CN107345911A - A kind of method of histamine in SERS qualitative and quantitative analysis rice fish tissue - Google Patents
A kind of method of histamine in SERS qualitative and quantitative analysis rice fish tissue Download PDFInfo
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- CN107345911A CN107345911A CN201710420397.3A CN201710420397A CN107345911A CN 107345911 A CN107345911 A CN 107345911A CN 201710420397 A CN201710420397 A CN 201710420397A CN 107345911 A CN107345911 A CN 107345911A
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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
- G01N30/02—Column 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Abstract
The method that the present invention discloses histamine in a kind of SERS qualitative and quantitative analysis rice fish tissue, including：Muddy flesh is made after taking meter fish tissue chopping, weighs several pieces equivalent rice fish tissue as sample, and the histamine standard solution for preparing various concentrations is added in each sample, and the sample of different time points is taken during storage as mark-on sample and prediction sample；Mark-on sample and prediction sample are added into trichloroacetic acid, extract solution is obtained after filtering；The content of histamine in the rice flesh of fish of prediction sample is determined using HPLC；Raman spectrum data collection is carried out after nanometer strengthener is added in the extract solution of mark-on sample, and utilizes raman characteristic peak corresponding to Density function theory histamine；Quantitative Analysis Model is established with the content of the peak intensity of raman characteristic peak and histamine, and Histamine concentrations in meter fish tissue are measured using the Quantitative Analysis Model.The present invention can quickly and accurately detect in the fresh flesh of fish concentration range in 1~150mg/L histamine content.
Description
Technical field
The present invention relates to histamine detection field in the flesh of fish, more particularly, to a kind of SERS qualitative, quantitative point
The method for analysing histamine in rice fish tissue.
Background technology
Biogenic amine (Biogenic Amine, BA) is a kind of non-volatile aliphatic, alicyclic or heterocycle nitrogenous had
The general name of machine compound, it has certain bioactivity, is widely present among organism and numerous food.Appropriate biology
Amine has important physiologically active in body, such as adjusts secretion, control blood pressure, the participation immune response of neurotransmitter.But
When it takes in too high or a large amount of accumulations in vivo may then toxicity be produced to body.Wherein, histamine (Histamine) is toxicity
Most strong biogenic amine.Research shows, when histamine content is in 8~40mg/100g in the flesh of fish of intake, can trigger slight poisoning
Reaction, and concentration may produce the serious toxic reaction such as vomiting, dizziness, diarrhoea, allergy more than 100mg/100g, even result in
Shock, threat to life.The national standard of the Ministry of Public Health of China issue《Fresh, jelly animality aquatic products sanitary standard》, it is specified that histamine contains
Amount：Mackerel≤100mg/100g, other fish≤30mg/100g；And European Union limits average content≤10mg/ of histamine in the flesh of fish
100g.In addition, U.S. FDA is also using the histamine content standard rotten as the flesh of fish is judged more than 50ppm.Therefore, quickly and accurately
The content of histamine in the flesh of fish is detected, to ensureing that the health and safety of flesh quality and consumer are significant.
Tradition for histamine in the flesh of fish detection method mainly have high performance liquid chromatography (HPLC), fluorescent quantitation, from
Sub- exchange chromatography, capillary electrophoresis and enzyme linked immunosorbent assay (ELISA) etc., although these method sensitivity are higher,
The shortcomings of pre-treatment is cumbersome, detection is time-consuming longer, instrument is inconvenient to carry and reagent price is expensive is tested with certain limitation
Property.SERS (Surface-Enhanced Raman Spectroscopy, SERS) is a kind of highly sensitive
Finger-print, " surface enhanced " refer to compound molecule be adsorbed onto some nanoscale rough metals (such as gold, silver, copper) surface or
In colloidal sol, its Raman signal is into the enhancing of geometry multiple, and therefore, SERS technologies can be realized to micro-example and monomolecular quick
Detection.Meanwhile SERS has the advantages that pre-treating method is simple, instrument is easy to carry and detection speed is fast, remained in agricultural product
Detection of hazardous material etc. is widely used in the detection of trace materials, environment in the rapid screening of agricultural chemicals, food.
