Homogeneous photoelectrochemical biosensor for microRNA based on target-responsive hydrogel coupled with exonuclease III and nicking endonuclease Nb.BbvCI assistant cascaded amplification strategy

MicroRNAs can serve as biomarkers for many cancers, so it is significant to develop simple and sensitive strategies for microRNAs detection. Photoelectrochemical (PEC) detection has the advantages of simple equipment and high sensitivity. But in conventional PEC DNA sensors, tedious immobilization procedures of photoactive materials and capture probes on electrode surfaces are inevitable. To overcome those limitations, a homogeneous PEC biosensor based on target-responsive hydrogels has been developed (miRNA-155 has been chosen as a model target). PEC signal molecules (TiO 2 nanoparticles, TiO 2 NPs) were embedded in DNA hydrogels formed by hyaluronic acid sodium salt, amine-modified DNA double strands, and polyethylenimine rich in amine groups. In the presence of the target, DNA double strands in hydrogel were nicked by endonuclease and TiO 2 NPs were released to the supernate and a high PEC response was obtained when collecting the supernate for PEC test, while almost no TiO 2 NPs released in the absence of the target. Thanks to the exonuclease III and nicking endonuclease Nb.BbvCI-assisted cascaded amplification strategy, the proposed biosensor exhibits high sensitivity toward miRNA-155 with a low detection limit of 0.41 fM and a wide linear range from 1.0 fM to 100 pM. Since this method circumvents tedious electrode modification procedures, the proposed technique exhibits the advantages of simplicity and good reproducibility. Moreover, the prepared hydrogels have outstanding storage stability, so that they can be prepared in advance and shorten detection time. This biosensing platform provides a versatile strategy for the construction of homogeneous PEC biosensors for the detection of diverse targets. Graphical abstract Photoelectrochemical detection techniques have been coupled with controlled release system to develop an immobilization-free microRNA biosensor. High sensitivity has been realized based on cascaded signal amplification strategy, and the proposed biosensor has been applied to detect the target in real sample with satisfied results. Since no tedious electrode modifications, the proposed homogeneous PEC sensor exhibits high reproducibility and good stability.