinity, water is added to be settled to
Analyte sample fluid is obtained by filtration through filter paper in 10.0mL, centrifuging and taking supernatant；
(2) external diazo-reaction:
NaNO is added in the analyte sample fluid that step (1) obtains2To its final concentration of 2.5-3.5 mol/L, HCl is added extremely
Its final concentration of 0.14-0.18 mol/L, is placed in 40-80 DEG C of water-bath, and after reacting 20-50 min, xanthine oxidase is added
Change enzyme, at pH=7.0-7.5,20-50 DEG C of reaction temperature, reacts 20-60 min, obtain the sample to be tested containing uric acid, wherein
Xanthine oxidase additive amount is 40-100 U/L；
(3) synthesis of Au@AgNPs
It is that 0.1M gold chloride is added in 100 mL ultrapure waters by the concentration of 0.2-0.3mL, under the conditions of magnetic agitation and oil bath
It is heated to boiling, then after being rapidly injected the sodium citrate solution that the concentration of 1.0 mL is 1wt%, mixed solution is flowed back 20-
40min, until solution colour becomes claret；After being gradually cooling to room temperature under stiring, resulting solution is passed through 0.22 μm
Ultrafiltration membrance filter takes filtered solution, obtains the AuNPs suspension containing the AuNPs particle that partial size is 40-50nm；Take 10mL
AuNPs suspension, under magnetic stirring be added 1.5mL concentration be 0.1M ascorbic acid solution, continue under magnetic stirring with
The silver nitrate solution that 1.5-4.5mL concentration is 1mM is added for the speed of 12-18 μ L/min until the color of solution becomes orange red,
Au@AgNPs solution is obtained after concentrated；
(4) SERS sample preparation
The Au@AgNPs solution that the sample to be tested containing uric acid of step (2) preparation and step (3) are prepared is by volume
The ratio of 1:1 mixes, and takes 3uL mixed solution drop on quartz plate, surveying its SERS signal intensity, according to uric acid Raman peak intensity with
Quantitative relationship between uric acid concentration, the concentration for calculating uric acid in acquisition sample to be tested are calculated according to diazo-reaction formula
Total purine content in aquatic products out.
Preferably, HCl concentration is 0.16 mol/L, NaNO in step (2)2Mass concentration is 3.0 g/L, and at 60 DEG C
Lower 40 min of water-bath；100 U/L of enzyme dosage, optimum temperature and time are 30 DEG C of 40 min of constant temperature, Optimal pHs 7.0.
Preferably, concentration step in step (3) specifically: take certain volume Au@AgNPs suspension original solution, 8000r/min
It is centrifuged 5min, after the supernatant for removing 3/4 volume of original solution, vibrates 2min on the oscillator, Au@AgNPs is made to suspend uniformly, i.e.,
Obtain the Au@AgNPs solution of 4 times of volumes of concentration.
Compared with the prior art, the advantages of the present invention are as follows: present invention firstly discloses one kind based on diazo-reaction and
The method that surface-enhanced Raman (SERS) measures purine total content in aquatic products, the adenine generated after aquatic products hydrolysis and bird
Purine can act on nitrite and generate diazonium salt, when diazonium salt heats in an acidic solution, hydrolysis occurs and generates phenol, utilizes
Adenine and guanine can be converted into respectively xanthine and hypoxanthine by the reaction.Then xanthine and hypoxanthine are in Huang
Final product uric acid is generated under the action of purine oxidase.Last uric acid combination Au@AgNPs, will show in Raman detector
Show characteristic signal, the relationship based on signal strength in standard curve and uric acid concentration, so that it may calculate the content of uric acid, purine is logical
Diazo-reaction is crossed, uric acid is converted to, the uric acid amount based on detection, so that it may it is counter to release total purine amount, to obtain indirectly
Total purine content in aquatic products has the advantage of fast detection speed, high sensitivity and high specificity.Obtained lowest detection inspection
Survey concentration is 0.005 mM, and recovery of standard addition is between the % of 100.0 % ~ 102.5.Therefore, this method can be used for total purine and contain
The qualitative and quantitative determination of amount, and sample treatment time-consuming is within 24 hours.Result of study shows diazo-reaction combination SERS
It can be used for the quick detection of total purine in aquatic products, this is provided for the fast quantitative analysis of total purine content in aquatic products
Reference frame shows that quickly detection has extensive use potentiality to SERS in aquatic product.