The content of the invention
It is an object of the invention to use SERS technology (Surface-Enhanced Raman
Spectroscopy, SERS) density functional theory (Density Functional Theory, DFT) is combined, establish accurate fast
The detection method of histamine content in the rice flesh of fish of speed.
Concrete technical scheme of the present invention is as follows：
The method of histamine, comprises the following steps in a kind of SERS qualitative and quantitative analysis rice fish tissue：
(1) muddy flesh is made after taking meter fish tissue chopping, weighs several pieces equivalent rice fish tissue as sample, and prepare difference
The histamine standard solution of concentration is added in each sample, taken during storage the samples of different time points as mark-on sample with
Predict sample；
(2) described mark-on sample and prediction sample are added into trichloroacetic acid, extract solution is obtained after filtering；
(3) content of histamine in the rice flesh of fish of HPLC measure prediction samples is utilized；
(4) Raman spectrum data collection is carried out after nanometer strengthener is added in the extract solution of mark-on sample, and is utilized close
Spend Functional Theory and calculate raman characteristic peak corresponding to histamine；
(5) Quantitative Analysis Model is established with the content of the peak intensity of the raman characteristic peak and histamine, and it is quantitative using this
Analysis model measures Histamine concentrations in meter fish tissue.
The present invention is using Shishou section fresh rice fish (Miichthys miiuy) muscle as carrier, the mould in a manner of additionally adding
Intend the change of flesh of fish histamine during storage, using Gold nanoparticle as substrate, utilize SERS technology combination density functional theories
(Density Functional Theory, DFT) carries out qualitative and quantitative analysis to histamine content in the flesh of fish.Rice fish dorsal used
Muscle shreds, and meat grinder is made uniform muddy flesh, prepares the histamine standard solution of various concentrations, adds various concentrations standard respectively
For liquid into each sample, institute divides mark-on sample and prediction sample using test specimen, and machine testing on SERS is used for after preparation.
Include as preferable, described histamine standard solution：It is respectively with 12% trichloroacetic acid configuration Histamine concentrations
0th, 0.25,1.0,5.0,10.0,15.0,20.0,25.0mg/L titers, for preparing SERS standard curves and HPLC standards
Curve.SERS instruments used are two poles that RamTracer-200-HS types Portable Raman spectrometer combines 785nm excitation wavelengths
Pipe frequency stabilization exciter.
In step (2), mark-on sample and prediction sample press 1：10 solid-liquid ratio adds trichloroacetic acid, after ultrasonic extraction
It is centrifuged, filters, and add trichloroacetic acid constant volume, produces extract solution.
As preferable, in step (3), the step of being determined using HPLC, is included：
(3.1) grease removal：Isometric n-hexane is added in the extract solution of prediction sample, fully shaking, removes organic phase；
(3.2) extract：Liquid, which adds NaCl, after grease removal makes solution saturation, adds NaOH solution, is added just after mixing
Centrifuged after butanol-chloroform mixed solution, fully vibration, take upper organic phase, extract is obtained after extraction, salt is added to extract
Acid, mix and dried up after nitrogen under water-bath, add dissolving with hydrochloric acid residue, obtain treating derivative solution；
(3.3) it is derivative：Treat that derivative solution adds saturation NaHCO to described3Solution and dansyl Cl derivative solution, drying
Saturation NaHCO is added afterwards3Solution, nitrogen blows away acetone under 40 DEG C of water-baths, adds ether and extracts, after stratification, in absorption
Clear liquid, methanol dissolution residual substance is added after ether extraction liquid is dried up by nitrogen, then be used for by membrane filtration, filtered fluid
HPLC is detected；
(3.4) HPLC condition determinations：Chromatographic column：Agilent ZORBAX SB-C18；Detector：Photodiode array
Detector；Mobile phase：A- methanol, B- ultra-pure waters；Flow velocity：0.3mL/min；Detection wavelength：254nm；Sample size：5μL；Column temperature：
30℃。
Include the silver nanoparticle colloidal sol or gold nano colloidal sol as substrate as preferable, described nanometer strengthener；
The preparation process of gold nano colloidal sol is：After high chlorauric acid solution is heated into boiling, citric acid three sodium solution is added,
Stir simultaneously, the color for the aurosol being prepared into is claret；After solution cooling, above-mentioned gold size solution is poured into centrifuge tube
In, outwell a small amount of supernatant after centrifugation, then toward ultra-pure water is added in centrifuge tube, mixed with sonic oscillation, obtain gold nano colloidal sol；
The preparation process of silver nanoparticle colloidal sol is：Silver nitrate solution is heated to seethe with excitement, concentration is progressively instilled in 2min is
1% citric acid three sodium solution, is stirred simultaneously, and now by transparent change light brown, the celadon liquid obtained after reaction is solution
Silver nanoparticle colloidal sol.
