On the basis of hybridized target microRNA (miRNA) strand initiated cleavage of hybridized deoxyribonucleic acid (DNA) capture probes (CPs) by a duplex-specific nuclease (DSN), a highly sensitive and selective label-free miRNA biosensor is developed in this article. Briefly, thiolated DNA CPs are immobilized onto a gold electrode through self-assembly. The electrode is then hybridized to a sample solution containing the target miRNA. The hybridized CPs in the miRNA-CP duplexes are simultaneously cleaved by the DSN, releasing the target miRNA strands back to the sample solution. The released target miRNA strands again hybridize with the remaining CPs on the electrode, thus forming an isothermal amplification cycle. The distinct difference in electrochemical impedance between a control and the DSN cleaved biosensor allows label-free detection of miRNA down to femtomolar levels. The mismatch discrimination ability of the DSN permits miRNA expression to be profiled with high selectivity. The exceptional amplification power of the DSN along with the simple assay protocol makes direct miRNA expression profiling possible in real-world samples with minimal or no sample pretreatments. Attempts are made in direct profiling circulating miRNAs in serum and miRNAs in total RNA extracted from cancer cells.

No abstract

No abstract

A simple and ultrasensitive label-free microRNA (miRNA) biosensor, based on hybridized miRNA-templated deposition of an insulating polymer film and electrochemical impedance spectroscopic detection, is described in this report. The biosensor is made of a monolayer of charge-neutral morpholino capture probes on an indium tin oxide (ITO)-coated glass slide. Upon hybridization, the neutral surface of the biosensor is converted to anionic by the hybridized miRNA strands. The deposition of the insulating polymer film, poly(3,3'-dimethoxybenzidine) (PDB), is then carried out by the horseradish peroxidase-catalyzed polymerization of 3,3'-dimethoxybenzidine in the presence of H(2)O(2). The cumulative nature of the PDB deposition process significantly enhances the sensitivity of the biosensor. Under optimized conditions, miRNA expression profiling can be performed label-freely from 5.0 fM to 2.0 pM with a detection limit of 2.0 fM. The biosensor is applied to the detection of circulating miRNAs in blood and miRNAs in total RNA extracted from cultured cells.

A simple and ultrasensitive colorimetric DNA assay based on the detection of the product of a ligation chain reaction (LCR) and the use of gold nanoparticles (AuNPs) as signal generators has been developed. During LCR, the AuNPs were ligated together, resulting in a distinct color change in real time after a sufficient number of thermal cycles. The cumulative nature of the protocol produced a detection limit of 20 aM with a selectivity factor of 10(3).