An enzyme-free and substrate-free electrochemical biosensor with robust porphyrin-based covalent-linked nanomaterial as nanoelectrocatalyst and efficient support for sensitive detection of uracil-DNA glycosylase.

We developed a novel electrochemical biosensor for uracil-DNA glycosylase (UDG) detection based on enzyme-free and substrate-free electrocatalytic signal amplification by porphyrin-based covalent-linked nanomaterial (OAPS-Por). This OAPS-Por could not only absorb much Thionine (Thi), but also possess obvious electrocatalytic activity toward the reduction of Thi without involvement of H2O2. Sequentially, the functionalized OAPS-Por with Thi, Au nanoparticles and single-stranded DNA (OAPS-Por/Thi@AuNPs-ssDNA) was ingeniously designed as the signal probe. Meantime, the hairpin DNA (hDNA) with four uracil bases was immobilized on AuNPs/GCE via an Au-S bond. When UDG was present, the uracil in hDNA was removed and hairpin structure was unfolded. Next, the signal probes binded with the unfolded hDNA by DNA hybridization. The Thi in signal probes could generated an original electrochemical signal, which could be further amplified and output due to the robust electrocatalytic activities of OAPS-Por toward Thi. As a result, the as-constructed electrochemical biosensor had a broad linear range from 0.005 to 1 U mL-1. It also exhibited a low detection limit of 6.97 × 10-4 U mL-1. Moreover, this biosensor could be used to assay the inhibition of UDG (UGI) and the UDG activity in real samples (HeLa cell lysates and human blood serums), and demonstrated great prospect in clinical diagnostics and biomedical research.