Above-mentioned silver nanoparticle colloidal sol and gold nano colloidal sol substrate are prepared using trisodium citrate heating reducing process, by making
Influence of the two kinds of reinforcing agents of silver nanoparticle and gold nano to SERS signal is characterized with transmission electron microscope.As a result show, Yin Na
Rice corpuscles diameter about 60nm, golden nanometer particle diameter 40nm, the particle size of two kinds of nano-particles are all more uniform.Gold nano colloidal sol
It is higher to the Raman signal intensity of histamine molecule, 953,992,1262,1106,1262,1317,1425,1593cm-1Place's enhancing
Positive effect, illustrate that gold nano substrate can effectively adsorb negative histamine molecule, obtain stronger signal effect, further preferably adopt
Strengthen substrate with gold nano.
In the present invention, the Raman spectrum analysis of histamine：Using B3LYP hydridization functional in DFT, B3LYP joins for Becke3 types
Number density functional model is to histamine (molecular formula C5H9N3) carry out theoretical Raman spectrum calculating, its molecular structure mainly by imidazole ring,
The groups such as C-N, C=N, C=C, C-C, C-H and N-H form.The Raman spectrum of histamine can be parsed according to functional group's eigen vibration frequency
Peak.Obtained histamine Raman spectrum is simulated by the Raman spectrum and use Density function theory of histamine standard items solid,
It can be seen that, spectral peak and Density function theory spectral peak relation.These spectral peaks can be as the raman characteristic peak of histamine.
As preferable, in step (4), 150 μ L nanometer strengtheners and 50mL NaCl sequentially added to each sample, are put
Enter supporting liquid sample pool and carry out spectra collection.
The processing such as smooth, baseline correction is carried out to the SERS of all samples, to remove noise and baseline
The influence of drift.Deduct rice fish blank SERS spectra signal, can recognize that 5 at histamine Molecular Raman characteristic peak (953,992,1106,
1262、1317cm-1), due to 1262cm-1It is higher to locate the peak intensity of characteristic peak, and is nearby influenceed without folded peak and miscellaneous peak, from this
The peak intensity of characteristic peak establishes the Quantitative Analysis Model of histamine content in rice fish.With 1262cm-1Locate histamine SERS characteristic peak peak intensities
The unitary linear standard curve made with Histamine concentrations is spent, Histamine concentrations are in 1~150mg/kg in the flesh of fish, linear equation y
=27.889x+106.25, coefficient R2=0.9806, linear dependence is good.
Using the SERS data equations established, the real content of histamine in the rice flesh of fish is predicted, and with HPLC institutes
Obtain actual value to be compared, obtain relative standard deviation and rate of recovery etc..Condition determination is as follows, chromatographic column：Agilent
ZORBAX SB-C18 (150mm × 2.1mm, 3.5 μm)；Detector：Photodiode array detector (DAD)；Mobile phase：A-
Methanol, B- ultra-pure waters；Flow velocity：0.3mL/min；Detection wavelength：254nm；Sample size：5μL；Column temperature：30℃.
The present invention is to be respectively provided with preferable testing result, and histamine molecule in 1~150mg/L to Histamine concentrations in the rice flesh of fish
Characteristic peak 1262cm-1Peak intensity and concentration are in good linear correlation, R in standard curve2=0.9806.Using what is established
The content of histamine, the predicted value of table SERS methods and state in rice fish sample after being placed 1,4,7 day at 4 DEG C of SERS-DFT model predictions
Measurement result (actual value) is basically identical obtained by mark method.The RSD of SERS instruments is between 2.59~4.72%, the rate of recovery of method
For 87.02~117.25%.Show accurately may be used using the method for histamine content in the SERS technology combination DFT methods detection rice flesh of fish
Lean on, a kind of new Research Thinking is provided for the quick detection of histamine in the flesh of fish.