Detailed description of the invention
Fig. 1 is various concentration NaNO2With various concentration HCl to diazo-reaction generation hypoxanthine and xanthine content
Influence；
Fig. 2 is optimal N aNO2, the HCl concentration lower reaction time to diazo-reaction generate hypoxanthine and xanthine content shadow
It rings；
Fig. 3 is optimal N aNO2, reaction temperature generates the shadow of hypoxanthine and xanthine content to diazo-reaction under HCl concentration
It rings；
Fig. 4 is influence of the dosage of xanthine oxidase to uric acid production rate；
Fig. 5 is influence of the differential responses temperature to uric acid production rate under certain xanthine oxidase dosage；
Fig. 6 is influence of the differential responses time to uric acid production rate under certain xanthine oxidase dosage；
Fig. 7 is the ultraviolet-visible absorption spectroscopy of the Au@AgNPs under silver nitrate solution Different adding amount；
Fig. 8 is the SERS spectra of the Au@Ag NPs under silver nitrate solution Different adding amount；
Fig. 9 is in 1073cm-1Locate influence of the silver nitrate solution Different adding amount to the SERS signal intensity value of Au@AgNPs；
Figure 10 is the uric acid solution S ERS spectrogram under Au@AgNPs solution difference extension rate；
Figure 11 is influence of the Au@Ag NPs solution difference extension rate to uric acid solution S ERS intensity value；
Figure 12 is SERS spectra figure of the Au@AgNPs in conjunction with various concentration uric acid solution；
Figure 13 is uric acid solution concentration and characteristic peak 631cm-1Locate the linear relationship chart of SERS intensity.
Specific embodiment
The present invention will be described in further detail below with reference to the embodiments of the drawings.
One, specific embodiment
1, material and instrument
Material and reagent: hairtail decaptitating truncates internal organ, is put into meat grinder and rubs, and removes fishbone；Shrimp: decaptitating, removes intestines at decladding
Gland takes muscle parts；Crab: decladding takes muscle parts, and meat gruel is made in homogeneous mixing respectively, saves in -20 DEG C of refrigerator freezings standby
With.
Purine (adenine, guanine, xanthine, hypoxanthine, purity >=99 %), uric acid (purity >=99 %) are by Shanghai
Aladdin biochemical technology Co., Ltd formulates.Gold chloride (HAuCl4, 98 %), L-AA (99%), 4- mercaptobenzoic acid
(4MBA, 99 %), xanthine oxidase (300 U/L) are purchased from Sigma-Aldrich company.Silver nitrate (AgNO3, 99.95%),
Sodium citrate (98%), sodium nitrite (NaNO2, 99 %), sodium hydroxide (NaOH, 99 %), hydrochloric acid (36.46 %), perchloric acid
(HClO4, 60 %) and (being that analysis is pure), phosphate buffer (pH 7.0) is prepared using deionized water before testing.
Instrument: high performance liquid chromatography (HPLC) uses 1260 series of high efficiency liquid chromatogram (HPLC) (Germany) of Agilent；
XploRA ONE high sensitivity Raman spectrometer.
2. the method for quickly detecting total purine content in aquatic products based on diazo-reaction and SERS, comprising the following steps:
(1) purine extracts
0.15-0.25 g aquatic products sample is weighed in centrifuge tube, the perchloric acid solution that 3.0 mL concentration are 10wt% is added, is vortexed
Stir 35-45 s, heating hydrolysis 50-70 min；Sample after hydrolysis sodium hydroxide is adjusted into pH to alkalinity, water is added to be settled to
Analyte sample fluid is obtained by filtration through filter paper in 10.0mL, centrifuging and taking supernatant；
(2) external diazo-reaction
NaNO is added in the analyte sample fluid that step (1) obtains2To its final concentration of 2.5-3.5 mol/L, be added HCl to its
Final concentration of 0.14-0.18 mol/L, is placed in 40-80 DEG C of water-bath, and after reacting 20-50 min, xanthine oxidation is added
Enzyme reacts 20-60 min, obtains the sample to be tested containing uric acid at pH=7.0-7.5,20-50 DEG C of reaction temperature, wherein yellow
Purine oxidase additive amount is 40-100 U/L；
(3) synthesis of Au@AgNPs
Used glassware chloroazotic acid is impregnated 1 hour first, is cleaned up, is dried for standby with ultrapure water.By 0.2-
The concentration of 0.3mL is that 0.1M gold chloride is added in 100 mL ultrapure waters, is heated to boiling under the conditions of magnetic agitation and oil bath
It rises, then after being rapidly injected the sodium citrate solution that the concentration of 1.0 mL is 1wt%, mixed solution is flowed back 20-40min, until molten
Liquid color becomes claret；After being gradually cooling to room temperature under stiring, by resulting solution by 0.22 μm of ultrafiltration membrance filter, take
Filtered solution obtains the AuNPs suspension containing the AuNPs particle that partial size is 40-50nm；10mL AuNPs suspension is taken, in magnetic
Power is added with stirring the ascorbic acid solution that 1.5mL concentration is 0.1M, continues under magnetic stirring with the speed of 12-18 μ L/min
The silver nitrate solution that addition 1.5-4.5mL concentration is 1mM becomes orange red up to the color of solution, and Au@is obtained after concentrated
AgNPs solution；
(4) SERS sample preparation
The Au@AgNPs solution that the sample to be tested containing uric acid of step (2) preparation and step (3) are prepared is by volume
The ratio of 1:1 mixes, and takes 3uL mixed solution drop on quartz plate, surveying its SERS signal intensity, according to uric acid Raman peak intensity with
Quantitative relationship between uric acid concentration, the concentration for calculating uric acid in acquisition sample to be tested are calculated according to diazo-reaction formula
Total purine content in aquatic products out.
