Label-free and sensitive assay for deoxyribonuclease I activity based on enzymatically-polymerized superlong poly(thymine)-hosted fluorescent copper nanoparticles

Deoxyribonuclease I (DNase I) is an important physiological indicator and diagnostic biomarker, but traditional methods for assessing its activity are time-consuming, laborious, and usually radioactive. Herein, by effectively combining the special functions of DNase I and terminal deoxynucleotidyl transferase (TdT), a simple, green, cost-effective, label-free and ultrasensitive assay for DNase I activity has been constructed based on superlong poly(thymine)-hosted copper nanoparticles (poly T-CuNPs). In this strategy, a 3′-phosphorylated DNA primer is designed to block TdT polymerization. After addition of DNase I, the primer could be digested to release 3′-hydroxylated fragments, which could further be tailed by TdT in dTTP pool with superlong poly T ssDNA for CuNPs formation. Fluorescence measurements and gel electrophoresis demonstrated its feasibility for DNase I analysis. The results indicated that with a size of 3–4   nm, the CuNPs templated by TdT-polymerized superlong poly T (>500 mer) had several advantages such as short synthetic time (<5   min), large Stokes shift (~275   nm) and intense red fluorescence emission . Under the optimal conditions, quantitative detection of DNase I was realized, showing a good linear correlation between 0.02 and 2.0   U/mL (R 2 =0.9928) and a detection limit of 0.02   U/mL. By selecting six other nucleases or proteins as controls, an excellent specificity was also verified. Then, the strategy was successfully applied to detect DNase I in diluted serum with a standard addition method, thus implying its reliability and practicability for biological samples. The proposed strategy might be promising as a sensing platform for related molecular biology and disease studies.