Brief description of the drawings
Fig. 1 is histamine schematic arrangement；
Fig. 2 is the Raman spectrum of histamine standard items solid；
Fig. 3 is to be used as the SERS spectra (a and b) of the histamine standard liquid of substrate using silver nanoparticle or gold nano respectively；Without
The histamine standard (c and d) of surface reinforcing agent processing and the Raman spectrogram of blank；
Fig. 4 is silver nanoparticle colloidal sol transmission electron microscope figure；
Fig. 5 is gold nano colloidal sol transmission electron microscope figure；
Fig. 6 is the Surface Enhanced Raman Scattering Spectrum of the histamine of various concentrations in rice fish；
A figures and B figures in Fig. 7 are 1262cm-1Locate the unitary line that histamine SERS characteristic peaks peak intensity makes with Histamine concentrations
Property standard curve.
Embodiment
Further explanation is made to technical scheme with reference to specific embodiment：
In the present embodiment, using Shishou section fresh rice fish (Miichthys miiuy) muscle as carrier, with the side additionally added
Formula simulates the change of flesh of fish histamine during storage, using Gold nanoparticle as substrate, utilizes SERS technology combination Density functionals
Theoretical (Density Functional Theory, DFT) carries out qualitative and quantitative analysis to histamine content in the flesh of fish.Specific steps
It is as follows：
1st, the preparation of sample
Mark-on sample：The chopping of meter fish dorsal muscle is taken, uniform muddy flesh is made in meat grinder, accurately weighs 17 parts of samples, every part
5g, the histamine standard solution (being prepared with 12% trichloroacetic acid) of various concentrations is prepared, adds various concentrations titer respectively
0.1mL into each sample, the histamine final concentration that makes each mark-on sample is respectively 1,5,20,40,60,80,100,120,140,
160、180、200、220、240、260、280、300mg/kg.Meanwhile to be not added with the fresh flesh of fish of histamine as dummy,
And confirm that it is free of histamine by " 2.5 " method.
Forecast sample：Each 10 parts of the rice flesh of fish placed at 4 DEG C 1,4,7 day is chosen, histamine is carried out with SERS models have been established
Content prediction, and actual value measure is carried out with National Standard Method, to examine the SERS method degrees of accuracy.
Histamine standard working solution：Be respectively 0 with 12% trichloroacetic acid configuration Histamine concentrations, 0.25,1.0,5.0,10.0,
15.0th, 20.0,25.0mg/L titers, for preparing SERS standard curves and HPLC standard curves.
2nd, the processing of sample
Mark-on sample (5g) and prediction sample (5g) press 1：10 solid-liquid ratio adds trichloroacetic acid (12%), 50 DEG C,
Ultrasonic extraction 5min, is during which stirred continuously under 60W power.5min, the double-deck quantitative filter paper of supernatant are centrifuged under 3600r/min
Filtering, adds trichloroacetic acid (12%) to be settled to 50mL, produces extract solution.The accurate extract solution 2mL that measures has plug glass examination in 15mL
Guan Zhong, adding appropriate NaCl (about 1.2g) makes solution saturation, adds 0.75mL 2mol/L NaOH solution, is vortexed after mixing
Add 2mL n-butanol fully shaking 5min, 1min is centrifuged under 2000r/min, take the μ L of supernatant (n-butanol phase) 200 on SERS
In sample bottle (2mL quartz bottle), nitrogen dries up under 60 DEG C of water-baths.The machine testing on SERS.(sample preparation time about 30 minutes)
Take the histamine standard working solution that two parts of concentration are 10.0mg/L, every part of 2mL, after same step process, on SERS
Machine testing, the determination for gold or silver nanoparticle substrate.
3rd, the measure of forecast sample actual value
The detection of actual value is slightly modified with reference to national standard (GB/T 5009.208-2008) method.
