Ratiometric electrochemical biosensor for ultrasensitive and highly selective detection of p53 gene based on nicking endonuclease-assisted target recycling and rolling circle amplification

Tumor suppressor p53 gene is one vital anticancer gene that plays critical role in the prevention, diagnosis, and prognosis evaluation of cancers. Specific recognition and sensitive detection of p53 gene is important for early cancer screening and diagnosis. A ratiometric electrochemical biosensor is developed for ultrasensitive detection of tumor suppressor p53 gene in this work. Hairpin capture probe (Fc-HP-SH) is labeled with ferrocene (Fc) at the 3 ' termini and thiol group at the 5 ' termini. Fc-HP-SH was self-assembled on the surface of gold electrode through Au-S binding, while 6-mercaptohexanol was used to block the unoccupied sites on electrode surface. At the presents of p53 gene and nicking endonuclease, p53 gene specifically hybridizes with the loop sequences of FcHP-SH to unwind the hairpin structure. Nicking endonuclease subsequently cleaves DNA duplex to release p53 gene for target recycling. Residual single-stranded oligonucleotide is prolonged with the help of phi29 DNA polymerase via rolling circle amplification, leading to the increased gathering of electroactive probe methylene blue (MB) on long polynucleotide. Such variations cause the alterations of the differential pulse voltammetry peak currents of Fc at + 0.32 V (vs. Ag/AgCl) and MB at - 0.25 V (vs. Ag/AgCl), resulting in an obvious enhancement of the ratiometric signal of two peak currents. Under optimum experimental conditions, this biosensor has a response that covers the 1 fM to 10 pM p53 gene concentration range, and the detection limit is 0.23 fM (for S/N = 3). This biosensor highly recognized p53 gene over its mutants and was successfully applied in human serum samples. This approach shows excellent reproducibility, acceptable stability, good selectivity, and ultrahigh sensitivity toward p53 gene detection.