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Preparation method of dumbbell-shaped DNA/copper nanoparticle fluorescent biosensor and application of dumbbell-shaped DNA/copper nanoparticle fluorescent biosensor in quantitative detection of ATP

A biosensor, dumbbell-type technology, applied in the field of fluorescence sensing preparation, can solve the problems of long time and high cost, and achieve the effects of low cost, low detection limit and high sensitivity

Active Publication Date: 2020-04-03
ANHUI NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Strategies for ATP detection have been developed, many based on host-guest, peptides, conjugated polymers, DNA / RNA aptamers and ATP-dependent ligation reactions, and even though some methods show very good analytical performance, they are usually Signal marking is involved, and the time is relatively long and the cost is high

Method used

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  • Preparation method of dumbbell-shaped DNA/copper nanoparticle fluorescent biosensor and application of dumbbell-shaped DNA/copper nanoparticle fluorescent biosensor in quantitative detection of ATP
  • Preparation method of dumbbell-shaped DNA/copper nanoparticle fluorescent biosensor and application of dumbbell-shaped DNA/copper nanoparticle fluorescent biosensor in quantitative detection of ATP
  • Preparation method of dumbbell-shaped DNA/copper nanoparticle fluorescent biosensor and application of dumbbell-shaped DNA/copper nanoparticle fluorescent biosensor in quantitative detection of ATP

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Effect test

Embodiment 1

[0041] A method for preparing a dumbbell-shaped DNA / copper nanoparticle fluorescent biosensor, comprising the following steps:

[0042] (1) Dissolve 50 μL of 1 μM 5’-end phosphorylated DS DNA solution in 34 μL of 10 mM MOPS buffer solution with pH=7.6, then add 1 μL of 350 U / μL T4 DNA ligase and 5 μL of ATP, and add PH=7.6 10mM MOPS buffer solution to the volume of the reaction system is 200μL, at room temperature, the ATP-triggered ligation reaction can occur within 50 minutes to obtain a dumbbell-shaped DNA solution;

[0043] The gene sequence of the phosphorylated DS DNA at the 5' end is:

[0044] 5'-PO 4- ATATATATATATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATATATATATATATATATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATATATATAT-3';

[0045] (2) Add 50 μL of the dumbbell-shaped DNA solution obtained in step (1) to 50 μL of 10 mM MOPS buffer solution with pH=7.6, and then add 50 μL of 0.8 mM CuSO 4 solution and 50 μL of 8mM sodium ascorbate solution, mixed evenly and reacted for 6 minutes, th...

Embodiment 2

[0047] A quantitative detection method for ATP, other is the same as embodiment 1, just add the ATP of different concentration respectively in the step (1), make the final concentration of ATP in the reaction system be respectively 0.1, 1, 10, 20, 50, 100, 200, 500, 1000, 5000, 10000nM;

[0048] Then test the fluorescence intensity of each dumbbell-shaped DNA / copper nanoparticle fluorescent biosensor obtained in step (2) respectively, and the excitation wavelength and emission wavelength of the fluorescence spectrophotometer are respectively 340nm and 630nm. Such as figure 2 As shown, with the ATP concentration in the range of 0-20nM as the abscissa, the fluorescence intensity value of each dumbbell-shaped DNA / copper nanoparticle fluorescent biosensor at 630nm is plotted on the ordinate to construct a standard curve, as image 3 As shown, the linear equation F=27.06C is obtained ATP +874.09, its correlation coefficient is R 2 =0.992;

[0049] According to the standard cur...

Embodiment 3

[0051] In order to verify the stability of the dumbbell-shaped DNA solution, the others are the same as in Example 1, except that the final concentration of ATP in the fixing step (1) is 100 nM, and in the dumbbell-shaped DNA solution obtained in step (1) of Example 1, add 5 μL of 5U / μL Exo I and 5μL 200U / μL Exo III, shear at room temperature for 60 minutes, and incubate at 80°C for 5 minutes after 60 minutes to terminate the degradation reaction. Then add 50μL 8mM sodium ascorbate to the above mixture, mix thoroughly, and then add 50μL 0.8mM CuSO 4 Add the above solution and mix well, and at room temperature for 6 minutes, detect the fluorescence intensity of the reaction system, and find that the obtained product, that is, the prepared biosensor, still has strong fluorescence, indicating that the dumbbell-shaped DNA structure will not be cleaved by exonuclease. In the presence of copper ions and ascorbic acid, fluorescent copper nanoparticles can still be formed, which has ...

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Abstract

The invention discloses a preparation method of a dumbbell-shaped DNA / copper nanoparticle fluorescent biosensor and application of the dumbbell-shaped DNA / copper nanoparticle fluorescent biosensor inquantitative detection of ATP. The method comprises the following steps: reacting DS DNA (Deoxyribonucleic Acid) with phosphorylated 5 '-terminal under the action of T4 DNA ligase and ATP (Adenosine Triphosphate); the dumbbell type DNA is prepared; the dumbbell-shaped DNA / copper nanoparticle fluorescent biosensor combined with the copper nanoparticles is further prepared on the basis of the dumbbell-shaped DNA; the concentration of the added adenosine triphosphate is increased; the fluorescence intensity of the dumbbell-shaped DNA / copper nanoparticle fluorescence biosensor is increased accordingly, so that the linear relation between the ATP concentration and the fluorescence intensity is constructed, high-sensitivity and high-specificity detection on adenosine triphosphate is achieved, and the dumbbell-shaped DNA / copper nanoparticle fluorescence biosensor has the advantages of being easy to operate, high in sensitivity and low in detection limit.

Description

technical field [0001] The invention belongs to the technical field of fluorescent sensing preparation, and in particular relates to a preparation method of a dumbbell-shaped DNA / copper nano particle fluorescent biosensor and its application in quantitative detection of ATP. Background technique [0002] Adenosine triphosphate (ATP) is a multifunctional nucleotide that is not only a universal energy source but also an extracellular signaling mediator involved in many biological processes including membrane ion channels, DNA replication, biosynthesis, and is also used As an indicator of cell viability and cell damage in organisms. Therefore, it is widely used in biochemical research and clinical diagnosis, and it is essential for the detection of ATP with high sensitivity and high selectivity. Strategies for ATP detection have been developed, many based on host-guest, peptides, conjugated polymers, DNA / RNA aptamers and ATP-dependent ligation reactions, and even though some m...

Claims

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Application Information

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IPC IPC(8): G01N21/64B82Y5/00B82Y15/00B82Y30/00
CPCG01N21/6428B82Y30/00B82Y15/00B82Y5/00
Inventor 王广凤陈纪华
Owner ANHUI NORMAL UNIV
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