DNA walking system integrated with enzymatic cleavage reaction for sensitive surface plasmon resonance detection of miRNA.

Abnormal expression levels of miRNA are associated with various tumor diseases, for example, glioma tumors are characterized by the up-regulation of miRNA-182. Surface plasmon resonance (SPR) assay for miRNA-182 from glioma patients was performed via DNA walking amplification strategy. The duplex between aminated swing arm DNA (swDNA) and block DNA (blDNA), and aminated track DNA (trDNA) with a biotin tag were tethered on the poly(ethylene glycol) (PEG)-modified chips. Upon formation of miRNA/blDNA duplex, the SPR signal decreased with the walking process of swDNA, as the biotinylated fragment of trDNA (biotin-TTGGAGT) was detached from the sensor surface caused by the nicking endonuclease Nb.BbvCI. Such a repeated hybridization and cleavage cycle occurred continuously and the detachment of more biotinylated fragments of trDNA from the chips led to the attachment of fewer streptavidin (SA) molecules and then smaller SPR signals. MiRNA-182 with concentrations ranging from 5.0 fM to 1.0 pM could be readily determined and a detection limit of 0.62 fM was achieved. The proposed method was highly selective and possessed remarkable capability for evaluating the expression levels of miRNA-182 in serum samples from healthy donors and glioma patients. The sensing protocol holds great promise for early diagnosis of cancer patients.