Electrochemical ultrasensitive detection of miRNA-21 via RAFT/ROMP dual polymerization amplification.

Abstract

The ultrasensitive detection of microRNAs in bodily fluids is crucial for early diagnosis of cancer. Herein, we design an electrochemical biosensor based on a dual-signal amplification strategy utilizing reversible addition-fragmentation chain transfer (RAFT) and ring-opening metathesis polymerization (ROMP) to achieve ultra-sensitive quantitative detection of miRNA-21. The sensing interface starts from a gold electrode carrying a thiol-immobilized peptide nucleic acid (PNA) capture probe. Once miRNA-21 hybridizes specifically with the PNA, we anchor the RAFT chain-transfer agent 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (CDTPA) onto the duplex by taking advantage of Zr⁴⁺ coordination chemistry. With the azo initiator 2,2'-azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride (VA-044), in solution, the monomer dicyclopentenyloxyethyl acrylate (DCPEA)-which bears a cyclopentene side group-undergoes RAFT polymerization, decorating the duplex with polymer chains that bristle with pendant cyclopentene rings. Subsequently, the ruthenium-based catalyst AquaMet ((4-((4-ethyl-4-methylpiperazin-1-ium-1-yl)methyl)-1,3-dimesitylimidazolidin-2-ylidene)dichloro(2-isopropoxybenzylidene) ruthenium(II)) chloride coordinates with the cyclopentene groups, catalyzing the ROMP of cyclopentene-based ferrocene (CFc), thereby forming a polymer brush rich in ferrocene electroactive molecules on the electrode surface, achieving efficient two-stage amplification of the electrochemical signal. Under optimized conditions, the sensor returns a linear response to miRNA-21 from 10^-17 M to 10^-13 M and reaches a detection limit as low as 4.72 aM. Signals from single-mismatched RNA, triple-mismatched RNA, and miRNA-320b were all strongly suppressed relative to the target, while the sensor also proved to be stable in one week and remarkably unfazed by complex biological backgrounds.