Method for ultra-sensitively detecting small molecular substances

A technology for sensitive detection of small molecular substances, applied in the biological field, can solve problems such as low sensitivity and inability to perform multiple detection, and achieve the effects of improving detection sensitivity, improving fluorescent labeling efficiency, and simple synthesis process

Inactive Publication Date: 2019-01-08
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ELISA is simple, economical, and has been widely used in automated operations, but ELISA has certain limitations: one is low sensitivity, and the other is that it cannot perform multiple detection

Method used

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  • Method for ultra-sensitively detecting small molecular substances
  • Method for ultra-sensitively detecting small molecular substances
  • Method for ultra-sensitively detecting small molecular substances

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The small molecular substances detected by the method of the present invention refer to substances that can be recognized by other substances through specific actions, such as antigens that can be recognized by antibodies, ligands that can be recognized by receptors, substrates that can be recognized by enzymes, and substances that can be recognized by antibodies. Polysaccharides recognized by lectins, biotin that can be recognized by avidin or streptavidin. Because the principle is the same, take the antibody-antigen immune reaction as an example to prove that there is a theoretical minimum value in the sensitivity (half-inhibitory concentration, IC50) of competitive immunoassay, and the sensitivity of competitive immunoassay can be improved by reducing the concentration of competitors and the concentration of detection antibodies, so that It is close to the theoretical limit.

[0027] Through mathematical modeling, a theoretical model was established to study competit...

Embodiment 2

[0058] Embodiment 2 The method for detecting small molecules of the present invention

[0059] The small molecular substances detected by the method of the present invention refer to substances that can be recognized by other substances through specific actions, such as antigens that can be recognized by antibodies, ligands that can be recognized by receptors, substrates that can be recognized by enzymes, and substances that can be recognized by other substances. Polysaccharides recognized by lectins, biotin that can be recognized by avidin or streptavidin. Because the principle is the same, this embodiment takes the detection of antibody-antigen immune reaction as an example to illustrate the method for ultrasensitive detection of small molecular substances of the present invention.

[0060] When the small molecule is a hapten, the method for detecting the hapten comprises the following steps:

[0061] 1. Preparation of hapten-labeled solid phase support

[0062] The solid ...

Embodiment 3

[0073] Embodiment 3 adopts the inventive method to detect aflatoxin B1

[0074] 1. Materials and Methods

[0075] Bovine serum albumin (BSA), bovine serum albumin-aflatoxin B1 conjugate (BSA-Aflatoxin B1), aflatoxin B1 acetonitrile solution, PBS buffer, tris(hydroxymethyl)aminomethane (Tris), chloride Sodium (NaCl), ethyl[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC), 2-(N-morpholine)ethanesulfonic acid (MES), Tween-20 were purchased from Sigma Corporation. Aflatoxin B1 monoclonal antibody (disclosed in Journal of Immunological Methods 329 (2008) 112-124). Dynabeads TM MyOne TM Carboxyl-modified magnetic beads (hereinafter referred to as carboxyl-modified magnetic beads) and Alexa 647 active ester was purchased from American Thermofisher Company.

[0076] 2. Specific methods for detecting aflatoxin B1

[0077] (1) Preparation of hapten-labeled magnetic beads

[0078] Take 100 μL of carboxy-modified magnetic beads (10 mg / mL, 1 μm in diameter) in a 1.5 mL ce...

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Abstract

The invention provides a method for ultra-sensitively detecting small molecular substances, and belongs to the field of biotechnology. To-be-detected small molecule substances in the method are one ormore than two of antigens, ligands, biotins, substrates and polysaccharides. The method comprises the following steps: respectively marking solid phase carriers by using various to-be-detected smallmolecules; respectively marking substances for recognizing the various to-be-detected small molecules through specific action by using all fluorescence probes to obtain fluorescent markers; adding thesolid phase carriers marked by all the to-be-detected small molecules and all the fluorescent markers into a sample solution for reaction; after the reaction is ended, separating the solid phase carriers and releasing the fluorescent probes combined to the surfaces of the solid phase carriers; detecting the fluorescent probes with a single molecular detection method, and calculating the concentrations of all the to-be-detected small molecules in the sample solution according to a standard working curve. By adopting the method for ultra-sensitively detecting the small molecular substances provided by the invention, various small molecular substances can be rapidly and ultra-sensitively detected.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for ultrasensitive detection of small molecular substances. Background technique [0002] Immunological detection generally refers to a detection method in which an antibody (or antigen) is used to detect an antigen (or antibody) through the specific recognition of the antibody and the antigen. This detection method through specific mutual recognition can also be extended to the detection of other substances, such as receptors and ligands, enzymes and substrates, lectins and polysaccharides. The most classic method of immunoassay is enzyme-linked immunosorbent assay (ELISA). ELISA is simple, economical, and has been widely used in automated operations. However, ELISA has certain limitations: one is low sensitivity, and the other is that it cannot perform multiple detections. Contents of the invention [0003] The purpose of the present invention is to provide...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N33/543G01N33/577G01N21/64
CPCG01N21/6428G01N33/533G01N33/543G01N33/54313G01N33/54326G01N33/54346G01N33/577G01N2021/6439
Inventor 王军王旭
Owner NANJING UNIV OF TECH
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