A highly sensitive electrochemical assay for microRNA expression profiling

Abstract

A simple and highly sensitive electrochemical assay for ligation-free and polymerase chain reaction (PCR)-free microRNA (miRNA) expression profiling is described in this work. The electrode used in the assay was made of a monolayer of stem-looped capture probes (CPs) comprising of a miRNA complementing region at one end and detection probes (DPs) receiving region at the other. It engaged an electrocatalytic reaction between electrochemically activated glucose oxidase (GOx) and glucose to enhance its sensitivity. Briefly, upon hybridizing to its target miRNA, the stem loop is unlocked exposing the DP receiving region. A subsequent hybridization with the DPs brought them together with an amplifier, the activated GOx, onto the electrode. The activated GOx exhibited excellent catalytic activity towards electrooxidation of glucose. MicroRNA detection could therefore be conducted in 60 mM glucose in phosphate-buffered saline. A detection limit of 4.0 fM and a linear calibration curve up to 10 pM were obtained under optimal conditions. The assay was applied to profile human let-7 miRNA expressions in cultured cancer cells.