Forecast sample obtains extract solution, (1) grease removal by " 2.4 " method：5mL accurately is measured in 15mL tool plug test tubes, is added
Enter isometric n-hexane, fully shaking 5min, remove organic phase, repeat twice.(2) extract：By liquid after above-mentioned grease removal
Adding appropriate NaCl (about 3.0g) makes solution saturation, adds 1.8mL 2mol/L NaOH solution, and 5.0mL is being added just after mixing
Butanol-chloroform (1:1) mixed solution, 5min is fully vibrated, centrifuges 10min in 3600r/min, take upper organic phase, repeat to extract
Take step 1 time.Combining extraction liquid simultaneously mixes, and takes 3mL extracts to add 0.2mL 1mol/L hydrochloric acid, mixes after 40 DEG C of water-baths
Lower nitrogen drying, adds 0.1mol/L hydrochloric acid 1.0mL dissolution residual substances, waits to derive.(3) it is derivative：Take and treat derivative solution 0.5mL
In 10mL tool plug test tubes, (10mg/mL acetone is molten for addition 1.5mL saturation NaHCO3 solution (now matching somebody with somebody) and 1.0mL dansyl Cls
Liquid) derivative solution, shakes and mixes after reacting 30min in 60 DEG C of baking ovens, centre is shaken 2 times.100 μ L-Glus are added after taking-up
Sodium (50mg/mL saturation NaHCO3 solution), concussion mix, and 60 DEG C of insulation 15min, add 1mL ultra-pure waters, the nitrogen under 40 DEG C of water-baths
Acetone is removed in air-blowing, adds 3mL ether and extracts, fully shaking 2min, after stratification, Aspirate supernatant (ether phase), repeats to extract
Take 2 times, merge ether extraction liquid, add 1.0mL methanol dissolution residual substances after nitrogen drying, 0.22 μm of membrane filtration, be used for
HPLC is detected.(sample preparation time about 5 hours)
The histamine standard working solution of 0.5mL various concentrations is taken to sequentially add 20.0 μ 0 in each 10mL tool plug test tubes respectively
(100mg/L) internal standard uses liquid, after above-mentioned " (3) are derivative " step process, is detected for HPLC.
HPLC condition determinations：Chromatographic column：Agilent ZORBAX SB-C18 (150mm × 2.1mm, 3.5 μm)；Detector：
Photodiode array detector (DAD)；Mobile phase：A- methanol, B- ultra-pure waters；Flow velocity：0.3mL/min；Detection wavelength：
254nm；Sample size：5μL；Column temperature：30℃；Elution program is shown in Table 1.
The gradient elution program table of table 1
4th, prepared by silver nanoparticle colloidal sol and gold nano colloidal sol substrate
Aurosol nanometer substrate is prepared using trisodium citrate heating reducing process, is slightly changed.Manufacturing process is as follows：By one
The high chlorauric acid solution (the high gold chlorides of 10mg are dissolved in 200mL ultra-pure waters) for determining concentration is poured into flask, is placed on constant temperature magnetic force and is stirred
Mix on device, temperature control 120 DEG C it is constant be heated to boiling after, be rapidly added certain density citric acid three sodium solution (20mg
Trisodium citrate is dissolved in 4mL ultra-pure waters), while quickly stirred with 100rpm rotating speeds, the color for the aurosol being prepared into is red for wine
Color.After solution cooling, above-mentioned appropriate gold size solution is poured into centrifuge tube, a small amount of supernatant, then past centrifugation are outwelled after centrifugation
Appropriate ultra-pure water is added in pipe, is mixed with sonic oscillation, gold size is kept in dark place after repeatedly purification.
Silver nanoparticle colloidal sol substrate prepares the trisodium citrate heating reducing process using Lee-Meisel, and manufacturing process is as follows：
Certain density silver nitrate solution (36mg silver nitrates are dissolved in 200mL ultra-pure waters) is poured into flask, is placed on temperature constant magnetic stirring
On device, high temperature is heated rapidly to seethe with excitement, and citric acid three sodium solution (the 60mg citric acids that concentration is 1% are progressively instilled in 2min
Trisodium is dissolved in 6mL ultra-pure waters), while stirred with 200r/min rotating speed, now solution is slowly by transparent change light brown, reaction
Celadon liquid is obtained after 25min, is kept in dark place.
