Electrochemical based detection of microRNA, mir21 in breast cancer cells

WOS: 000308455700031PubMed ID: 22776181In 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 (alpha-NAP, the product). alpha-NAP was oxidized at +0.23 V vs Ag/AgCl and this signal was measured by Differential Pulse Voltammetry (DPV). The signals obtained from alpha-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. (C) 2012 Elsevier B.V. All rights reserved.Turkish State Planning Organization (DPT)Turkiye Cumhuriyeti Kalkinma Bakanligi [06/DPT/001]We would like to acknowledge Ege University Pharmaceutical Sciences Research Center (FABAL) for enabling us to use its laboratory instruments. This study was financially supported by Turkish State Planning Organization (DPT) with the research project number 06/DPT/001. We also thank Dr. Metiner Tosun and Dr. Cigdem Selli Karakaya for providing us the total RNA which was extract from 293-HEK human embryonic kidney cell line. We also would like to thank Dr. Y. Selamet for FT-IR interpretation