Split aptamer sensors for ATP detection and their applications

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

Active Publication Date: 2021-01-29
SHANGQIU NORMAL UNIVERSITY
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AI Technical Summary

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 have been reported to improve sensitivity using split-ATP aptamers and ThT

Method used

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  • Split aptamer sensors for ATP detection and their applications
  • Split aptamer sensors for ATP detection and their applications
  • Split aptamer sensors for ATP detection and their applications

Examples

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Embodiment 1

[0039] 1 Experimental part

[0040] 1.1 Instruments and reagents

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

[0042] The DNA sequence used in the present invention was purchased from Shanghai Sangon Bioengineering Co., Ltd., see the sequence listing. Dissolve DNA solids with ultrapure water and prepare a stock solution with a concentration of 100 μM. Thioflavin T (ThT) was purchased from Sigma-Aldrich Company, dissolved in ultrapure water, the concentration of the mother solution was 1 mM, and stored at 4°C in the dark. Magnesium chloride and potassium chloride are commercially available. The buffer solution used in the experiment was 10mM Tris-HCl (30mM MgCl 2 ,pH=6.0). Human serum used for sample determination was obtain...

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Abstract

The invention discloses a split aptamer sensor for ATP detection and an application thereof, belonging to the technical field of biochemistry. The present invention modifies G-rich sequences to the ends of the two split ATP aptamer fragments. ThT interacts with two G-rich ATP-split aptamers to form a more highly fluorescent complex than the intact aptamer / ThT complex. Since the G-rich sequence can increase the fluorescence signal, the sensitivity of the new method can be improved. With the help of the G-rich sequence, the detection limit of the ATP aptamer sensor of the present invention is 5 nM. The method has a good linear relationship for ATP from 100 nM to 120 μM. Compared with the complete aptamer based on ThT-substituted ATP detection previously reported, the aptamer sensor of the present invention is more sensitive, has a wider dynamic linear range, and significantly improves the selectivity to ADP and AMP. This aptasensor can be used to detect ATP in complex biological serum samples.

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 fluorescent sensor and its application, and belongs to the technical field of biochemistry. Background technique [0002] Aptamers are single-stranded RNA or DNA molecules selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX) to specifically bind targets with high affinity, and 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, aptamers have many advantages, such as high specificity, simple synthesis, relatively easy labeling, relatively small size, non-immunogenicity, good stability, relatively fast, cheap production, and extremely High accuracy and reproducibility. At the same time, when designing novel biosensors with high detection sensitivity ...

Claims

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Application Information

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Patent Type & Authority Patents(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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