Silver nanoclusters-assisted triple-amplified biosensor for ultrasensitive methyltransferase activity detection based on AuNPs/ERGO hybrids and hybridization chain reaction

Abstract In the paper, a triple-amplified biosensor was built for the purpose of ultrasensitive methyltransferase (Dam MTase) activity detection and inhibitor screening based on in-situ synthesized silver nanoclusters (AgNCs) as signal probes to provide amplified current signal coupling with gold nanoparticles/electrochemical reduced graphene oxide (AuNPs/ERGO) hybrids and hybridization chain reactions (HCR) amplification strategy. For biosensor preparation, the AuNPs/ERGO hybrids were firstly generated on the electrode surface by electrochemical co-reduction method, then the ds-DNA structures which comprised the specific recognition sequences (5′-G-A-T-C-3′) of the Dam MTase and the restrictive endonuclease DpnI were formed on the electrode by the hybridization of the assistant DNA with the capture DNA. The presence of Dam MTase methylated partial ds-DNA structures on the electrode which could be digested by DpnI and could not undergo HCR process. The unmethylated ds-DNA structures underwent HCR process to further hybridize with DNA1 and DNA2 to form ds-DNA superstructures as effective template for AgNCs in-situ synthesis. Oxidation peak current of silver was obtained as signal output for Dam MTase detection. Integrated synthesis and detection in one as well as combined metal nanocluster with DNA superstructures, this biosensor showed a good performance for Dam MTase activity detection with a detection limit of 0.0073 U/mL. Compared with reported biosensor, the designed biosensor showed high sensitivity and had potential in clinical diagnosis as well as inhibitor screening.