CN113075196A - Method for rapidly detecting biogenic amine in food by surface enhanced Raman scattering - Google Patents
Method for rapidly detecting biogenic amine in food by surface enhanced Raman scattering Download PDFInfo
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- 150000001412 amines Chemical class 0.000 title claims abstract description 41
- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 title claims abstract description 39
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- 238000000034 method Methods 0.000 title claims abstract description 35
- 235000013305 food Nutrition 0.000 title claims abstract description 18
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 claims abstract description 117
- KIDHWZJUCRJVML-UHFFFAOYSA-N putrescine Chemical compound NCCCCN KIDHWZJUCRJVML-UHFFFAOYSA-N 0.000 claims abstract description 98
- 229960001340 histamine Drugs 0.000 claims abstract description 55
- 239000005700 Putrescine Substances 0.000 claims abstract description 48
- 238000001514 detection method Methods 0.000 claims abstract description 44
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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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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a method for rapidly detecting biogenic amine in food by surface enhanced Raman scattering; according to the invention, biogenic amine and o-phthalaldehyde are subjected to chemical derivatization to form a derivative with fluorescence, and after the derivative is compounded with gold nanoparticles, surface enhanced Raman scattering detection is carried out, the gold nanoparticles play a role in enhancing Raman and a role in quenching fluorescence, the Raman scattering intensity of the biogenic amine derivative is greatly improved, and meanwhile, new characteristic peaks appear, and biogenic amine capable of carrying out the reaction comprises histamine and putrescine, but can be respectively detected due to the respective characteristic peaks; the method is applied to detection and analysis of histamine and putrescine in food, and the result accords with the determination method of biogenic amine in GB5009.208-2016 food safety national standard food; the detection limit of histamine and putrescine is 1mg/L, and the method has the characteristics of strong specificity, simple operation, rapidness and the like.
Description
Technical Field
The invention relates to the technical field of chemical analysis and detection, in particular to a method for rapidly detecting biogenic amine in food by surface enhanced Raman scattering.
Background
Biogenic amines are nitrogen-containing low molecular weight organic bases which can be classified into aliphatic amines, aromatic amines and heterocyclic amines according to the difference of chemical structures, and are mainly derived from decarboxylation of free amino acids or amination and transamination of aldehydes and ketones. Trace biogenic amine plays a key role in controlling cell growth, differentiation and gene regulation, but when the biogenic amine content exceeds a threshold value, adverse effects can be caused to human health, for example, when the phenylethylamine exceeds 3mg/kg, migraine can be caused, and when the histamine exceeds 100 mg/kg, serious toxic reaction can be generated, even the life is threatened. Therefore, it is of great significance to establish a rapid, effective and reliable biological detection method. Currently, methods for measuring histamine in foods include biological methods, thin layer chromatography, liquid chromatography, fluorescence methods, spectrophotometry, and the like.
Due to the advantages of high sensitivity, high resolution, capability of providing abundant structural information, no need of high vacuum detection conditions and the like, the Surface Enhanced Raman Scattering (SERS) spectrum can realize qualitative and quantitative detection and interface stress analysis, and is widely applied to the aspects of environmental protection, food monitoring, sample trace analysis and the like. The noble metal nanoparticle sol has the advantages of simple preparation, high SERS activity and the like, and becomes one of the most common and most researched substrates in the SERS enhanced substrate at present. The signal enhancement effect is achieved by preparing different SERS substrate enhancers, the method is a hotspot in current research, SERS detection reports about biogenic amine are less, and the current method is only limited to use of SiO2Study of @ Au @ Ag as a Raman enhancer. The reason may be that the biological amines themselves have weak SERS signals, while enhancers are relatively poorly studied.
Disclosure of Invention
The invention aims to provide a method for rapidly detecting biogenic amine in food by surface enhanced Raman scattering, which comprises the steps of chemically deriving biogenic amine and o-phthalaldehyde to form a derivative with fluorescence, compounding the derivative with gold nanoparticles, and performing surface enhanced Raman scattering detection, wherein the Raman scattering intensity is greatly improved due to the formation of a rigid planar structure with a larger resonance system; the method is applied to detection and analysis of histamine and putrescine in food, and the result accords with the determination method of biogenic amine in GB5009.208-2016 food safety national standard food; the detection limit of histamine and putrescine is 1mg/L, and the method has the characteristics of strong specificity, simple operation, rapidness and the like.
