Label-Free Nuclease Assay with Long-Term Stability

Nature endows us with a unique toolbox of highly specific enzymes, while their detection is of great importance in biological processes. The label-free assay based on DNA-templated CuNPs is widely accepted for enzyme assay owning to its simple procedure, fast kinetic, high quantum yield, and large Stokes shift. A challenge in the application of them is the low fluorescent signal stability of DNA-templated CuNPs, whose signal sharply decreases in a few minutes after formation. In this work, a long-term stable nuclease assay is proposed by utilizing the elemental mass spectrometry detection of CuNPs. The high sensitivity was also realized, thanks to a great number of copper isotopes (Cu-63 and Cu-65) intrinsically incorporated in CuNPs. The experimental conditions, including the length of polyT ssDNA template, the concentration of polyT template, the concentration of Cu2+, the sodium ascorbate concentration, the copper reduction reaction time, and the Exonuclease I (Exo I) digestion reaction time, were investigated in detail. The dynamic range of the Exo I concentration from 0.1 U/mL to 20 U/mu l, was obtained using inductive coupled plasma mass spectrometry (ICPMS) Cu-63 signal, with a detection limit (3 sigma) of 0.029 U/mL. The ICPMS Cu-63 signal remained unchanged for at least 18 days. The spiked-recovery assay in RPMI 1640 cell medium also demonstrated the reliability of the proposed nuclease assay.