Method for constructing alkaline/surfactant/polymer compound system detecting probe

A ternary composite, detection probe technology, applied in the field of biosensing, to achieve the effects of improving detection sensitivity, reducing costs, and avoiding molecular markers

Inactive Publication Date: 2015-04-08
JIANGSU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although there are currently reports on the use of aptamers to construct detection probes, constructing a ternary system, utilizing the recognition of small molecules by aptamers in the ternary system, and using the peroxidase analog unit to realize the label-free determination of the detection signal, Combining with nucleic acid chain reaction to amplify the signal, the construction of detection probes has not been reported for the detection of small molecules.

Method used

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  • Method for constructing alkaline/surfactant/polymer compound system detecting probe
  • Method for constructing alkaline/surfactant/polymer compound system detecting probe

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Add 10 μL of DNA H to 50 μL of a certain concentration of ochratoxin A solution 1 , (H 1 The DNA sequence is GATCGGGTGT GGGTGGCGTA AAGGGAGCAT CGGACACGCC ACCCACAC) aqueous solution, so that H 1 The final concentration of 1nM, then add 10μL CaCl 2 solution and 10 μL KCl solution to make CaCl 2 The final concentration of KCl was 10 mM, and the final concentration of KCl was 20 mM, and then 10 μL of hemin solution was added to make the concentration of hemin 150 nM, mixed evenly, and reacted for 10 minutes. Then add 10 μL of another DNA, namely H 2 (H 2 The DNA sequence is CCACACCCGA TCCTGGGAGG GAGGGAGGGG TGTGGGTGGC G) aqueous solution, so that H 2 The final concentration was 1nM, mixed evenly, and reacted for 1 hour. Then add 80 μL containing H 2 o 2 Tetramethylbenzidine solution, (after adding the H in the solution 2 o 2 Concentration: 0.03%, tetramethylbenzidine concentration: 0.2 μg / mL) and mix evenly, react for 1 hour, then add 50 μL of 1M hydrochloric acid, ...

Embodiment 2

[0027] Add 10 μL of DNA H to 50 μL of a certain concentration of ochratoxin A solution 1 , (H 1 The DNA sequence is GATCGGGTGT GGGTGGCGTA AAGGGAGCAT CGGACACGCC ACCCACAC) aqueous solution, so that H 1 The final concentration of 50nM, then add 10μL CaCl 2 solution and 10 μL KCl solution to make CaCl 2 The final concentration of KCl was 10 mM, and the final concentration of KCl was 20 mM, and then 10 μL of hemin solution was added to make the concentration of hemin 150 nM, mixed evenly, and reacted for 10 minutes. Then add 10 μL of another DNA, namely H 2 (H 2 The DNA sequence is CCACACCCGA TCCTGGGAGG GAGGGAGGGG TGTGGGTGGC G) aqueous solution, so that H 2 The final concentration is 50nM, mix well, and react for 1 hour. Then add 80 μL containing H 2 o 2 Tetramethylbenzidine solution, (after adding the H in the solution 2 o 2 Concentration: 0.03%, tetramethylbenzidine concentration: 0.2 μg / mL) and mix evenly, react for 1 hour, then add 50 μL of 1M hydrochloric acid, measu...

Embodiment 3

[0030] Add 10 μL of DNA H to 50 μL of a certain concentration of ochratoxin A solution 1 , (H 1 The DNA sequence is GATCGGGTGT GGGTGGCGTA AAGGGAGCAT CGGACACGCC ACCCACAC) aqueous solution, so that H 1 The final concentration is 100nM, then add 10μL CaCl 2 solution and 10 μL KCl solution to make CaCl 2 The final concentration of KCl was 10 mM, and the final concentration of KCl was 20 mM, and then 10 μL of hemin solution was added to make the concentration of hemin 150 nM, mixed evenly, and reacted for 10 minutes. Then add 10 μL of another DNA, namely H 2 (H 2 The DNA sequence is CCACACCCGA TCCTGGGAGG GAGGGAGGGG TGTGGGTGGC G) aqueous solution, so that H 2 The final concentration is 100nM, mix well, and react for 1 hour. Then add 80 μL containing H 2 o 2 Tetramethylbenzidine solution, (after adding the H in the solution 2 o 2 Concentration: 0.03%, tetramethylbenzidine concentration: 0.2 μg / mL) and mix evenly, react for 1 hour, then add 50 μL of 1M hydrochloric acid, mea...

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Abstract

The invention discloses a method for constructing an alkaline/surfactant/polymer compound system detecting probe, and belongs to the field of biological sensing. A detection probe capable of sensitively detecting to-be-detected molecules such as ochratoxin A is designed by using the established method. The method is characterized by amplifying a detection signal by using a nucleic acid base interworking and nucleic acid chain continuous complementary principle, and belonging to the field of biological sensing. The method comprises the following steps that firstly, after the to-be-detected molecule acts with a nucleic acid chain H1, a configuration of the nucleic acid chain H1 is changed, so that the to-be-detected molecule is in complementary pairing with a base of a nucleic acid chain H2 to ensure that a configuration of the nucleic acid chain H2 is changed, the changed nucleic acid chain H2 can be also in complementary pairing with a base of the nucleic acid chain H1, thus the purpose that a single detection molecule initiates nucleic acid chain reaction is realized. Finally, when chlorhematin exists, peroxidase enzyme simulation sequence on the nucleic acid chain H2 can catalyze hydrogen peroxide to oxidize tetramethyl benzidine, and a colored solution is produced when a hydrochloric acid solution exists. A detection curve of the solution can be obtained according to a relationship between absorbance of the solution at a certain wavelength and the concentration of the to-be-detected molecule.

Description

technical field [0001] The patent of the present invention relates to a technology for constructing a detection probe of a ternary complex system, which can be used for sensitive detection of ochratoxin A. The feature is that the detection signal is amplified by using aptamers, nucleic acid base mutual matching and continuous complementation of nucleic acid chains, and belongs to the field of biosensing. Background technique [0002] Ochratoxin A is a common mycotoxin widely found in moldy fruits and grains. Its toxicity is mainly renal toxicity, liver toxicity, immunotoxicity, teratogenic toxicity and carcinogenicity. [0003] At present, the detection methods of ochratoxin A in food are mostly biological identification method, chemical analysis method and instrumental analysis method. Among them, the biological identification method is mainly used to qualitatively determine the presence or absence of mycotoxins, and its disadvantage is that the sensitivity is not high. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31
Inventor 王承克王坤董晓娅刘倩
Owner JIANGSU UNIV
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