The method comprises the following specific steps:
(1) adding histamine standard solutions and putrescine standard solutions with different concentrations, 100 mu L of 1mol/L NaOH solution and 500 mu L of 5mol/L o-phthalaldehyde solution into a 5mL colorimetric tube with a plug, uniformly mixing, standing for 5min to form a biogenic amine derivative, then adding 100 mu L of gold nano solution, diluting to 5mL by using acetic acid-sodium acetate buffer solution with pH of 4, carrying out vortex mixing for 1-2 min, standing for 5-10 min, carrying out Raman spectrum detection by using a portable Raman spectrometer, drawing a standard curve, and obtaining a regression equation; determining 1632cm according to the molecular structure of biogenic amine and its derivatives, Raman peak position attribution, and linear relationship between biogenic amine concentration and peak area-1The characteristic peak is used as the discrimination basis for detecting putrescine by surface enhanced Raman scattering, 1185cm-1The characteristic peak can be used as the discrimination basis for detecting histamine by surface enhanced Raman scattering;
(2) sample processing
Cheese sample: putting 1-5 g of mashed cheese sample into 5-10 mL of 0.1mol/L HCl, uniformly mixing, putting into a microwave oven, treating for 3min under 500MHz power, completing acidolysis, cooling, transferring into a centrifuge tube, adding 0.5mL of Carrez solution I and 0.5mL of Carrez solution II respectively to remove protein, carrying out vortex mixing for 1min, centrifuging for 5min, taking out supernatant, and testing;
the Carrez solution I is prepared by dissolving 10.6g of potassium hexachloroferricyanide in 100mL of deionized water; carrez II was prepared by adding deionized water to 100mL of 29.9g of zinc acetate and 3mL of acetic acid;
fish and other samples: treating according to GB5009.208-2016 method;
(3) and (3) sample determination: adding 2mL of the solution to be detected in the step (2) into a 5mL colorimetric tube with a plug, and addingAdding 100 mu L of 1mol/L NaOH solution and 500 mu L of 5mol/L o-phthalaldehyde solution, uniformly mixing, and standing for 5min to form the biogenic amine derivative; then adding 100 mu L of gold nano solution, diluting to 5mL by using acetic acid-sodium acetate buffer solution with the pH value of 4, mixing for 1-2 min in a vortex mode, standing for 5-10 min, and then adding gold nano solution at the concentration of 316 cm-1And 941 cm-1And performing surface enhanced Raman scattering detection.
The gold nano solution is prepared as follows: adding 150mL of 2.2mmol/L citric acid solution into a three-neck flask with a condenser, stirring for 15min at 100 ℃, adding 1mL of 25mmol/L chloroauric acid solution, stirring for 10min under reflux, gradually changing the color of the solution from yellow to blue-gray and then to light pink, cooling the solution to 90 ℃, adding 1mL of 60mmol/L citric acid solution and 1mL of 25mmol/L chloroauric acid solution, stirring for 30min under reflux, changing the solution to wine-red, cooling to 90 ℃, adding 1mL of 60mmol/L citric acid solution and 1mL of 25mmol/L chloroauric acid solution, stirring for 30min under reflux, deepening the color, immediately cooling the solution to room temperature in an ice-water bath, and obtaining the gold nano-solution.
The Raman spectrum detection is carried out under the conditions of 785nm exciting light, 500mW laser power and 10s scanning time, and the characteristic peaks of the putrescine derivative are as follows: 348cm-1、617cm-1、812cm-1、1632cm-1Characteristic peaks of histamine derivatives: 326cm-1、434cm-1、1185cm-1、1555cm-1Characteristic peaks shared by histamine and putrescine: 1053cm-1、1118cm-1、1329 cm-1。
The detection limit of the method for histamine and putrescine is 1 mg/L.
The detection limit of biogenic amine in food by the method is lower than 2.5 mg/kg.
The invention has the advantages that:
1. the invention utilizes biogenic amine and o-phthalaldehyde to carry out chemical derivatization to form a derivative with fluorescence, the derivative has a compound with a large resonance choke system rigid plane structure, so that the Raman scattering intensity is greatly improved, and the putrescine and histamine derivatives have respective characteristic peaks and provide basis for detection;
2. the gold nanoparticles synthesized by the method play double roles, and firstly, the gold nanoparticles are used as a Raman reinforcing agent to reinforce Raman spectrum signals and are also used as a fluorescence quenching agent to quench the fluorescence of the biogenic amine fluorescence derivative, so that Raman detection signals are improved, and background interference is reduced;
3. the novel biological histamine SERS detection method established by the invention can simultaneously detect putrescine and histamine, other biological amines do not have the reaction, the detection sensitivity is high, and the limit of detection of the histamine and the putrescine is 1 mg/L;
4. compared with the method of pre-column derivation combined with HPLC adopted by the national standard, the method has the characteristics of high sensitivity, strong specificity, simple and rapid operation and the like.
