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Cracking aptamer sensor for ATP (Adenosine Triphosphate) detection and application thereof

An aptamer sensor and aptamer technology, applied in the field of biochemistry, can solve the problems of poor fluorescence enhancement, increased ThT fluorescence, weak binding, etc., and achieve the effects of wide dynamic linear range, significant signal amplification, and wide linear range.

Active Publication Date: 2019-03-01
SHANGQIU NORMAL UNIVERSITY
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Problems solved by technology

However, the traditional split aptamer strategy cannot be directly used to improve label-free nucleic acid aptamers based on Thioflavin T (ThT) substitution detection of ATP, because the fluorescence enhancement effect of the split ATP aptamers on ThT is far less than that of the intact ones. aptamer, mainly due to the very weak binding between the split ATP aptamer and ThT, resulting in no significant increase in ThT fluorescence
No methods for improving sensitivity using split ATP aptamers and ThT have been reported so far

Method used

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  • Cracking aptamer sensor for ATP (Adenosine Triphosphate) detection and application thereof
  • Cracking aptamer sensor for ATP (Adenosine Triphosphate) detection and application thereof
  • Cracking aptamer sensor for ATP (Adenosine Triphosphate) detection and application thereof

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Embodiment 1

[0039] 1 Experimental part

[0040] 1.1 Instruments and reagents

[0041] Hitachi F-7000 fluorescence spectrophotometer; Chirascan circular dichrograph; ultrapure water (Mill-Q pure water meter, conductivity> 18MΩ.cm); KS-300D ultrasonic cleaner (Ningbo Kesheng Instrument Factory); Lei Magnetic PHS-3C acidity meter; McAry (SQ) Eq-001 centrifuge.

[0042] The DNA sequence used in the present invention was purchased from Shanghai Shenggong Biological Engineering Co., Ltd., see the sequence table. Dissolve the solid DNA with ultrapure water and prepare a mother liquor with a concentration of 100 μM. Thioflavin T (ThT) was purchased from Sigma-Aldrich, dissolved in ultrapure water, the concentration of the mother liquor was 1 mM, and stored at 4°C in the dark. Magnesium chloride and potassium chloride are commercially available products. The buffer solution used in the experiment is 10mMTris-HCl (30mM MgCl 2 , pH = 6.0). The human serum used for sample determination was obtained fro...

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Abstract

The invention discloses a cracking aptamer sensor for ATP (Adenosine Triphosphate) detection and application thereof and belongs to the technical field of biochemistry. According to the cracking aptamer sensor, G-enriched sequences are modified to tail ends of two cracked ATP aptamer segments. ThT and two G-enriched ATP cracking aptamers have a mutual effect to form a fluorescence compound which is better than a complete aptamer / ThT compound. The G-enriched sequences can be used for improving a fluorescence signal, so that the sensitivity of a novel method is improved. With the help of the G-enriched sequences, the detection limit of the ATP aptamer sensor is 5 nM. The method has a good linear relation on ATP from 100 nM to 120 muM. Compared with a ThT substitution based complete aptamer for the ATP detection, which is reported before, the aptamer sensor provided by the invention is more sensitive and has a wider dynamic linear range; the selectivity to ADP (Adenosine Diphosphate), AMP(Adenosine Monophosphate) and the like is obviously improved. The aptamer sensor can be used for detecting the ATP in a complicated biological serum sample.

Description

Technical field [0001] The invention relates to a split aptamer sensor for ATP detection and its application, in particular to a label-free split ATP aptamer based on a fluorescence sensor and its application, and belongs to the technical field of biochemistry. Background technique [0002] Nucleic acid aptamers are single-stranded RNA or DNA molecules that are selected in vitro through the systematic evolution of exponential enrichment (SELEX) ligands to specifically bind targets with high affinity. They provide a practical alternative to antibodies The method is used for the detection of nucleic acids, proteins, small molecules and even whole cells. Compared with antibodies, nucleic acid aptamers have many advantages, such as higher specificity, simple synthesis, relatively easy labeling, relatively small size, non-immunogenicity, good stability, relatively fast speed, cheap production, and extremely High accuracy and reproducibility. At the same time, when designing a new ty...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N33/573
CPCG01N21/6428G01N33/5735G01N2021/6432
Inventor 王永祥耿凤华马雨徐茂田瞿鹏张银堂
Owner SHANGQIU NORMAL UNIVERSITY
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