Highly sensitive label-free fluorescence determination of lymphotropic virus DNA based on exonuclease assisted target recycling amplification and in-situ generation of fluorescent copper nanoclusters

Abstract

Specific and sensitive detection of target DNA plays a significant role in clinical diagnosis and biomedical research. Herein, a sensitive ratio fluorescence biosensor was constructed based on in-situ generation of fluorescent copper nanoclusters (CuNCs) on the DNA modified graphene quantum dots (GQDs) for the detection of HTLV-I DNA. Firstly, large amount of AT rich single DNA (o-DNA) can be released from the designed hairpin DNA in the presence of trace target DNA via exonuclease III assisted target recycling amplification strategy. Then, the released o-DNA hybridized with the complementary DNA1 which was modified on the surface of GQDs, and the formed hybridized DNA with blunt 3′-hydroxylated terminus that couldn’t be digested by exonuclease I could act as the template for the generation of fluorescent CuNCs. While in the absence of target DNA, the o-DNA couldn’t be released, thus the DNA1 with 3′overhang ends could be degraded by exonuclease I, which resulted in the in-situ generation of fluorescent CuNCs on the surface of GQDs was blocked owing to the lack of DNA template. The GQD fluorescence in the GQD-CuNC nanohybrid was almost uninfluenced during the whole process. Therefore, with the GQD serving as the reference and CuNC acting as the reporter signal, there was an excellent linear relationship between the change of fluorescence intensity ratio (F595/F445) and the concentration of HTLV-I DNA in the range of 20 pM-12 nM with a detection limit of 10 pM. Furthermore, the biosensor exhibited satisfactory results for monitoring the presence of HTLV-I DNA in human serum.