Fluorescein marker and organic phosphorus pesticide homogeneous phase multi-residue immune detection method based on fluorescence polarization

An immunological detection method and organophosphorus pesticide technology, which is applied in the field of food safety detection, can solve problems such as complex and time-consuming operations, and achieve simple pre-treatment, convenient operation, and good results

Inactive Publication Date: 2011-10-05
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to provide a fluorescent marker, which can be applied...

Method used

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  • Fluorescein marker and organic phosphorus pesticide homogeneous phase multi-residue immune detection method based on fluorescence polarization
  • Fluorescein marker and organic phosphorus pesticide homogeneous phase multi-residue immune detection method based on fluorescence polarization
  • Fluorescein marker and organic phosphorus pesticide homogeneous phase multi-residue immune detection method based on fluorescence polarization

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Preparation method of fluorescent marker

[0044] The preparation method of the fluorescent marker comprises the following steps:

[0045] Step (1): Preparation of ethylenediamine-derivatized fluorescein isothiocyanate (EDF-FITC)

[0046] EDF-FTIC was synthesized by optimizing the conditions referring to literature (Nistor et al. Anal. Chim. Acta, 2001, 426, 185–195). Specifically, 20 mg (0.3 mmol) of ethylenediamine and 11.7 mg (0.03 mmol) of fluorescein isothiocyanate (FITC) were dissolved in 5 mL and 1 mL of methanol (containing 1% v / v of triethylamine) solution, respectively. middle. The FITC solution was added dropwise to the ethylenediamine solution, and stirred for 1 hour at room temperature in the dark. After concentrating the reaction solution by rotary evaporation, powdered ethylenediamine-derivatized fluorescein isothiocyanate (EDF-FITC) was obtained.

[0047] Step (2): Activation of organophosphorus pesticide hapten

[0048]10 mg (40 mmol) org...

Embodiment 2

[0053] Example 2 Sample pretreatment

[0054] Before the test, the sample needs to be processed briefly, and the pre-treatment methods are as follows:

[0055] (1) Environmental water samples: If there are impurities, filter them with a microporous membrane with a pore size of 0.45 μm to remove particulate impurities. If there is no impurity, it can be directly used for detection.

[0056] (2) Fruit and vegetable samples: take 15mL centrifuge tubes respectively, add 2 g of chopped fruit or vegetable samples to each tube, add 5 mL of anhydrous methanol, shake and extract for 10 min, centrifuge at room temperature at 5000 rpm / min for 10 min, and absorb the upper layer The clarified solution was diluted with borate buffer for detection.

Embodiment 3

[0057] Embodiment 3 Drawing of standard curve

[0058] Borate buffer (50 mmol / L, pH 8.5) was used as a diluent for all samples. Add 90 μL of 15 nmol / L fluorescent marker, 20 μL of serial concentrations of organophosphorus pesticide standards and 90 μL of 200-fold diluted organophosphorus pesticide multispecific antibody into a 96-well fluorescent microwell plate, and pass the fluorescence polarization after 10 mins The immunoanalyzer measures the fluorescence polarization value (mP). Take the mP value as the ordinate, and the logarithmic value of the standard organophosphorus pesticide concentration as the abscissa, and use the four-parameter logarithmic function of originPro 7.5 software for curve fitting:

[0059] (1)

[0060] Among them, A and D represent the fluorescence polarization value (mP) at the minimum and maximum drug concentration respectively, and C is the midpoint concentration. When the concentration of the standard substance is equal to C, the fluorescen...

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Abstract

The invention discloses a fluorescein marker and an organic phosphorus pesticide homogeneous phase multi-residue immune detection method based on fluorescence polarization. In the invention, the fluorescence polarization value to be detected can be generated by mixing a sample to be detected, a fluorescence tracer and an organic phosphorus pesticide multi-specificity antibody and combining the fluorescence tracer and the organic phosphorus pesticide multi-specificity antibody, the fluorescence polarization value of the mixture is measured and a standard curve is established through a series of organic phosphorus pesticide standards with known concentrations, and the concentration of the organic phosphorus pesticide in the sample to be detected is calculated by utilizing the standard curve. The method disclosed by the invention has the advantages of easy pre-treatment of the sample, no need for washing separation in the reaction process, detection time of less than 3-5min, convenience for operation, capability of simultaneously detecting 20 organic phosphorus pesticides, sensitivity between 0.003-1.0mg/kg, and capability of simultaneously detecting 42 samples by using 96 micro-plates (each sample is paralleled twice), and is a fast, simple and convenient organic phosphorus pesticide high flux detection method with high sensitivity.

Description

technical field [0001] The invention belongs to the technical field of food safety detection, and relates to a fluorescein marker and a homogeneous multi-residue immunological detection method for organophosphorus pesticides based on fluorescence polarization. Background technique [0002] Organophosphorus pesticides (organophosphorus pesticides) are a class of broad-spectrum pesticides. After the use of organochlorine pesticides was banned, organophosphorus pesticides became the most widely used pesticides in my country, accounting for about 70% of the total amount of pesticides. Due to the potential toxicity of organophosphorus pesticides to humans and animals, its large-scale use has caused the residues of vegetables, food and environmental water in my country to seriously exceed the standard, which not only directly poisons the human body, but also pollutes the ecological environment. Food poisoning accidents caused by excessive pesticide residues occur frequently. There...

Claims

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Application Information

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IPC IPC(8): C09K11/06C07F9/18C07F9/17G01N33/577G01N21/21
Inventor 孙远明徐振林雷红涛王强沈玉栋杨金易王弘
Owner SOUTH CHINA AGRI UNIV
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