阅读说明：本技术 一种基于pei的赭曲霉毒素a的快速检测方法 (PEI-based rapid detection method for ochratoxin A ) 是由 李胎花 黄磊 宫磊 胡润涛 于 2019-09-20 设计创作，主要内容包括：本发明公开了一种基于PEI的赭曲霉毒素A(OTA)的快速检测方法,属于赭曲霉毒素A检测领域。该方法包括以下步骤：在含0.05％PEI的MES缓冲液中,加入不同标准浓度的OTA,以OTA浓度为横坐标,以相对荧光值为纵坐标作标准曲线,计算回归方程及相关系数；将待测样品加到含PEI的MES缓冲液中,将测得的相对荧光值代入回归方程中,得到待测样品中的赭曲霉毒素A浓度。MES缓冲液的浓度为20mmol/L,pH为6。本发明基于表面活性剂PEI对OTA荧光光谱的增强作用,对OTA进行快速检测,操作步骤简单,材料廉价且易于获取；抗干扰性强,特异性好,灵敏度高,OTA浓度的检测下限为0.056nmol/L。(The invention discloses a PEI (polyetherimide) -based rapid detection method for ochratoxin A (OTA), and belongs to the field of ochratoxin A detection. The method comprises the following steps: adding OTA with different standard concentrations into MES buffer solution containing 0.05% PEI, taking the concentration of the OTA as an abscissa and taking a relative fluorescence value as an ordinate to take a standard curve, and calculating a regression equation and a correlation coefficient; and adding the sample to be detected into a MES buffer solution containing PEI, and substituting the measured relative fluorescence value into a regression equation to obtain the concentration of ochratoxin A in the sample to be detected. The MES buffer concentration was 20mmol/L and the pH was 6. The method is based on the enhancement effect of the PEI (polyetherimide) surfactant on the OTA fluorescence spectrum, the OTA is rapidly detected, the operation steps are simple, the material is cheap and is easy to obtain; strong anti-interference performance, good specificity and high sensitivity, and the detection lower limit of the OTA concentration is 0.056 nmol/L.)

1. A rapid detection method of ochratoxin A based on PEI is characterized by comprising the following steps:

1) adding ochratoxin A with different standard concentrations into a MES buffer solution containing PEI, taking the concentration of the ochratoxin A as an abscissa and taking a relative fluorescence value as an ordinate to take a standard curve, and calculating a regression equation and a correlation coefficient;

2) and adding the sample to be detected into a MES buffer solution containing PEI, and substituting the measured relative fluorescence value into a regression equation to obtain the concentration of ochratoxin A in the sample to be detected.

2. The rapid detection method of PEI based ochratoxin a according to claim 1, wherein in step 1), the concentration of PEI contained in MES buffer is 0.001% -0.1%.

3. The rapid detection method of PEI based ochratoxin a according to claim 2, wherein in step 1), the concentration of PEI in MES buffer is 0.05%.

4. The rapid detection method of PEI based ochratoxin a according to claim 2, wherein in step 1), MES buffer has a concentration of 20mmol/L and pH of 6.

5. The method for rapidly detecting ochratoxin A based on PEI as claimed in claim 1, wherein in the step 1), the relative fluorescence value is the difference between the fluorescence value of ochratoxin A detected at the emission wavelength of 450nm and the excitation wavelength of 390nm and the fluorescence value of ochratoxin A without the ochratoxin A.

6. The rapid detection method of PEI based ochratoxin a according to claim 1, wherein in step 2), the regression equation is 1.2809x + 1.3856.

7. The rapid detection method of ochratoxin A based on PEI as claimed in claim 1, wherein the detection interval of the concentration of ochratoxin A is 0-100 nmol/L.

8. The rapid detection method of ochratoxin A based on PEI as claimed in claim 1, wherein the lower limit of detection of ochratoxin A concentration is 0.056 nmol/L.

9. Use of a method according to any one of claims 1 to 8 for the detection of ochratoxin a.

Technical Field

The invention belongs to the field of ochratoxin A detection, and particularly relates to a PEI-based rapid detection method for ochratoxin A.

Background

Ochratoxin (ochratoxin) is a secondary metabolite produced by some species of aspergillus and penicillium, and belongs to mycotoxins, and comprises seven similar compounds, namely ochratoxin A (OTA), ochratoxin B (OTB), ochratoxin C (OTC) and the like. OTA is one of ochratoxins which has the strongest toxicity, the widest distribution and the greatest harm to human bodies, animals and plants, in animal husbandry, animals eating feed polluted by OTA can cause the body damage of livestock and poultry, the productivity is influenced, and health problems can be caused when people eat food polluted by OTA by mistake. OTA has carcinogenicity, teratogenicity, hepatotoxicity, nephrotoxicity and genotoxicity, and at the same time has serious damage to the nervous and immune systems of the human body.

