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Enzyme-linked immunoassay kit for detecting coumarin and application thereof

An enzyme-linked immunosorbent reagent and coumarin technology, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of complex and expensive instruments and equipment, tedious and time-consuming processing, and high detection costs, and achieve simple pretreatment process and fast detection speed , the effect of low detection cost

Active Publication Date: 2018-12-07
ZHENGZHOU TOBACCO RES INST OF CNTC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the instrumental analysis method can achieve accurate qualitative and quantitative analysis, it requires complex and expensive instruments and professional operators. In addition, the sample pretreatment is cumbersome and time-consuming, the detection time is long, and the detection cost is high, which has limitations for the development of on-site detection work.

Method used

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  • Enzyme-linked immunoassay kit for detecting coumarin and application thereof
  • Enzyme-linked immunoassay kit for detecting coumarin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of Detecting Coumarin Kit Reagent

[0033] 1. Synthesis and identification of coumarin hapten

[0034] Take 1.00 g of coumarin, add 80 mL of chlorobenzene and stir to dissolve, add 2.50 g of tert-butanol hydroperoxide, stir and mix well, add 3-(1,3-dioxa-2-yl) propanal 0.89 g, stirred at 100°C for 24 h. After stopping the reaction, add 100 mL of cold water, extract 3 times with 100 mL of ethyl acetate, wash with 50 mL of water, dry the organic phase with anhydrous sodium sulfate and evaporate to dryness, and 60 mL of petroleum ether / dichloromethane (v / v, 1 / 1 ) recrystallized to obtain 1.70 g of acetal coumarin compound with a yield of 94.00%.

[0035]Take 1.70 g of acetal coumarin, add 20 mL of acetonitrile to dissolve, add 10 mL of 1 mol / L dilute hydrochloric acid, stir vigorously at room temperature for 4 hours, stop the reaction, rotary evaporate, remove acetonitrile, add 50 mL of water, add 1 mol / L NaOH to adjust When the pH value reaches 6,...

Embodiment 2

[0063] Example 2 The formation of the enzyme-linked immunosorbent assay kit for detecting coumarin

[0064] Set up the ELISA kit for detecting coumarin, so that it includes the following components:

[0065] (1) A microtiter plate coated with coumarin-conjugated antigen;

[0066] (2) 6 bottles of coumarin standard solution, the concentrations are 0 μg / L, 2.5 μg / L, 5 μg / L, 10 μg / L, 25 μg / L, 50 μg / L;

[0067] (3) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0068] (4) Coumarin-specific antibody working solution;

[0069] (5) The substrate chromogenic solution is composed of A liquid and B liquid, A liquid is carbamide peroxide, and B liquid is tetramethylbenzidine;

[0070] (6) The stop solution is 2 mol / L sulfuric acid;

[0071] (7) The washing solution has a pH value of 7.4, contains 0.5% to 1.0% Tween-20, 0.01‰ to 0.03‰ sodium azide preservative, and 0.1 to 0.3 mol / L phosphate buffer, and the percentage is weight volume percentage;

[0072] (8) T...

Embodiment 3

[0073] Example 3 Detection of coumarin in tobacco

[0074] 1. Sample pretreatment

[0075] Weigh 1.0±0.05 g of crushed tobacco leaf sample into a polystyrene centrifuge tube; add 10 mL of 50% methanol solution, and use a homogenizer to crush it sufficiently to obtain a sample liquid; pipette 50 μL of sample liquid and 450 μL of The ionized water was mixed and waited for inspection.

[0076] 2. Detection with kit

[0077] Add 50 μL / well of coumarin standard solution or pre-treated sample solution to the microwells of the enzyme plate coated with coumarin-conjugated antigen, and then add 50 μL / well of coumarin-specific antibody working solution, Gently oscillate to mix well, cover the plate with a cover film and place it in a light-proof environment at 25°C for 30 minutes of reaction; carefully peel off the cover film, dry the liquid in the well, wash thoroughly with washing solution 250 μL / well for 4- 5 times, each interval of 10 s, pat dry with absorbent paper; add horserad...

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Abstract

The invention provides an enzyme-linked immunoassay kit for detecting coumarin and an application thereof. The kit comprises an enzyme-labeled plate coated with a coating antigen, a coumarin standardsolution, an enzyme-labeled secondary antibody, a coumarin-specific antibody, a substrate color developing solution, a stop solution, a washing solution and a complex solution. The coating antigen isa coumarin-coupled antigen. The enzyme-labeled secondary antibody is an enzyme-labeled goat anti-mouse anti-antibody. The coumarin-specific antibody is prepared through a coumarin-conjugated antigen as an immunogen. The coumarin-conjugated antigen is obtained through coupling a coumarin hapten to a carrier protein. The coumarin hapten is obtained through acetalization of coumarin. The enzyme-linked immunoassay kit can be used for detecting the content of coumarin in tobacco and tobacco flavors and fragrances, is easy to operate, has a low cost and high sensitivity and realizes on-site monitoring and screening of a large amount of samples.

Description

technical field [0001] The invention relates to enzyme-linked immunosorbent detection technology, in particular to an enzyme-linked immunosorbent assay kit for detecting coumarin and its application, which can qualitatively and quantitatively detect the residual amount of coumarin in tobacco, tobacco products, and tobacco flavors and fragrances . technical background [0002] Coumarin-based compounds are a class of natural products with an aromatic odor and a benzoα-pyrone structure as the core. They are widely distributed in the plant kingdom and can be divided into simple coumarin-based compounds and furanocoumarin-based compounds according to their structures. Compounds, pyranocoumarin-based compounds and other coumarin-based compounds, etc. Coumarin, also known as o-hydroxycinnamic acid lactone and 1,2-benzopyrone, is a natural plant secondary product and a lactone compound widely present in nature. It was discovered in 1820 Found in Tonka Bean, mainly exists in plants...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535G01N33/537G01N33/577
CPCG01N33/535G01N33/537G01N33/577
Inventor 陈黎刘惠民何方洋崔海峰赵乐崔华鹏樊美娟蔡君兰崔廷婷贾云祯
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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