Photoelectrochemical biosensor for highly sensitive detection of microRNA based on duplex-specific nuclease-triggered signal amplification

A selective and sensitive photoelectrochemical assay was proposed for microRNA detection based on the isothermal cycle hybridization amplification of microRNA-21 that originated from the specific cleavage activity of duplex-specific nuclease (DSN) toward a DNA probe in DNA-RNA double helix and in situ enzymatic production of electron donor of ascorbic acid. After the biotin-functionalized DNA probe hybridized with complementary target microRNA-21, the hybridized DNA probe could be cleaved by DSN and microRNA-21 was released back to the incubation solution. The released microRNA could be further hybridized with the remaining single-strand DNA probe; thus, the isothermal cycle hybridization amplification would be formed. As a result, little avidin-alkaline phosphatase conjugate could be captured due to the release of biotin originating from the DSN cleavage. The distinct photocurrent change between a control biosensor and the DSN cleavage biosensor achieved label-free microRNA-21 detection with the linear range from 1 to 500 fM. The fabricated biosensor showed high detection selectivity even for one-base mismatched sequence. The attempt was carried out to directly assay the expression level change of microRNA-21 in total RNA extracted from chicken fibroblast cells infected with subgroup J avian leukosis virus.