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Preparation method of fluorescence resonance system for rapid detection of ATP in mitochondria

A fluorescence resonance and mitochondrial technology, which is applied in the field of biotechnology and rapid diagnostic testing, can solve the problems of less detection 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 in mitochondria
  • Preparation method of fluorescence resonance system for rapid detection of ATP in mitochondria
  • Preparation method of fluorescence resonance system for rapid detection of ATP in mitochondria

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Embodiment Construction

[0018] The present invention is described in detail below in conjunction with accompanying drawing.

[0019] Preparation method of fluorescence resonance system for rapid detection of ATP in mitochondria,

[0020] Step 1: prepare a Cy3 fluorescently labeled ATP aptamer and MTS fusion polypeptide, the ATP aptamer is a 3'-Cy3 modified DNA aptamer, the concentration is 0.2uM-1uM;

[0021] Step 2: Coupling the QDs with the ATP aptamer complementary chain and the MTS fusion polypeptide to obtain the mitochondrial localized QDs molecular probe. Ligand complementary strand is 5'-NH 2 -(CH 2 ) 12 Modified DNA, the concentration is 10-500uM;

[0022] Step 3: React the mitochondrial-localized QDs molecular probe obtained in step 2 with the Cy3 fluorescently labeled ATP-aptamer aptamer to obtain a fluorescence resonance system after the interaction between the ATP-aptamer aptamer and the complementary chain;

[0023] Step 4: Incubate the fluorescent resonance molecular probe system ...

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Abstract

The invention provides a preparation method of a fluorescence resonance system for rapid detection of ATP in mitochondria. The method comprises the following steps: step 1, providing a Cy3 fluorescence-labeled ATP-aptamer and an MTS sequence; step 2, performing a coupling reaction for QDs with a complementary strand of the ATP-aptamer and the MTS to obtain QDs molecular probes, wherein QDs is water-soluble quantum dots on carboxyl surface (COOH-QDs) and has a concentration of 1 [mu]M; 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 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 also realizes real-time monitoring for changes of ATP concentration in mitochondrial through specific transmembrane peptides-mitochondrial targeting fusion sequence (MTS), 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 fields of biotechnology and rapid diagnosis and detection, in particular to a preparation method of a fluorescence resonance system for rapidly detecting the concentration of ATP in mitochondria, which is suitable for the application of mitochondrial function in cell biology. 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 often used as an indicato...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 李政彭年才刘小龙
Owner XI AN JIAOTONG UNIV
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