研究目的
To develop a fluorometric method for the determination of the activity of the enzyme T4 polynucleotide kinase phosphatase (T4 PNKP) using DNA-templated copper nanoclusters (CuNCs) as fluorescent indicators.
研究成果
The developed fluorometric method using DNA-templated CuNCs offers a simple, label-free, and sensitive approach for detecting T4 PNKP activity with a low detection limit and high specificity. It holds potential for applications in clinical diagnosis and drug discovery.
研究不足
The method requires working in the UV range, making it prone to interferences by biomatter in complex samples like blood/serum, cells, marine water, and wastewaters.
1：Experimental Design and Method Selection:
The method involves the hybridization of a short 3′-terminus phosphorylated DNA strand with a long DNA strand to form a partially double-stranded DNA (dsDNA) substrate. T4 PNKP dephosphorylates the substrate to generate long dsDNA, which then templates the synthesis of fluorescent CuNCs. The fluorescence intensity is measured to quantify T4 PNKP activity.
2：Sample Selection and Data Sources:
DNA strands were custom-designed and synthesized. T4 PNKP and other enzymes were obtained from commercial sources.
3：List of Experimental Equipment and Materials:
Includes DNA strands, enzymes (T4 PNKP, Klenow Fragment polymerase), deoxyribonucleoside triphosphates (dNTPs), SYBR Green I, sodium ascorbate (Vc), CuSO4, and a F-4600 Hitachi Fluorometer.
4：Experimental Procedures and Operational Workflow:
The process involves incubation of T4 PNKP with the DNA substrate, polymerization with KF polymerase and dNTPs, synthesis of CuNCs, and fluorescence measurement.
5：Data Analysis Methods:
Fluorescence intensity at 570 nm was recorded to quantify T4 PNKP activity.
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