A kit for detecting ATP with nucleic acid aptamer and its detection method

A nucleic acid aptamer and kit technology, which is applied to a kit for detecting ATP with nucleic acid aptamer and the field of detection thereof, can solve the problems of immune antibody preparation, stability, cost limitations, etc., and achieves poor stability and low loss. Live, high-precision effects

Active Publication Date: 2019-03-15
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Commonly used fluorescence polarization / fluorescence anisotropy analysis methods to detect small molecules often require the use of immune antibodies as recognition reagents to construct detection methods, but immune antibodies have limitations in preparation, stability, and cost

Method used

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  • A kit for detecting ATP with nucleic acid aptamer and its detection method
  • A kit for detecting ATP with nucleic acid aptamer and its detection method
  • A kit for detecting ATP with nucleic acid aptamer and its detection method

Examples

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Embodiment 1

[0037] Example 1: Using nucleic acid aptamer AD25 or AD25-10P to detect ATP based on fluorescence anisotropy (fluorescence polarization)

[0038] The nucleic acid aptamer of this embodiment is a nucleic acid molecule with the sequence shown in SEQ ID NO.1 (abbreviated as AD25) or a nucleic acid molecule with the sequence shown in SEQ ID NO.2 (abbreviated as AD25-10P).

[0039] Incubate 10 nM TMR-labeled ATP, 5 μM nucleic acid aptamer AD25 (or AD25-10P) and different concentrations of ATP in the binding buffer solution, and then measure the fluorescence anisotropy value (r) or fluorescence polarization value (P) of TMR (Temperature is measured under 10 ℃), the result is as follows figure 2 and image 3 shown. The sequence of the nucleic acid aptamer AD25 is SEQ ID NO.1 (5'-CCT GGG GGA GTATTG CGG AGG AAG G-3'). The sequence of the nucleic acid aptamer AD25-10P is SEQ ID NO.2 (5'-ACT CAG CCGGCC TGG GGG AGT ATT GCG GAG GAA GGC CGG CTG AGT-3').

[0040] from figure 2 It can ...

Embodiment 2

[0041] Example 2: Detection of ATP in diluted urine samples based on fluorescence anisotropy using aptamer AD25-10P

[0042] Incubate 10nM TMR-labeled ATP, 5μM AD25-10P, and different concentrations of ATP in urine samples diluted 20 times with binding buffer solution, and then measure the fluorescence anisotropy value (r) of TMR (measured at 10°C) , subtract the fluorescence anisotropy value corresponding to the blank sample to obtain the change value of the fluorescence anisotropy Δr. The results show that the ATP in the diluted urine sample can be detected, and the relationship between the change value Δr of the fluorescence anisotropy and the change of the ATP concentration can be found in Figure 4 .

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Abstract

The invention provides a kit for detecting an ATP with a nucleic acid aptamer and a detection method thereof. The kit comprises a nucleic acid aptamer having a sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2, and an ATP labeled by Tetra-Methyl Rhodamine (TMR). The kit achieves detection of the ATP based on fluorescence polarization (fluorescence anisotropy) of the nucleic acid aptamer, and has the advantages of simple operation, fast detection, high sensitivity, high accuracy and good reproducibility, and is insusceptible to fluorescence intensity fluctuation and fluorescence photobleaching. Compared with such methods as chromatography, no expensive instrument is needed and the operation steps are simple; and moreover, determination can be achieved just by simple sample mixing.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a kit for detecting ATP with a nucleic acid aptamer and a detection method thereof. Background technique [0002] Adenosine triphosphate (ATP) is an important active molecule in organisms and plays a variety of functions. ATP is a high-energy phosphate compound that stores and releases energy through conversion with adenosine diphosphate (ADP), providing energy for the life activities of cells. In the physiological function of cells, ATP plays a key role in the regulation of cell metabolism and biological reactions. ATP is also involved in various physiological functions such as building protein molecules, actively transporting ions, and nerve electrical signal transduction. Monitoring ATP can be used to indicate cell viability and cell damage. Moreover, the content of ATP is often used to determine the activity of microorganisms, the freshness of fish...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11
CPCG01N33/5735
Inventor 赵强李亚飘孙琳琳
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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