Raman spectrometer is using silicon wafer (Si) in 520 cm before testing every time-1Peak carries out instrumental correction as reference peaks.Table
Face enhances Raman spectrum acquisition unless otherwise specified, and 785 nm of optical maser wavelength, laser intensity is set as 50 %, power 120
MW, 50 × eyepiece, 60 s of time for exposure, the spectral region of detection are 200 to 3500 cm-1, resolution ratio is 1 cm-1.Sample
Parameter setting is consistent in detection process, and each sample is at least repeated 3 times, to obtain accurate Raman spectrum result.
Two, comparing result is analyzed
1, the experimental condition optimization of diazo-reaction
1.1、NaNO2Influence with HCl dosage, time and temperature to diazo-reaction
Adenine, guanine various concentration NaNO2With generation hypoxanthine and xanthine, NaNO under the action of HCl2With
The dosage of HCl is a most important influence factor in diazotising method, therefore to be optimized to the dosage of the two.Pass through Dan Yin
Element experiment optimizes the enzymatic hydrolysis condition of diazo-reaction and xanthine oxidase.To HCl and NaNO in diazo-reaction2's
Experiment is optimized in additional amount, hydrolysis temperature (20,40,60,80,100 DEG C) and time (10,20,30,40,50 min).
As a result as shown in Figure 1, in NaNO2, HCl concentration when being respectively under conditions of 3.00 mol/L, 0.16 mol/L,
The conversion reaction is complete.Based on NaNO2, HCl concentration be respectively 3.00 mol/L, 0.16 mol/L under conditions of, test is ground
Study carefully the hypoxanthine and xanthine generated when reacting 30 min to diazo reaction under 55 DEG C of different times of constant temperature and different temperatures
The influence of content, as a result as shown in Fig. 2, when reaching 40 min between when reacted, the content of two kinds of purine reaches stable state, says
Bright reaction is to convert completely.Therefore 40 min of diazo-reaction selection of time.It can visually see from Fig. 3 and work as reaction temperature
After reaching 60 DEG C, the content of purine tends towards stability, so selecting bath temperature for 60 DEG C.
The experimental condition optimization of 1.2 xanthine oxidases
Since hypoxanthine and xanthine can be converted into uric acid under xanthine oxidase effect, in test to enzyme dosage (40,
60,80,100,120 U/L), reaction temperature (20,30,40,50 DEG C) and time (20,30,40,50,60 min) carries out
Optimization experiment.
Best enzyme dosage is confirmed to guarantee purine to be completely converted into uric acid, as a result as shown in figure 4, with enzyme dosage
Increase, the uric acid content of generation is gradually increased, and after enzyme dosage reaches 100 U/L, the content of uric acid does not continue to rise.Cause
This, selects 100 U/L as the optimum amount of the enzyme.
Hypoxanthine and xanthine generate uric acid under the action of xanthine oxidase.According to the xanthine oxidation bought
Enzyme obtains the optimal pH of the oxidizing ferment 7.0 ~ 7.5.Effect of vigor when temperature is to enzyme effect is maximum, secondly the best enzyme of confirmation
Action time.The influence of different temperatures and time to enzyme effect when experiment investigation xanthine oxidase concentration is 50.0 U/L.
