Electrochemical based detection of microRNA, mir21 in breast cancer cells

In this work, a novel electrochemical microRNA (miRNA) detection method based on enzyme amplified biosensing of mir21 from cell lysate of total RNA was demonstrated. The proposed enzymatic detection method was detailed and compared with the conventional guanine oxidation based assay in terms of detection limit and specificity. For the detection of mir21, capture probes and/or cell lysates were covalently attached onto the pencil graphite electrode (PGE) by coupling agents of N-(dimethylamino)propyl-N’-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS). Having immobilized the capture probe onto the surface of PGE, hybridization was achieved with a biotinylated (from its 3′ end) complementary target. Extravidin labeled alkaline phosphatase (Ex-Ap) binds to the biotinylated target due to the interaction between biotin–avidin and the enzyme converts electro-inactive alpha naphtyl phosphate (the substrate) to electro-active alpha naphtol (α-NAP, the product). α-NAP was oxidized at +0.23V vs Ag/AgCl and this signal was measured by Differential Pulse Voltammetry (DPV). The signals obtained from α-NAP oxidation were compared for the probe and hybrid DNA. The specificity of the designed biosensor was proved by using non-complementary sequences instead of complementary sequences and the detection limit of the assay was calculated to be 6pmol for cell lysates. ► Enzyme based electrochemical detection of miRNA(mir21) was developed. ► Total RNA containing miRNA was isolated from breast cancer cell lysates. ► Oxidation signal of the enzymatic reaction product, alpha naphtol, was measured. ► Covalently immobilized capture probes/real samples were used as recognition layer. ► A Picomole range of detection was reached as biomarker for microRNA.