A kind of preparation method of dumbbell-shaped dna/copper nanoparticle fluorescent biosensor and its application in quantitative detection of ATP
A biosensor, dumbbell-shaped technology, applied in the preparation of dumbbell-shaped DNA/copper nanoparticle fluorescent biosensors, the application field of quantitative detection of ATP, can solve the problems of high cost and long time, and achieve low cost and low detection limit , the effect of high sensitivity
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Embodiment 1
[0041] A preparation method of a dumbbell-shaped DNA / copper nanoparticle fluorescent biosensor, comprising the following steps:
[0042] (1) Dissolve 50 μL of 1 μM 5′-phosphorylated DS DNA solution in 34 μL of 10 mM MOPS buffer solution with pH=7.6, then add 1 μL of 350U / μL T4 DNA ligase and 5 μL of ATP, and supplement with pH=7.6 10mM MOPS buffer solution to the volume of the reaction system is 200μL, at room temperature, the ligation reaction initiated by ATP can occur in 50 minutes, and the dumbbell-shaped DNA solution is obtained;
[0043] The gene sequence of the DS DNA phosphorylated at the 5' end is:
[0044] 5’-PO 4-ATATATATATATTTTTTTTTTTTTTTTTTTTTTTTTTTTTATATATATATATATATATATATTTTTTTTTTTTTTTTTTTTTTTTTTTTTATATATATATAT-3';
[0045] (2) Add 50 μL of 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 The solution and 50 μL of 8mM sodium ascorbate solution were mixed evenly and reacted...
Embodiment 2
[0047] A quantitative detection method of ATP, the other is the same as in Example 1, except that different concentrations of ATP are added in step (1), so that the final concentrations of ATP in the reaction system are respectively 0.1, 1, 10, 20, 50, 100, 200, 500, 1000, 5000, 10000nM;
[0048] Then, the fluorescence intensity of each dumbbell-shaped DNA / copper nanoparticle fluorescent biosensor obtained in step (2) was tested respectively, and the excitation wavelength and emission wavelength of the fluorescence spectrophotometer were 340 nm and 630 nm, respectively. like figure 2 As shown, take the ATP concentration in the range of 0-20nM as the abscissa, and 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 shown in image 3 As shown, the linear equation F=27.06C is obtained ATP +874.09 with a correlation coefficient of R 2 =0.992;
[0049] Accor...
Embodiment 3
[0051] In order to verify the stability of the dumbbell-shaped DNA solution, the rest are the same as in Example 1, except that the final concentration of ATP in the fixation step (1) was 100 nM, and 5 μL of 5U was added to the dumbbell-shaped DNA solution obtained in the step (1) of Example 1. / μL Exo I and 5 μL 200U / μL Exo III, sheared for 60 minutes at room temperature, and incubated at 80°C for 5 minutes after 60 minutes to stop the degradation reaction. Then 50 μL of 8 mM sodium ascorbate was added to the above mixture, mixed well, and then 50 μL of 0.8 mM CuSO was added 4 The above solution was added and mixed well, and the fluorescence intensity of the reaction system was detected for 6 minutes at room temperature. It was found that the obtained product, the prepared biosensor, still had strong fluorescence, indicating that the dumbbell-shaped DNA structure would not be cleaved by the exonuclease. In the presence of copper ions and ascorbic acid, fluorescent copper nano...
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