Drawings
FIG. 1 is a SERS plot of histamine and putrescine and derivatives of example 1, in which: 1. Au-NPs + o-phthalaldehyde, 2, Au-NPs + histamine and putrescine (1: 5) histamine derivatives, 3, Au-NPs + histamine derivatives, 4, Au-NPs + putrescine histamine derivatives;
FIG. 2 is a graph of fluorescence quenching of histamine derivatives by Au-NPs, in which: 1. histamine + Au-NPs phthalaldehyde fluorescence spectrum, 2, histamine + phthalaldehyde;
FIG. 3 is a Raman spectrum of putrescine in Raman spectrum detection, wherein 1-5 are concentration 1.0mg/L, 10mg/L, 20mg/L, 25mg/L and 30mg/L respectively;
FIG. 4 is a Raman spectrum detection standard curve of putrescine;
FIG. 5 is a Raman spectrum of histamine detection by Raman spectroscopy, wherein 1-6 are concentrations of 1.0mg/L, 2.5mg/L, 5mg/L, 10mg/L, 15mg/L and 20mg/L, respectively;
FIG. 6 is a Raman spectrum standard curve of histamine determination.
Detailed Description
The technical solutions of the present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto.
Example 1: determination of biogenic amines in cheese samples
(1) Preparation of gold nanoparticles
Adding 150mL of 2.2mmol/L citric acid solution into a three-neck flask with a condenser, stirring for 15min at 100 ℃, adding 1mL of 25mmol/L chloroauric acid solution, stirring for 10min under reflux, gradually changing the color of the solution from yellow to blue-gray and then to light pink, cooling the solution to 90 ℃, adding 1mL of 60mmol/L citric acid solution and 1mL of 25mmol/L chloroauric acid solution, stirring for 30min under reflux, changing the solution to wine-red, cooling to 90 ℃, adding 1mL of 60mmol/L citric acid solution and 1mL of 25mmol/L chloroauric acid solution, stirring for 30min under reflux, deepening the color, immediately cooling the solution to room temperature in an ice-water bath to obtain a gold nano solution;
(2) SERS detection of histamine and putrescine standard solutions
Adding a putrescine standard solution, a histamine standard solution, 100 mu L of a 1mol/L NaOH solution and 500 mu L of a 5mol/L o-phthalaldehyde solution into a 5mL colorimetric tube with a plug, uniformly mixing, and standing for 5min to form a biogenic amine derivative; adding 100 mu L of gold nano solution, diluting to 5mL with acetic acid-sodium acetate buffer solution with pH4, wherein the putrescine concentration is 1.0mg/L, 10mg/L, 20mg/L, 25mg/L and 30mg/L, and the histamine concentration is 1.0mg/L, 2.5mg/L, 5mg/L, 10mg/L, 15mg/L and 20 mg/L; vortex mixing for 1min, standing for 5min, scanning for 10s under 785nm exciting light and laser power of 500mW, performing Raman spectrum detection on the liquid to be detected by using a portable Raman instrument, and drawing a standard curve to obtain a regression equation; determining 1632cm according to the molecular structure of biogenic amine and its derivatives, Raman peak position attribution, and linear relationship between biogenic amine concentration and peak area-1The characteristic peak is the discrimination basis of histamine detection by putrescine, 1185cm-1The characteristic peak is used as the discrimination basis for detecting histamine by SERS;
determination of the detection wave number of the SERS spectrum of histamine and putrescine and SERS analysis of a standard substance: as shown in fig. 1, the characteristic peaks of putrescine derivatives: 348cm-1、617cm-1、812cm-1、1632cm-1Characteristic peaks of histamine derivatives: 326cm-1、434cm-1、1185cm-1、1555cm-1Characteristic peaks shared by histamine and putrescine: 1053cm-1、1118cm-1、1329 cm-1;
FIG. 2 shows that the addition of gold nanosoluts to histamine fluorescence derivatization results in significant quenching of fluorescence; according to the molecular structure and Raman peak position assignment of biogenic amine and derivatives thereof, andthe linear relation between the concentration of the substance amine and the peak area is determined to be 1632cm-1The characteristic peak is the discrimination basis of putrescine detection, 1185cm-1The characteristic peak can be used as a discrimination basis for detecting histamine by SERS, the linear regression equation is shown in table 1 and figures 3-6, and the obvious Raman spectrum peak still exists when the concentration of histamine and putrescine is 1 mg/L. Therefore, the detection concentration of the standard solution of histamine and putrescine in the method reaches 1 mg/L;
(3) preparation of Carrez solution I: 10.6g of potassium hexachloroferricyanide are dissolved in 100mL of deionized water; preparation of Carrez II solution: adding deionized water to 100mL of 29.9g of zinc acetate and 3mL of acetic acid to obtain the zinc acetate-containing aqueous solution;