OTA is classified as a possible human carcinogen by the International agency for research on cancer (IARC), and health organizations around the world make detailed regulations on the amount of OTA ingested by humans. Due to the extreme toxicity of OTA, it is important to determine the content of OTA in food products.

Several methods for quantitative analysis and detection of OTA have been established at present: including Thin Layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC), ultraviolet-visible spectroscopy (UV-Vis), Mass Spectrometry (MS), or immunoassay, etc. However, most of these methods are expensive in equipment, high in requirements for operators, simple in operation, low in price, but insufficient in sensitivity.

OTAs themselves have optical properties of ultraviolet/visible absorption, fluorescence, quantum yield, and Stokes shift. The position and intensity of the fluorescence spectrum of the same OTA in different solvents can be obviously different, if the solvents and the fluorescent substances form a complex or the solvents change the ionization state of the fluorescent substances, the fluorescence spectrum also changes, and when the surfactant is added into the solution of the fluorescent substances, the fluorescence spectrum of the fluorescent substances is enhanced based on the solubilization principle, deprotonation and other effects of the surfactant, so that the sensitization effect is generated on the fluorescence spectrum of the fluorescent substances.

Surfactants have been widely used in various fields, but they have been studied little in the field of analytical detection of substances, particularly in the field of analytical detection of OTA.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to solve the technical problem of providing a PEI-based rapid detection method for ochratoxin A, which comprises the steps of adding a sample to be detected into 20mmol/L of PEI-containing pH6MES buffer solution, substituting the measured relative fluorescence value into a regression equation, and obtaining the concentration of the ochratoxin A in the sample to be detected. The method has the advantages of simple operation steps, cheap and easily-obtained materials, and capability of quickly and accurately detecting the concentration of the OTA; the anti-interference performance is strong when detecting OTA, and the specificity and the sensitivity are high.

In order to solve the technical problems, the invention adopts the technical scheme that:

a rapid detection method of ochratoxin A based on PEI comprises the following steps:

1) adding ochratoxin A with different standard concentrations into a MES buffer solution containing PEI, taking the concentration of the ochratoxin A as an abscissa and taking a relative fluorescence value as an ordinate to take a standard curve, and calculating a regression equation and a correlation coefficient;

2) and adding the sample to be detected into a MES buffer solution containing PEI, and substituting the measured relative fluorescence value into a regression equation to obtain the concentration of ochratoxin A in the sample to be detected.

In the step 1), the concentration of PEI in the MES buffer solution is 0.001% -0.1%.

In the step 1), the concentration of PEI in the MES buffer solution is 0.05%.

In step 1), the concentration of MES buffer was 20mmol/L and the pH was 6.

In the step 1), the relative fluorescence value is the difference between the fluorescence value of ochratoxin A measured at the emission wavelength of 450nm and the excitation wavelength of 390nm and the fluorescence value without ochratoxin A.

In step 2), the regression equation is 1.2809x + 1.3856.

Furthermore, the detection interval of the ochratoxin A concentration is 0-100 nmol/L.

Further, the lower limit of detection of the ochratoxin A concentration is 0.056 nmol/L.

The application of the method in detection of ochratoxin A.

Has the advantages that: compared with the prior art, the invention has the following technical advantages:

1) the method is based on the enhancement effect of the PEI (polyetherimide) surfactant on the OTA fluorescence spectrum, can be used for rapidly detecting the OTA, and has the advantages of simple operation steps, cheap and easily-obtained materials, rapidness, accuracy and the like; expensive equipment is not needed, and the requirement on operators is not high;

2) the invention has strong anti-interference performance and high specificity when detecting OTA;

3) the lower limit of OTA detection is 0.056nmol/L, and the sensitivity is high.

Drawings

FIG. 1 is a graph of the fluorescence spectra of different concentrations of surfactant with 100nmol/L OTA at pH6, pH7.4, pH 9; in the figure, A: no surfactant; b: 0.005% PEI; c: 0.005% CTAB; d: 0.01% PDDA; the fixed emission wavelength is 450nm, and the slit width is 6 multiplied by 6 nm;

FIG. 2 is a fluorescence spectrum of different concentrations of PEI with 1. mu. mol/L OTA at pH 6; the fixed excitation wavelength was 390 nm; the width of the slit is 6 multiplied by 6 nm;

FIG. 3 is pH 6; a standard curve graph of the concentration and fluorescence intensity of OTA in 0.05% PEI in 20mmol/L MES buffer;

FIG. 4 is a graph showing the specificity of 100nmol/L OTA in MES buffer at pH620 mmol/L; the fixed emission wavelength is 450 nm; the width of the slit is 6 multiplied by 6 nm;

FIG. 5 is a fluorescence spectrum of the labeling experiment of different concentrations of OTA on barley corn flour; the fixed excitation wavelength was 390 nm; the slit width was 6X 6 nm.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to be limiting.