Ultrasensitive Biosensors Detecting m⁶A in Blood: Achieving Early Screening and Typing of Tumors

Abstract

N 6 -methyladenosine (m 6 A) modification is one of the most widespread RNA modifications in eukaryotes and is involved in cancer development and progression by regulating oncogene expression. Herein, a reticulated rolling circle amplification (RCA) cascade reaction was used to construct a novel electrochemical biosensor for ultrasensitive detection of m 6 A, employing ferrocene-tyramine (Fc-Tyr) molecules as electroactive probes. In this strategy, the RCA cascade reaction not only amplifies specific circular DNA in the designed template to reduce the binding with similar nucleic acid sequences but also generates a long ssDNA through multiple repetitions to capture a large number of electrochemical signal probes and achieve the amplification of electrochemical biosensing signals.The developed biosensor demonstrated high selectivity and sensitivity toward m 6 A in the range of 0.5 pM-150 nM, with a detection limit of 14.07 fM. Meanwhile, total RNA extracted from cell samples was analyzed for m 6 A expression levels using the developed biosensor and a commercial colorimetric immunoassay, the biosensor and immunoassay showed consistent results. In addition, m 6 A levels in clinical serum samples were assessed using the developed electrochemical biosensor, which showed that m 6 A expression was much lower in healthy individuals than in cancer patients, therefore the biosensor is promising for cancer typing. This study provides a new method for rapid and convenient tumor marker detection in clinical practice, as well as a new idea for sensitive detection of other biomolecules.