As a result as shown in figure 5, when temperature is 30 DEG C, the content of the uric acid generated at this time reaches maximum, illustrates enzyme under the conditions of the temperature
Activity reach maximum, as shown in fig. 6, the time influences less enzyme effect, therefore select 30 DEG C of water-baths 40 under pH=7.0
Min is as enzyme effect condition.
2, the optimization of SERS experiment condition
In 50mL conical flask, the 10mL AuNPs suspension of preparation is added, it is anti-bad that 1.5mL 0.1M is added under magnetic stirring
Hematic acid solution；A certain amount of 1mM silver nitrate solution is added in above-mentioned solution with the speed of 15 μ L/min under magnetic stirring,
With the addition of silver nitrate, the color of solution can gradually become it is orange red, continue stir 30min.By the way that different amounts of nitre is added
Sour silver (0.5,1.0,1.5,2.0,2.5,3.5,4.5mL) changes the thickness of silver-colored shell.
As a result as shown in fig. 7, gold kind has a maximum absorption peak at 520nm, but with the addition of silver nitrate solution
Amount is gradually increased, and the maximum absorption band at 520nm occurs blue shift and is gradually increased, but when silver nitrate solution additional amount increases to
The reason of when 4.5mL, absorption peak of the Au@AgNPs at 400nm is had the advantage peak, is influenced may be that the silver-colored shell of synthesis is too thick.
Optimal SERS reinforcing effect in order to obtain, test is with the MBA(of 1mM to mercaptobenzoic acid) to the Au of synthesis
The stability of AgNPs carries out Raman detection.As a result acquired as shown in figure 8, when the additional amount of silver nitrate solution is 3.5mL
SERS signal it is most strong.As shown in figure 9, when the additional amount with silver nitrate is gradually increased, in 1073 cm-1The intensity of characteristic peak
It is gradually increased, but when additional amount reaches 4.5mL, the intensity of characteristic peak declines.Therefore selection silver nitrate solution additional amount is
3.5mL carries out Raman detection to synthesize Au@AgNPs.
Figure 10 and Figure 11 reflects shadow of the Au@Ag NPs solution to the SERS spectra of uric acid solution of different extension rates
It rings.By can be seen that characteristic peak has corresponding intensity value everywhere in Figure 10.By obtaining the increase with extension rate in Figure 11,
For SERS signal in downward trend after first rising, and when extension rate is 4 times, obtained SERS signal is maximum.Dilution
The excessively high and too low SERS signal that will affect sample of multiple, when extension rate is excessively high, Au@Ag NPs does not fill in conjunction with sample
Point；When extension rate is too low, Au@AgNPs can reunite, so that the binding ability with analyte weakens, so that SERS signal
It dies down.Therefore 4 times are set by the extension rate of Au@AgNPs in the experiment of uric acid detection limit.
Three, method validation
Uric acid standard solution (0.1 g/L is equivalent to 0.6 mM): 0.0100 g uric acid is accurately weighed, 6 mL 0.1mol/L are dissolved in
In sodium hydroxide solution, 100 mL are settled to distilled water, are stored at 4 DEG C.With standard solution successively dilute prepare 0.5 mM,
The uric acid solution of 0.4 mM, 0.3 mM, 0.2 mM, 0.1 mM, 0.06 mM, 0.01 mM, 0.005 mM.Take Au@AgNPs solution
With the uric acid solution of various concentration with the mixing of volume ratio 1:1 ratio, take 3uL mixed solution drop on quartz plate, surveying its SERS letter
Number.
As shown in figure 12, as the concentration of uric acid solution reduces, corresponding raman characteristic peak 631cm-1Also with gradually dropping
It is low, SERS analysis is carried out to uric acid aqueous solution between 0.6 ~ 0.005mM, is obtained between uric acid Raman peak intensity and its concentration
Quantitative relationship.As shown in figure 13, within the scope of 0.6 ~ 0.005mM, obtained linear equation is y=20278.9x-832.259,
Related coefficientR 2 =0.9803, obtained minimal detectable concentration is 0.005 mM, and recovery of standard addition is 100.0% ~ 102.5%, such as
Shown in table 1.By the rate of recovery and precision test, this method is reproducible, and accuracy is high.
The obtained rate of recovery of the application Raman peak intensity of table 1 and predicted value experiment
。
Four, sample analysis
Aquatic products after pretreatment, measure its uric acid content after new method conversion according to specific embodiment.And use efficient liquid phase
Chromatography directly measures the content of four kinds of purine, further demonstrates the accuracy of this method.As shown in table 2, two methods it
Between be not significantly different, illustrate that the applicability of new method in this research is acceptable.Compared with reference HPLC method, opened
The significant cost for reducing chemicals and equipment of the method for hair simultaneously reduces the analysis required time.
