Plasmonic gold nanocube-enhanced SERS biosensor based on INDEBT and click chemistry coupled with exonuclease III-assisted cycling amplification for sensitive detection of ribonuclease H activity.

Ribonuclease H (RNase H) is widely involved in key biological processes such as DNA replication, RNA transcription, and DNA repair, and it is emerging as an important therapeutic target for various diseases. In this study, a plasmonic gold nanocube-enhanced surface-enhanced Raman scattering (SERS) biosensor was developed for highly sensitive detection of RNase H activity by simultaneously integrating instant dehydration in butanol (INDEBT) for SERS tags preparation, copper-free click chemistry for SERS tag conjugation and exonuclease III (Exo III)-assisted target cyclic amplification. The INDEBT method enabled a simpler and highly efficient preparation of SERS tags. Additionally, click chemistry for SERS tags conjugation to the bio-sensing platform circumvented nanoparticle aggregation risk caused by high salt buffer solution necessary for DNA hybridization method in the previous studies. While the sharp geometric features of AuNCs for effective SERS enhancement and Exo III-assisted cyclic amplification stragety significantly enhanced the detection sensitivity. Under optimized experimental conditions, the proposed method exhibited high sensitivity and good selectivity, achieving a limit of detection (LOD) as 3.89 × 10^-7 U μL^-1, which was lower than most reported values in the literature. Furthermore, this method was successfully applied to the detection of RNase H activity in real cellular extracts.

Copyright © 2026 Elsevier B.V. All rights reserved.