A microRNA biosensor based on direct chemical ligation and electrochemically amplified detection

An ultrasensitive microRNA (miRNA) biosensor that utilizes direct chemical ligation and electrochemical signal amplification is described in this work. MicroRNAs in total RNA are directly ligated with an isoniazid-substituted osmium complex, Os(dmpy)(2)(IN)C1(+) (dmpy = 4,4'-dimethyl-2,2'-bipyridine, IN = isoniazid) through a condensation reaction. Gel electrophoretic tests reveal that Os(dmpy)(2)(IN)Cl+ is successfully tagged onto miRNAs. Following hybridization with pre-immobilized complementary capture probes, the resulting biosensor shows excellent electrocatalytic activity towards the oxidation of ascorbic acid. An increase in sensitivity of 2000-fold over direct voltammetry is obtained in electrochemically amplified amperometry, allowing ultrasensitive detection of miRNA. Under optimized experimental conditions, a detection limit of 800 fM and linear current-concentration relationship up to 300 pM are obtained. The biosensor is applied to the quantitation of miRNA in total RNA extracted from HeLa cells. (c) 2006 Elsevier B.V. All rights reserved.