High performance liquid chromatography carries out under the following conditions: chromatographic column: Agilent ZORBAX SB-Cl8(4.6 mm×250
Mm, 5 μm)；Mobile phase: H3PO4-KH2PO4Buffer (7.35 mM, pH 3.8)；Flow velocity: 1.0 mL/min；Column heater: 30
℃；Injection rate: 20 μ L；Ultraviolet detection wavelength: 254 nm.
Raman spectrometer is using silicon wafer (Si) in 520 cm before testing every time-1Peak carries out instrumental correction as reference peaks.Table
Face enhances Raman spectrum acquisition unless otherwise specified, and 785 nm of optical maser wavelength, laser intensity is set as 50 %, power 120
MW, 50 × eyepiece, 60 s of time for exposure, the spectral region of detection are 200 to 3500 cm-1, resolution ratio is 1 cm-1.Sample
Parameter setting is consistent in detection process, and each sample is at least repeated 3 times, to obtain accurate Raman spectrum result.
Table 2 compares the testing result of two methods
。
In conclusion being converted into hypoxanthine and Huang through diazo-reaction using adenine and guanine in this research method
Purine, then uric acid is converted into through xanthine oxidase.Using Au@Ag NPs as active substances, using SERS measurement converted product
The total content of purine can be obtained in uric acid.The SERS spectra for acquiring uric acid in the fish extracting solution of 0.005 ~ 0.6 mM range, obtains
Out in 631 cm-1The characteristic peak at place has good linear relationship (R with the standard curve that corresponding Raman peaks intensity value is established2
=0.9803).Obtained lowest detection detectable concentration is 0.005 mM, and recovery of standard addition is between 100.0% ~ 102.5%.By
In two methods, there was no significant difference, this method can preferably replace traditional HPLC method carry out the qualitative of total purine content and
Quantitative determination.Result of study shows that diazo-reaction combination SERS can be used for the quick detection of total purine in aquatic products, this be
The fast quantitative analysis of total purine content in aquatic products provides reference frame, shows that SERS is fast in aquatic products fish product
Speed detection, which has, is widely applied potentiality.
Above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art it is common
Within the essential scope of the present invention, the variations, modifications, additions or substitutions made also should belong to protection of the invention to technical staff
Range.
Claims (3)
1. a kind of method for quickly detecting total purine content in aquatic products based on diazo-reaction and SERS, it is characterised in that including
Following steps:
(1) purine extracts
0.15-0.25 g aquatic products sample is weighed in centrifuge tube, the perchloric acid solution that 3.0 mL concentration are 10wt% is added, is vortexed
Stir 35-45 s, heating hydrolysis 50-70 min；Sample after hydrolysis sodium hydroxide is adjusted into pH to alkalinity, water is added to be settled to
Analyte sample fluid is obtained by filtration through filter paper in 10.0mL, centrifuging and taking supernatant；
(2) external diazo-reaction:
NaNO is added in the analyte sample fluid that step (1) obtains2To its final concentration of 2.5-3.5 mol/L, be added HCl to its
Final concentration of 0.14-0.18 mol/L, is placed in 40-80 DEG C of water-bath, and after reacting 20-50 min, xanthine oxidation is added
Enzyme reacts 20-60 min, obtains the sample to be tested containing uric acid at pH=7.0-7.5,20-50 DEG C of reaction temperature, wherein yellow
Purine oxidase additive amount is 40-100 U/L；
(3) synthesis of Au@AgNPs
It is that 0.1M gold chloride is added in 100 mL ultrapure waters by the concentration of 0.2-0.3mL, under the conditions of magnetic agitation and oil bath
Be heated to boiling, then be rapidly injected 1.0 mL concentration be 1wt% sodium citrate solution after, reflow treatment 20-40min, until
Solution colour becomes claret；After being gradually cooling to room temperature under stiring, resulting solution is passed through into 0.22 μm of ultrafiltration membrance filter,
Filtered solution is taken, the AuNPs suspension containing the AuNPs particle that partial size is 40-50nm is obtained；10mL AuNPs suspension is taken,
The ascorbic acid solution that 1.5mL concentration is 0.1M is added under magnetic agitation, continues under magnetic stirring with the speed of 12-18 μ L/min
The silver nitrate solution that 1.5-4.5mL concentration is 1mM is added for degree until the color of solution becomes orange red, and Au@is obtained after concentrated
AgNPs solution；
(4) SERS sample preparation
The Au@AgNPs solution that the sample to be tested containing uric acid of step (2) preparation and step (3) are prepared is by volume
The ratio of 1:1 mixes, and takes 3uL mixed solution drop on quartz plate, surveying its SERS signal intensity, according to uric acid Raman peak intensity with
Quantitative relationship between uric acid concentration, the concentration for calculating uric acid in acquisition sample to be tested are calculated according to diazo-reaction formula
Total purine content in aquatic products out.
