A highly sensitive magnetic separation-assisted electrochemical sensor for detection of m6A-microRNA-17-5p based on rolling circle amplification and N3-kethoxal labeling strategy at LIG electrodes

Background: N6-methyladenosine (m6A) is a prevalent epigenetic modification in eukaryotic RNA and exerts critical regulatory effects on the progression of cancers and Other human disorders. Recent studies have revealed a close association between methylation levels in MicroRNA (miRNA) and human cancers, emphasizing the diagnostic potential of m6A-modified miRNAs for gastrointestinal (GI) cancers. However, the low abundance of m6A in miRNAs has hindered further research, highlighting the need for highly sensitive and selective detection methods capable of analyzing m6A in complex biological samples.

Results: We developed an innovative biosensor based on a dual amplification strategy-combining rolling circle amplification (RCA) with N₃-kethoxal nucleic acid labeling-to enable ultrasensitive detection of m6A-microRNA-17-5p (m6A-RNA) at laser-induced graphene (LIG) electrodes. In the proposed strategy, m6A-RNA is quickly captured from the sample matrix using immunomagnetic beads and binds to the RCA primer-template complex. RCA is initiated under mild isothermal conditions, yielding long guanine-rich single-stranded DNAs (ssDNAs) that can be labeled with N₃-kethoxal. Subsequently, a copper-free click reaction between N₃-kethoxal and DBCO-biotin generates numerous binding sites for streptavidin-labeled horseradish peroxidase (SA-HRP), thereby catalyzing the electrochemical reaction system. The developed sensor demonstrated excellent sensitivity in the linear range of 0.1 pM-10 nM with a low detection limit of 10.1 fM, and performed well in selectivity, stability and reproducibility.

Significance: This study established a novel electrochemical signal amplification strategy, advancing the development of m6A modification analysis. Furthermore, the magnetic separation-assisted LIG electrode platform enables efficient target isolation from complex matrices while promoting device miniaturization. Successful detection of total RNA from MIA PaCa-2 cells and colorectal Cancer (CRC) tissues demonstrates the method's applicability in clinical diagnostics, offering new avenues for GI Cancer research and point-of-care biomarker testing.

Electrochemical biosensor; Laser-induced graphene electrode; MicroRNA; N(3)-kethoxal; N6-methyladenosine; Rolling circle amplification.