A strategy for electrochemical biosensing based on dendritic HCR amplification for detection of RNA m5C and m6A methylation

Abstract

Sensitive and efficient detection of RNA methylation sites is considered an integral part of epigenetic assessment. Contrary to previous studies on the detection of RNA m5C or m6A methylation alone, this study innovatively designed a biosensor capable of detecting RNA m5C or m6A methylation at the same time. Substances with specific electrochemical reactions such as ferrocene (FC) or methylene blue (MB) were modified onto the hairpin probe. When the hairpin probe was activated under specific conditions, it triggered a dendritic nonlinear hybridization chain reaction (HCR), which resulted in signal amplification. Gold (Au) and iron tetraoxide (Fe₃O₄) composite nanomaterials were employed as the linking materials: the carboxylated ends of Fe₃O₄ were connected to an antibody that specifically recognizes the m5C and m6A methylation sites, while Au nanoparticle ends adhering to the carboxylated Fe₃O₄ surface are connected to the HCR enhanced signal amplifier. The m5C antibody was linked to Fc-containing HCR amplification products by this material, and similarly, the m6A antibody was linked to MB-containing amplifiers. Thus, the dandelion complex, a multifunctional body with methyl recognition and signal amplification, was formed. The capture probe immobilized on the surface of the gold electrode by Au-S recognized the target RNA sequence by base complementary pairing. Upon addition of the dandelion complex, due to its multi-functionality, the amplified signals were carried to the electrode by the antigen-antibody recognition mechanism, generating a current signal. The positions and heights of the current signal peaks enabled rapid determination of target RNA methyl modification sites and their abundance. The detection limits of this biosensor for RNA m5C and m6A were 4.68×10^-16 M and 1.10×10^-15 M, respectively, and the linear range was from 10^-15 mol/L to 10^-8 mol/L. The sensor developed in this study has the advantages of cost-effectiveness, ease of fabrication, and fast response time, and has the potential to be promoted for RNA methylation detection.