Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A kind of ELISA kit for detecting coumarin and its application

An enzyme-linked immunosorbent reagent and coumarin technology, 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, high detection costs, etc., to achieve efficient detection methods, fast detection speed, The effect of low detection cost

Active Publication Date: 2021-03-12
ZHENGZHOU TOBACCO RES INST OF CNTC +1
View PDF4 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of ELISA kit for detecting coumarin and its application
  • A kind of ELISA kit for detecting coumarin and its application
  • A kind of ELISA kit for detecting coumarin and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of Detecting Coumarin Kit Reagent

[0034] 1. Synthesis and identification of coumarin hapten

[0035] 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%.

[0036]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

[0064] Example 2 The formation of the enzyme-linked immunosorbent assay kit for detecting coumarin

[0065] Set up the ELISA kit for detecting coumarin, so that it includes the following components:

[0066] (1) A microtiter plate coated with coumarin-conjugated antigen;

[0067] (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;

[0068] (3) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0069] (4) Coumarin-specific antibody working solution;

[0070] (5) The substrate chromogenic solution is composed of A liquid and B liquid, A liquid is carbamide peroxide, and B liquid is tetramethylbenzidine;

[0071] (6) The stop solution is 2 mol / L sulfuric acid;

[0072] (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;

[0073] (8) T...

Embodiment 3

[0074] Example 3 Detection of coumarin in tobacco

[0075] 1. Sample pretreatment

[0076] 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.

[0077] 2. Detection with kit

[0078] Add 50 μL / well of coumarin standard solution or pretreated sample solution to the microwells of the microplate 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 dark 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, with an interval of 10 s each time, pat dry with absorbent paper; add hors...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides an ELISA kit for detecting coumarin and its application. The kit includes: ELISA plate coated with the original coating, coumarin standard solution, enzyme-labeled secondary antibody, coumarin-specific antibody, substrate chromogenic solution, stop solution, washing solution, reconstitution solution; The coating is originally a coumarin-coupled antigen, the enzyme-labeled secondary antibody is an enzyme-labeled goat anti-mouse anti-antibody, and the coumarin-specific antibody is prepared by using a coumarin-coupled antigen as an immunogen ; The coumarin-coupled antigen is obtained by coupling a coumarin hapten with a carrier protein, and the coumarin hapten is obtained by acetalization of coumarin. The enzyme-linked immunosorbent assay kit provided by the invention can be used to detect the content of coumarin in tobacco and tobacco flavors and fragrances, and has the advantages of simple operation, low cost, high sensitivity, on-site monitoring and suitable for screening of a large number 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 . [0002] technical background [0003] 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 i...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com