Facile and Sensitive Fluorescence Assay of DNA Polymerase Activity Using Cu²⁺ and Ascorbate as Signal Developers

Abstract

We report herein a label-free and turn-on fluorescence approach for detecting DNA polymerase activity. In the presence of DNA polymerase, the DNA primer could be elongated to produce one long double-stranded DNA (dsDNA). Then, the produced dsDNA acted as the template for in-situ synthesizing fluorescent copper nanoclusters (CuNCs) via Cu 2 +ascorbate reaction (10 min at room temperature), and the fluorescent intensity of the CuNCs was used to quantify the activity of DNA polymerase. In this strategy, cheap CuSO 4 and ascorbate acted as fluorescence developers, so this method is low-cost and easy to carry out. Interestingly, based on the sequence-dependent fluorescence of DNA-templated CuNCs, we rationally designed the sequence of DNA substrate to decrease the background and increase the signal of this assay. Therefore, this proposed method exhibited a high sensitivity toward DNA polymerase, and the limit of detection was 3.7 × 10 -7 U/μL for Klenow Fragment (KF) polymerase. The strategy suggests a "mix-and-detect" homogenous assay format without precipitation, separation and washing. The whole process (including enzyme reaction and synthesizing CuNCs) can be completed in a single tube. This fluorescence method is lowcost and simple, and shows potential application in some biomedical studies and clinical diagnostics.

Ligand functionalized copper nanoclusters for versatile applications in catalysis, sensing, bioimaging, and optoelectronics

“…They showed that in the absence of MNase, dsDNA with the aid of sodium ascorbate served as a template for the formation of Cu NCs with excitation/emission peaks located at 340/570 nm. The fluorescence turn-on strategy based on DNA-templated synthesis of Cu NCs was also used for the activity detection of enzymes like T4 polynucleotide kinase phosphatase, 43 DNA polymerase 231 and uracil-DNA glycosylase (UDG). 46 Both dsDNA and ssDNA can act as a template for the synthesis of Cu NCs.…”