(4) pretreatment of cheese samples: putting a mashed 3g cheese sample into 8mL of 0.1mol/L HCl, uniformly mixing, putting into a microwave oven, treating for 3min under 500MHz power, completing acidolysis, cooling, transferring into a centrifuge tube, adding 0.5mL of Carrez solution I and 0.5mL of Carrez solution II respectively, removing protein, carrying out vortex mixing for 1min, centrifuging for 5min at 4000r/min, taking out a supernatant, and testing;
(5) sample detection, standard recovery rate and precision test
Adding 2mL of the solution to be detected in the step (4) into a 5mL colorimetric tube with a plug, adding 100 mu L of 1mol/L NaOH solution and 500 mu L of 5mol/L o-phthalaldehyde solution, uniformly mixing, and standing for 5min to form the biogenic amine derivative; then adding 100 μ L gold nanometer solution, diluting to 5mL with acetic acid-sodium acetate buffer solution with pH of 4, vortex mixing for 1min, standing for 5min, and standing at 316 cm-1And 941 cm-1Performing surface enhanced Raman scattering detection;
performing blank surface enhanced Raman scattering detection on the treated cheese sample liquid, wherein the putrescine derivative is 1632cm-1Has a characteristic peak and a histamine derivative at 1185cm-1The characteristic peak is not changed, but the matrix interference reduces the spectrum identification of the SRES of the histamine and the putrescine, the content of the histamine in the sample is 5.5mg/kg, and the putrescine is not detected; through a standard recovery rate test, namely adding 2.5mg/L of histamine and putrescine standard solution during extraction of biogenic amine in a blank sample solution to be detected, performing SERS detection, and still obviously identifying, wherein the detection limit of the detection method on histamine and putrescine in cheese is 2.5 mg/kg;
TABLE 1 Linear equation, correlation coefficient, relative standard deviation, Linear Range
(6) Method specificity investigation: the method detects the specificity of the system of the method by coexisting histamine, putrescine and other biogenic amines (spermine, spermidine, tyramine, phenylenediamine, octopamine, tryptamine, cadaverine, butanediamine, putrescine, 5-hydroxytryptamine and o-phthalaldehyde), wherein the concentration of the histamine and the putrescine in the system is 1mg/kg, the concentration of an interference substance is 10mg/kg, and the result shows that only the histamine and the putrescine have obvious sensitization effect, other substances hardly have sensitization effect, and the method has good selection specificity.
TABLE 2 sample histamine and putrescine normalized recovery and RSD (n = 3)
"-" was not detected.
Example 2: determination of histamine in squid sample
(1) Preparing a gold nano solution: the same as example 1;
(2) SERS detection of a histamine and putrescine standard solution and SERS analysis of a standard product: the same as example 1;
(3) accurately weighing 10g (accurate to 0.01 g) of a squid sample after homogenization, placing the squid sample into a 100mL conical flask with a plug, adding 20mL of 5% trichloroacetic acid solution, oscillating and extracting for 30min, transferring the squid sample into a 50mL centrifugal tube with a plug, centrifuging at 5000r/min for 10min, transferring supernatant into a 50mL volumetric flask, extracting residues once again by using 20mL of 5% trichloroacetic acid solution, combining the supernatant, diluting the residues to a scale by using 5% trichloroacetic acid to obtain an extracting solution; transferring 10mL of extracting solution into a 25mL test tube with a plug, adding 0.5 g of sodium chloride, performing vortex oscillation until the sodium chloride is completely dissolved, adding 10mL of n-hexane, performing vortex oscillation for 5min, standing for layering, removing an upper-layer organic phase, adding 10mL of n-hexane into a lower-layer sample solution, and performing grease removal once again to obtain a solution to be detected;
(4) measuring the content of histamine and putrescine in the squid sample: in the same manner as in example 1, the histamine content of the sample was 6.1mg/kg, and putrescine was not detected.
Example 3: determination of histamine in hairtail
(1) Preparing gold nano-particles: the same as example 1;
(2) SERS detection of a histamine and putrescine standard solution, determination of SERS spectrum detection wave number of the histamine and putrescine and SERS analysis of the standard: the same as example 1;
(3) pretreatment of hairtail samples: same as example 2, step (3);
(5) determination of histamine and putrescine content in hairtail samples: in the same manner as in example 1, histamine was not detected and putrescine was not detected.