5th, Raman spectrum gathers
Before Raman spectrum data collection, rectified an instrument with acetonitrile, using 785nm excitation wavelengths.Parameter is as follows：Power is
200mW, 200~3300cm-1 of scanning range, optical resolution 2cm-1, time of integration 10s, gather 3 times and take average spectral value.
Histamine solid gathers：Histamine powder is flattened on quartz plate with slide, gathered with supporting microscope stage.
SERS is gathered：150 μ L nanometer strengtheners, 50mL NaCl are sequentially added into the quartzy bottle containing each sample, is put into and matches somebody with somebody
Cover liquid sample pool and carry out spectra collection.
6th, data analysis and processing
All data analyses are based on MATAB R2014a, Gaussian.v09, OMNIC v8.2, Origin v8.0, SPSS
V17.0 platforms are completed.
7th, the Raman spectrum analysis of histamine
Using B3LYP hydridization functional in DFT, B3LYP is Becke3 shape parameter Density functional models to histamine (molecular formula
C5H9N3) theoretical Raman spectrum calculating is carried out, its molecular structure is shown in Fig. 1, mainly by imidazole ring, C-N, C=N, C=C, C-C, C-H
Formed with groups such as N-H.The raman spectra of histamine can be parsed according to functional group's eigen vibration frequency.
Fig. 2 is the Raman spectrum of histamine, and wherein Fig. 2 (a) is the Raman spectrum of histamine standard items solid, and Fig. 2 (b) is to use
Density function theory simulates obtained histamine Raman spectrum.Can be seen that from figure, spectral peak 321,376,647,811,1032,
1099、1376、1432、1477cm-1With Density function theory spectral peak 319,379,648,811,1030,1098,1291,
1377、1433、1478cm-1It is basically identical.The spectral peak of histamine is belonged to, the results are shown in Table 2.Wherein 321,376cm-1For group
Amine molecule middle skeleton table out-of-plane bending vibration, spectral peak 647,811cm-1It is the outer ring vibration of histamine molecular surface, spectral peak 1032cm-1
For deformation vibration in the C-H surfaces of histamine molecule, spectral peak 1099cm-1For the C-N stretching vibrations of histamine molecule, 1376,1477cm-1For ring stretching vibration, spectral peak 1432cm-1It is that the ring vibration of histamine molecule and the table in-plane bending vibration of N-H groups are made jointly
With.These spectral peaks can be as the raman characteristic peak of histamine.
The raman spectra ownership of the histamine molecule of table 2
a Calvulated wavenumbers at B3LYP/6-31G(DFT).
bVs=very strong；S=strong；M=medlum；W=weak.
υ=stretching；δ=in-plane bending；γ=out of plane bending.
8th, different nanometer enhancing substrates compare the enhancing effect of histamine standard items molecule
Fig. 3 (a) and (b) are to be used as the SERS spectra of the histamine standard liquid of substrate using silver nanoparticle or gold nano respectively；Fig. 3
(c) and (d) is the Raman spectrogram of the histamine standard and blank handled without surface reinforcing agent respectively.It can be seen that from figure, no table
The histamine standard of face reinforcing agent processing, only occur in its Raman spectrogram five very weak characteristic peaks of intensity (432,749,844,
939、1339cm-1).And in SERS spectra figure obtain 953,992,1030,1106,1186,1262,1317,1425,
1593cm-1The histamine Raman peaks at place are stronger, and the Spectra peak recognition of these characteristic peaks the results are shown in Table 2.
Two kinds of reinforcing agents of silver nanoparticle and gold nano are characterized to SERS signal by using transmission electron microscope (TEM)
Influence.As a result show, Nano silver grain diameter about 60nm, golden nanometer particle diameter 40nm, the particle size of two kinds of nano-particles
It is all more uniform.Gold nano colloidal sol is higher to the Raman signal intensity of histamine molecule, 953,992,1262,1106,1262,
1317、1425、1593cm-1It is obvious to locate enhancing effect, illustrates that gold nano substrate can effectively adsorb negative histamine molecule, obtains more
Strong signal effect, follow-up study is using gold nano enhancing substrate.Because the Raman enhancement effect of variety classes material differs greatly,
And different enhancing substrates are different to the enhancement effect of same substance.In studying from now on, it can further inquire into and be suitable for histamine
Nanometer enhancing substrate, to improve the sensitivity of method, improve detection least concentration.
