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ATP chemiluminiscence detection method based on enzyme digestion assisted label-free aptamer sensor

A chemiluminescence detection and aptamer sensor technology, applied in the field of analytical chemistry, can solve problems such as poor application effect, cumbersome detection steps, unstable results, etc., to achieve fully automatic detection, shorten detection time, and meet the effects of rapid detection

Active Publication Date: 2020-12-11
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to address the deficiencies and difficulties of the prior art, and provide an ATP chemiluminescence detection method assisted by enzyme digestion without labeling aptamer sensors, so as to solve the problem of poor sensitivity, unstable results, and poor application effect of the ATP detection method in the prior art. good technical flaw
[0006] Another technical problem to be solved by the present invention is that the ATP chemiluminescence detection method based on the aptamer technology in the prior art generally requires additional markers to improve the luminescence sensitivity, resulting in tedious detection steps.

Method used

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  • ATP chemiluminiscence detection method based on enzyme digestion assisted label-free aptamer sensor
  • ATP chemiluminiscence detection method based on enzyme digestion assisted label-free aptamer sensor
  • ATP chemiluminiscence detection method based on enzyme digestion assisted label-free aptamer sensor

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Experimental program
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Effect test

Embodiment 1

[0036] 1 Experimental principle

[0037] In this experiment, carboxyl magnetic microspheres were used as the separation carrier to construct an ATP aptamer sensor on the surface of the magnetic microspheres. The positive correlation between the amount of ATP aptamer immobilized on the surface of the magnetic microspheres and the ATP concentration and the G base in the ATP aptamer sequence and The transient derivatization reaction of the chemiluminescence reagent PG realizes the enzyme cleavage-assisted chemiluminescence detection of ATP. First, the amino aptamer is immobilized on the surface of the carboxyl magnetic microsphere through the aminocarboxyl binding reaction; then; finally, the magnetic microsphere is separated by a magnetic separator, and the amount of the ATP aptamer fixed on the surface of the microsphere is detected by using a chemiluminescence method, thereby Indirect quantitative detection of ATP. When no ATP exists (i.e. blank group), the ATP aptamers immob...

Embodiment 2

[0056] A method for detecting ATP chemiluminescence based on an enzyme-assisted unlabeled aptamer sensor, comprising the following steps:

[0057] 1) Immobilizing the amino-modified aptamer on the surface of the carboxyl magnetic microspheres through an aminocarboxyl reaction to obtain a magnetic microsphere-aptamer complex;

[0058] 2) Add the sample to be tested to the magnetic microsphere-capture probe complex, so that the aptamer can specifically bind to ATP, so that the ATP capture can be indirectly connected to the surface of the magnetic microsphere, and the solid phase can be taken after the reaction to obtain the ATP aptamer sensor;

[0059] 3) Mix the ATP aptamer sensor described in step 2) with 50 μL of 1-fold concentration Exo T buffer solution and a certain unit amount of Exo T solution, and then detect the CL value of the luminescence signal.

[0060] On the basis of the above technical solutions, the following conditions are met:

[0061] Step 1) includes the ...

Embodiment 3

[0069] A method for detecting ATP chemiluminescence based on an enzyme-assisted unlabeled aptamer sensor, comprising the following steps:

[0070] 1) Immobilizing the amino-modified aptamer on the surface of the carboxyl magnetic microspheres through an aminocarboxyl reaction to obtain a magnetic microsphere-aptamer complex;

[0071] 2) Add the sample to be tested to the magnetic microsphere-capture probe complex, so that the aptamer can specifically bind to ATP, so that the ATP capture can be indirectly connected to the surface of the magnetic microsphere, and the solid phase can be taken after the reaction to obtain the ATP aptamer sensor;

[0072] 3) Mix the ATP aptamer sensor described in step 2) with 50 μL of 1-fold concentration Exo T buffer solution and a certain unit amount of Exo T solution, and then detect the CL value of the luminescent signal.

[0073] On the basis of the above technical solutions, the following conditions are met:

[0074] Step 1) includes the f...

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Abstract

The invention provides an ATP chemiluminiscence detection method based on an enzyme digestion assisted label-free aptamer sensor, which comprises the following steps: immobilizing an aptamer on the surface of a magnetic microsphere, and specifically binding the aptamer with adenosine triphosphate, then, degrading the single-stranded DNA specifically and gradually into a free basic group from the 3' end by utilizing exonuclease T, and reducing a background signal remarkably based on the exonuclease, so that a quantitative relation between the quantity of the aptamer detected on the surface of the magnetic microsphere and the concentration of the ATP to be detected is obtained; and finally, carrying out chemiluminiscence detection on the aptamer sensor. Based on the principle that guanine base (G) on an ATP aptamer chain and a chemiluminescence reagent benzoyl formaldehyde (PG) are subjected to an instantaneous derivatization reaction to generate chemiluminescence, the marking link of other chemiluminescence markers is omitted, the operation steps are simplified on the basis of ensuring the sensitivity, the detection time is shortened, and the detection efficiency is improved. In addition, the used chemiluminescence analysis method is wide in linear range, simple in equipment and easy to operate, so that full-automatic detection is expected to be realized.

Description

technical field [0001] The present invention relates to the technical field of analytical chemistry, and further relates to nucleic acid aptamer technology and exonuclease that can significantly reduce background signals, and in particular to an ATP chemiluminescent detection method based on an enzyme-assisted unlabeled aptamer sensor. Background technique [0002] Adenosine triphosphate (ATP) exists in biological cells and is the main source of energy for living cells. It can control a variety of cellular metabolic processes such as biosynthetic reactions, movement, and cell division, thereby enabling energy transduction. The amount of ATP always maintains a dynamic balance in the cell, so it can be used as an indicator of cell damage, vitality and proliferation. Abnormal levels of ATP in organisms are closely related to various pathogenesis, mainly including hypoxia, ischemia, hypoglycemia, Alzheimer's disease and Parkinson's disease. ATP is also specially used as an indi...

Claims

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Application Information

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IPC IPC(8): G01N21/76
CPCG01N21/76
Inventor 严喜鸾赵坤肖义陂刘杰束庆磊
Owner NANCHANG UNIV
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