Example 4: determination of histamine content of sausage sample
(1) Preparing gold nano-particles: the same as example 1;
(2) SERS detection of a histamine and putrescine standard solution, determination of SERS spectrum detection wave number of the histamine and putrescine and SERS analysis of the standard: the same as example 1;
(3) pretreatment of sausage samples: the same as example 2;
(5) measuring the content of histamine and putrescine in the sausage sample: as in example 1, the sample had a histamine content of 5.3mg/kg and a putrescine content of 8.1 mg/kg.
The results of comparison tests of examples 1-4 by the method of GB5009.208-2016 are shown in Table 3, the relative error is within +/-5, and the method has reliability.
TABLE 3 test results of the inventive method and HPLC method
Claims (4)
1. A method for rapidly detecting biogenic amine in food by surface enhanced Raman scattering is characterized by comprising the following steps:
(1) adding histamine standard solution and putrescine standard solution with different concentrations, 100 mu L of 1mol/L NaOH solution and 5mol/L o-phthalaldehyde solution into a 5mL colorimetric tube with a plug500 mu L, standing for 5min after uniform mixing to form a biogenic amine derivative, then adding 100 mu L of gold nano solution, diluting to 5mL by using acetic acid-sodium acetate buffer solution with the pH of 4, carrying out vortex mixing for 1-2 min, standing for 5-10 min, carrying out Raman spectrum detection by using a portable Raman instrument, drawing a standard curve, and obtaining a regression equation; determining 1632cm according to the molecular structure of biogenic amine and its derivatives, Raman peak position attribution, and linear relationship between biogenic amine concentration and peak area-1The characteristic peak is used as the discrimination basis for detecting putrescine by surface enhanced Raman scattering, 1185cm-1The characteristic peak is used as the discrimination basis for detecting histamine by surface enhanced Raman scattering;
(2) sample processing
Cheese sample: putting 1-5 g of mashed cheese sample into 5-10 mL of 0.1mol/L HCl, uniformly mixing, putting into a microwave oven, treating for 3min under 500MHz power, completing acidolysis, cooling, transferring into a centrifuge tube, adding 0.5mL of Carrez solution I and 0.5mL of Carrez solution II respectively to remove protein, carrying out vortex mixing for 1min, centrifuging for 5min, taking out supernatant, and testing;
fish and other samples: treating according to GB5009.208-2016 method;
(3) and (3) sample determination: adding 2mL of the solution to be detected in the step (2) into a 5mL colorimetric tube with a plug, adding 100 mu L of 1mol/L NaOH solution and 500 mu L of 5mol/L o-phthalaldehyde solution, uniformly mixing, and standing for 5min to form the biogenic amine derivative; then adding 100 mu L of gold nano solution, diluting to 5mL by using acetic acid-sodium acetate buffer solution with the pH value of 4, mixing for 1-2 min in a vortex mode, standing for 5-10 min, and then adding gold nano solution at the concentration of 316 cm-1And 941 cm-1And performing surface enhanced Raman scattering detection.
2. The method for rapidly detecting biogenic amine in food by surface enhanced Raman scattering according to claim 1, wherein the gold nano solution is prepared by the following steps: adding 150mL of 2.2mmol/L citric acid solution into a three-neck flask with a condenser, stirring for 15min at 100 ℃, adding 1mL of 25mmol/L chloroauric acid solution, stirring for 10min under reflux, gradually changing the color of the solution from yellow to blue-gray and then to light pink, cooling the solution to 90 ℃, adding 1mL of 60mmol/L citric acid solution and 1mL of 25mmol/L chloroauric acid solution, stirring for 30min under reflux, changing the solution to wine-red, cooling to 90 ℃, adding 1mL of 60mmol/L citric acid solution and 1mL of 25mmol/L chloroauric acid solution, stirring for 30min under reflux, deepening the color, immediately cooling the solution to room temperature in an ice-water bath, and obtaining the gold nano-solution.
3. The method for rapidly detecting biogenic amines in food by surface enhanced Raman scattering according to claim 1, wherein: carrez solution I is prepared by dissolving 10.6g of potassium hexachloroferricyanide in 100mL of deionized water; carrez II was prepared by adding deionized water to 100mL of 29.9g of zinc acetate, 3mL of acetic acid.
4. The method for rapidly detecting biogenic amines in food by surface enhanced Raman scattering according to claim 1, wherein: the Raman spectrum detection was performed under conditions of 785nm excitation light, a laser power of 500mW, and a scanning time of 10 seconds.
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