An ingenious double-amplified fluorometric genosensor based on hybridization chain reaction and enzymatic signal for the sensitive detection of piRNA–651

Small noncoding RNAs known as P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs/piRs) have attracted significant interest because of their pivotal role in the regulation of various biological processes, including carcinogenesis. Recently, piR–651 has been reported to be overexpressed in human sera and solid cancer tissues, such as breast, gastric, and lung cancers. Early diagnosis can alleviate the cancer burden and reduces the mortality rates. However, conventional detection methods of piRNAs are time-consuming and costly, limiting their clinical applications. In the present work, we report for the first time a fluorometric biosensor for highly sensitive and specific detection of piR–651. An ingenious combination of the target-catalyzed hairpin assembly (CHA) and enzymatic signal amplification yielded a high fluorescence signal. Indeed, the presence of piR–651 triggered the CHA process between biotin-modified hairpin 1 (Bt-H1) and hairpin 2 (H2), leading to the cyclic reuse of piR–651 and producing a large amount of H1–H2 hybrid. The CHA product was captured by a hairpin 3 (H3) immobilized on magnetic beads (MBs) for easy magnetic separation. Streptavidin-Horseradish peroxidase (Sterp-HRP) was used to reveal the CHA product by catalyzing the oxidation of o–phenylenediamine (o–PDA) substrate into fluorescent 2,3–diaminophenazine (DAP), excited at 450 nm and the emission was measured at 570 nm. The fluorescence intensity was found to be positively proportional to the piR-651 concentrations. An experimental design approach was used to optimize the main influential parameters of the bioassay. Under the optimal conditions, the biosensor exhibited a linear response to piR-651 from 5 fM to 200 fM with a detection limit of 0.1 fM, while effectively discriminating other piRs. It was also successfully applied to the determination of piR-651 levels in spiked human serum. Our newly developed fluorometric biosensor offers a highly sensitive and specific detection of piR-651, enabling its potential application in early cancer diagnosis and facilitating the study of its role in various cancer types.