Label-free polygonal-plate fluorescent-hydrogel biosensor for ultrasensitive microRNA detection

Abstract Traditional methods for microRNA detection are always dependent on large-scale instruments, labelled DNAs or cumbersome processes, which cannot meet the requirements of on-site detection. To overcome above limitations, we proposed a label-free polygonal-plate fluorescent-hydrogel (PPFH) biosensor for on-site detection of microRNA. In our study, all experimental procedures were easy to handle and performed at a constant temperature without any precision instruments or labelled probes. The target miR-21 initiated the rolling circle amplification (RCA) reaction to produce extensive elongate DNA chains, which offered a facile, economical and efficient strategy to construct a new type of DNA hydrogel called polygonal-plate hydrogel. The RCA reaction endpoint can be judged by naked eye in unconstrained environment. Moreover, this turn-on fluorescent biosensor worked spontaneously by integrating thioflavin T (ThT) with the whole G-quadruplex which was obtained by hybridizing RCA products with a pair of split G-quadruplex probes. The fluorescence intensity of the system was in good linear correlation with the miR-21 concentration in the dynamic range of 0.5-50 nM and the limit of detection was calculated to be 5 pM. The feasibility was also confirmed by detecting spiked serum samples. These results exhibited high stability and dependable detection capability of the PPFH biosensor and provided sufficient support for the practical on-site detection. Moreover, a surprising finding showed the morphological structure of polygonal plates can be controlled by adjusting RCA reaction time and miR-21 concentration. Overall, the PPFH biosensor is friendlier and more affordable compared with traditional methods and makes much progress in on-site microRNA detection.