9th, in rice fish histamine SERS testing result qualitative and quantitative analysis
The SERS of all samples carries out the processing such as smooth, baseline correction, is floated with removing noise and baseline
The influence of shifting.Due to the non-grease removal of extract solution so that substantial amounts of miscellaneous peak signal occur in SERS results, deduct Fig. 6 (q) rice fish blank
SERS spectra signal, can substantially identify histamine Molecular Raman characteristic peak at 5 (953,992,1106,1262,1317cm-1), with
Concentration reduces, and the strong signal of spectral peak gradually weakens, and when Histamine concentrations are 1mg/L in the flesh of fish, can still efficiently identify.Cause
This, chooses the foundation that this 5 characteristic peaks differentiate as histamine qualitative, quantitative in the rice flesh of fish, and Histamine concentrations are in 1~150mg/kg
Inside there is preferable identification signal.Histamine maximum level in European Union's regulation fresh fish (non-mackerel) meat<100mg/kg, the method can reach
To the requirement of qualitative and quantitative analysis.Meanwhile extract solution nitrogen after extracting n-butyl alcohol dries up, and can reach the purpose of concentration histamine,
Whole process is simple to operate, does not need the steps such as grease removal, derivative, is only the 1/10 of National Standard Method the time required to pre-treatment.
Due to 1262cm-1It is higher to locate the peak intensity of characteristic peak, and is nearby influenceed without folded peak and miscellaneous peak, from this feature peak
Peak intensity establishes the Quantitative Analysis Model of histamine content in rice fish.Fig. 7 (A) and Fig. 7 (B) is with 1262cm-1It is special to locate histamine SERS
The unitary linear standard curve that peak peak intensity makes with Histamine concentrations is levied, Histamine concentrations are in 1~150mg/kg in the flesh of fish, linearly
Equation is y=27.889x+106.25, coefficient R2=0.9806, linear dependence is good.
10th, model accuracy is verified
Using the SERS data equations established, the real content of histamine in the rice flesh of fish is predicted, and and National Standard Method
Gained actual value is compared.As a result as table 3, the SERS parallel determinations 6 times of each sample, relative standard deviation (RSD) are
2.59~4.72%.Gained predicted value and actual value are basically identical, and the rate of recovery is 87.02~117.25%.Prompting uses SERS
Histamine content in the rice flesh of fish can fast and accurately be detected with reference to DFT.
The predicted value and actual value of 3 meters of histamine in the flesh of fish of table
The foregoing is only the preferable implementation example of the present invention, be not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.
Claims (8)
1. a kind of method of histamine in SERS qualitative and quantitative analysis rice fish tissue, it is characterised in that including with
Lower step：
(1) muddy flesh is made after taking meter fish tissue chopping, weighs several pieces equivalent rice fish tissue as sample, and prepare various concentrations
Histamine standard solution add each sample in, the sample of different time points is taken during storage as mark-on sample and prediction
Sample；
(2) described mark-on sample and prediction sample are added into trichloroacetic acid, extract solution is obtained after filtering；
(3) content of histamine in the rice flesh of fish of HPLC measure prediction samples is utilized；
(4) Raman spectrum data collection is carried out after nanometer strengthener is added in the extract solution of mark-on sample, and it is general using density
Raman characteristic peak corresponding to letter theoretical calculation histamine；
(5) Quantitative Analysis Model is established with the content of the peak intensity of the raman characteristic peak and histamine, and utilizes the quantitative analysis
Model measures Histamine concentrations in meter fish tissue.
2. the method for histamine in SERS qualitative and quantitative analysis rice fish tissue as claimed in claim 1, it is special
Sign is that described histamine standard solution includes：Be respectively 0 with 12% trichloroacetic acid configuration Histamine concentrations, 0.25,1.0,
5.0th, 10.0,15.0,20.0,25.0mg/L titers, for preparing SERS standard curves and HPLC standard curves.
