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Preparation method of fluorescence resonance system for rapid detection of ATP

A fluorescence resonance and system technology, applied in the fields of biotechnology and rapid diagnostic detection, can solve the problem of less detection involved, and achieve the effects of shortening detection time, reducing cost, and improving sensitivity

Active Publication Date: 2013-07-17
XI AN JIAOTONG UNIV
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Problems solved by technology

At present, many ATP detection methods are mostly suitable for in vitro ATP detection, and less involved in the detection of ATP in the mitochondria of living cells.

Method used

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  • Preparation method of fluorescence resonance system for rapid detection of ATP
  • Preparation method of fluorescence resonance system for rapid detection of ATP
  • Preparation method of fluorescence resonance system for rapid detection of ATP

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preparation example Construction

[0022] A method for preparing a fluorescence resonance system for rapid detection of ATP, comprising the following steps:

[0023] Step 1: Prepare a Cy3 fluorescently labeled ATP-aptamer aptamer; the ATP-aptamer aptamer is a 3'-Cy3-modified DNA aptamer with a concentration of 0.2uM-1uM, and its sequence information is: 5'- ACCTGGGGGAGTATTGCGGAGGAAGGT-Cy3-3';

[0024] Step 2: Coupling reaction between QDs and ATP-aptamer aptamer complementary strands to obtain QDs molecular probes, QDs are water-soluble quantum dots (COOH-QDs) on the carboxyl surface, the concentration is 1uM; ATP aptamer complementary strands are 5'-NH 2 -(CH 2 ) 12 Modified DNA, the concentration is 10-500uM, its sequence information is: 5'-NH 2 -(CH 2 ) 12 -ACCTTCCTCCGCAATACTCCCCCAGGT-3' (Probe DNA, DNAp).

[0025] Step 3: react the QDs molecular probe obtained in step 2 with the Cy3 fluorescence-labeled ATP aptamer to obtain a fluorescence resonance system for detecting ATP after the ATP aptamer inte...

Embodiment 1

[0034]Add 32.5uL PB buffer (10mM, pH7.4) into the reactor, add 6.3uLCOOH-QDs300rpm, 5min, shake and mix; add 2.9uL EDC solution (10mg / mL, 10mM PB buffer, pH7.4) Activate with 3.3uL sulfo-NHS solution (10mg / mL, 10mM PB buffer, pH7.4) for 30min; add 5uL100uM DNAp, continue to shake and mix evenly, 300rpm, 25℃, 2h, the molar concentration ratio of each reactant is: QDs: DNA:EDC:sulfo-NHS=1:10:3000:3000; add 2uL100uM ATP aptamer, hybridize for 30min at 37°C; add 200uL PB buffer (10mM, pH7.4) to the reaction sample, concentrate and purify by ultrafiltration tube 5 Once, the final product was redissolved in 50uL PB buffer (10mM, pH7.4); the fluorescence value was detected by FLS920 fluorescence spectrophotometer; different concentrations of ATP (0-10uM) were added to the final product in turn, 45°C, 30min, Cool to room temperature, add 200uL PB buffer (10mM, pH7.4), concentrate and purify by ultrafiltration tube 5 times, measure the change of fluorescence intensity of the final prod...

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Abstract

The invention provides a preparation method of a fluorescence resonance system for rapid detection of ATP. The method comprises the following steps: step 1, preparing a Cy3 fluorescence-labeled ATP-aptamer; step 2, performing a coupling reaction for QDs with a complementary strand of the ATP-aptamer to obtain QDs molecular probes; and step 3, reacting the QDs molecular probes obtained in step 2 with the Cy3 fluorescence-labeled ATP-aptamer to obtain the fluorescence resonance system for detecting ATP after interaction of the ATP aptamer and the complementary strand. According to the invention, the method utilizes characteristics of specific binding of the ATP and the aptamer thereof as well as a resonance energy transfer technology, and thus realizes rapid detection of the ATP and real-time monitoring for changes of ATP concentration in mitochondria, thereby providing a novel method for detection and monitoring of the ATP in vitro and in vivo.

Description

technical field [0001] The invention relates to the field of biotechnology and rapid diagnosis and detection, in particular to a preparation method of a fluorescence resonance system for rapid detection of ATP, which is suitable for the rapid detection of low-concentration ATP and the application of mitochondrial function in cell biology and the like. technical background [0002] Adenosine triphosphate (ATP) is present in all living organisms from microorganisms to cells of higher animals and plants. The main function of ATP in the cell body is to provide energy and participate in the metabolism of fat, protein, sugar and nucleic acid in the body. It is an important source of energy for the body and plays an irreplaceable role in maintaining the normal function of the organism. As the most important energy molecule, ATP plays an important role in various physiological and pathological processes of cells, and can be used as an important marker of cell activity. ATP is also ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 李政彭年才刘小龙
Owner XI AN JIAOTONG UNIV
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