A kind of total purine content in aquatic products is quickly detected based on diazo-reaction and SERS 2. according to claim 1
Method, it is characterised in that: HCl concentration is 0.16 mol/L, NaNO in step (2)2Mass concentration is 3.0 g/L, and at 60 DEG C
Lower 40 min of water-bath；100 U/L of enzyme dosage, hydrolysis temperature and time are 30 DEG C of 40 min of constant temperature, enzymatic hydrolysis pH is 7.0.
A kind of total purine content in aquatic products is quickly detected based on diazo-reaction and SERS 3. according to claim 1
Method, it is characterised in that concentration process in step (3) specifically: take certain volume Au@AgNPs suspension original solution, 8000r/min
It is centrifuged 5min, after the supernatant for removing 3/4 volume of original solution, vibrates 2min on the oscillator, Au@AgNPs is made to suspend uniformly, i.e.,
Obtain the Au@AgNPs solution of 4 times of volumes of concentration.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110618124A (en) * | 2019-09-17 | 2019-12-27 | 宁波大学 | Method for detecting content of tyramine in aquatic product based on azo coupling reaction and surface enhanced resonance Raman scattering |
| CN114113506A (en) * | 2021-12-20 | 2022-03-01 | 合肥工业大学 | Method for judging freshness of meat by quantitatively detecting hypoxanthine |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1854730A (en) * | 2005-04-18 | 2006-11-01 | 清华大学 | Method for diagnosing gout and hyperuicemia by serum analysis |
| CN102288592A (en) * | 2011-05-18 | 2011-12-21 | 福建师范大学 | Method for quantitative detection of uric acid based on surface enhanced Raman spectroscopy (SERS) technology |
| CN106855508A (en) * | 2017-01-06 | 2017-06-16 | 北京物资学院 | A kind of method for detecting hypoxanthine content in fish |
-
2019
- 2019-04-25 CN CN201910336482.0A patent/CN110082336A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1854730A (en) * | 2005-04-18 | 2006-11-01 | 清华大学 | Method for diagnosing gout and hyperuicemia by serum analysis |
| CN102288592A (en) * | 2011-05-18 | 2011-12-21 | 福建师范大学 | Method for quantitative detection of uric acid based on surface enhanced Raman spectroscopy (SERS) technology |
| CN106855508A (en) * | 2017-01-06 | 2017-06-16 | 北京物资学院 | A kind of method for detecting hypoxanthine content in fish |
Non-Patent Citations (5)
| Title |
|---|
| AKSHAYA K. SAMAL ET AL.: "Size Tunable Au@Ag Core−Shell Nanoparticles: Synthesis and Surface-Enhanced Raman Scattering Properties", 《LANGMUIR》 * |
| FU-HSIANG KO ET AL.: "Au–Ag core–shell nanoparticles with controllable shell thicknesses for the detection of adenosine by surface enhanced Raman scattering", 《SENSORS AND ACTUATORS B》 * |
| LU PEI ET AL.: "Au-Ag Core-Shell Nanospheres for Surface-Enhanced Raman Scattering Detection of Sudan I and Sudan II in Chili Powder", 《JOURNAL OF NANOMATERIALS》 * |
| 冯晓晶: "嘌呤及其代谢产物尿酸检测方法研究和应用", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 郑彬等: "表面增强拉曼光谱快速检测尿液中的尿酸", 《光谱学与光谱分析》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110618124A (en) * | 2019-09-17 | 2019-12-27 | 宁波大学 | Method for detecting content of tyramine in aquatic product based on azo coupling reaction and surface enhanced resonance Raman scattering |
| CN114113506A (en) * | 2021-12-20 | 2022-03-01 | 合肥工业大学 | Method for judging freshness of meat by quantitatively detecting hypoxanthine |
| CN114113506B (en) * | 2021-12-20 | 2023-06-20 | 合肥工业大学 | Method for judging freshness of meat by quantitatively detecting hypoxanthine |
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