3. the method for histamine in SERS qualitative and quantitative analysis rice fish tissue as claimed in claim 1, it is special
Sign is, in step (2), mark-on sample and prediction sample press 1：10 solid-liquid ratio adds trichloroacetic acid, after ultrasonic extraction
It is centrifuged, filters, and add trichloroacetic acid constant volume, produces extract solution.
4. the method for histamine in SERS qualitative and quantitative analysis rice fish tissue as claimed in claim 3, it is special
Sign is, in step (3), the step of being determined using HPLC is included：
(3.1) grease removal：Isometric n-hexane is added in the extract solution of prediction sample, fully shaking, removes organic phase；
(3.2) extract：After grease removal liquid add NaCl make solution saturation, add NaOH solution, after mixing add n-butanol-
Centrifuged after chloroform mixed solution, fully vibration, take upper organic phase, extract is obtained after extraction, added hydrochloric acid to extract, mix
Conjunction dries up after nitrogen under water-bath, adds dissolving with hydrochloric acid residue, obtains treating derivative solution；
(3.3) it is derivative：Treat that derivative solution adds saturation NaHCO to described3Solution and dansyl Cl derivative solution, add after drying
Enter saturation NaHCO3Solution, nitrogen blows away acetone under 40 DEG C of water-baths, adds ether and extracts, Aspirate supernatant after stratification,
Methanol dissolution residual substance is added after ether extraction liquid is dried up by nitrogen, then is examined by membrane filtration, filtered fluid for HPLC
Survey；
(3.4) HPLC condition determinations：Chromatographic column：Agilent ZORBAX SB-C18；Detector：Photodiode array detection
Device；Mobile phase：A- methanol, B- ultra-pure waters；Flow velocity：0.3mL/min；Detection wavelength：254nm；Sample size：5μL；Column temperature：30℃.
5. the method for histamine in SERS qualitative and quantitative analysis rice fish tissue as claimed in claim 1, it is special
Sign is that described nanometer strengthener includes the silver nanoparticle colloidal sol or gold nano colloidal sol as substrate；
The preparation process of gold nano colloidal sol is：After high chlorauric acid solution is heated into boiling, citric acid three sodium solution is added, simultaneously
Stirring, the color for the aurosol being prepared into is claret；After solution cooling, above-mentioned gold size solution is poured into centrifuge tube, from
Outwell a small amount of supernatant after the heart, then toward ultra-pure water is added in centrifuge tube, mixed with sonic oscillation, obtain gold nano colloidal sol；
The preparation process of silver nanoparticle colloidal sol is：Silver nitrate solution is heated to seethe with excitement, it is 1% that concentration is progressively instilled in 2min
Citric acid three sodium solution, stir simultaneously, now for solution by transparent change light brown, the celadon liquid obtained after reaction is silver nanoparticle
Colloidal sol.
6. the method for histamine in SERS qualitative and quantitative analysis rice fish tissue as claimed in claim 1, it is special
Sign is, in step (4), using B3LYP hydridization functional in DFT, B3LYP is Becke3 shape parameter Density functional models to group
Amine carries out theoretical Raman spectrum calculating, and the Raman signatures spectral peak of histamine can be parsed according to functional group's eigen vibration frequency.
7. the method for histamine in SERS qualitative and quantitative analysis rice fish tissue as claimed in claim 6, it is special
Sign is, in step (4), sequentially adds 150 μ L nanometer strengtheners and 50mL NaCl to each sample, is put into supporting liquid-like
Product pond carries out spectra collection.
8. the method for histamine in SERS qualitative and quantitative analysis rice fish tissue as claimed in claim 7, it is special
Sign is that carrying out smooth, baseline correction to the SERS sampled is handled, and removes noise and baseline drift, with
1262cm-1Locate histamine SERS characteristic peaks peak intensity with the unitary linear standard curve of Histamine concentrations making as quantitative analysis mould
Type, linear equation y=27.889x+106.25, coefficient R2=0.9806.
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