Journal of The Electrochemical Society, 163 (6) B227-B233 (2016) B227 0013-4651/2016/163(6)/B227/7/$33.00 © The Electrochemical Society Label-Free Electrochemical Detection of MicroRNA-122 in Real Samples by Graphene Modified Disposable Electrodes Tugba Kilic, a,b,c Merve Kaplan, d Sibel Demiroglu, d Arzum Erdem, b, z and Mehmet Ozsoz e, z a Department of Biomedical Engineering, Faculty of Engineering and Architecture, Izmir Katip Celebi University, 35620 Cigli, Turkey b Department of Analytical Chemistry, Faculty of Pharmacy, Ege University, 35100 Bornova, Turkey c Swiss Federal Institute of Technology Lausanne, EPFL, Integrated Systems Laboratory, 1015 Lausanne, Switzerland d Department of Nanotechnology, Institute of Science, Gediz University, 35665 Seyrek, Turkey e Department of Biomedical Engineering, Faculty of Engineering and Architecture, Gediz University, 35665 Seyrek, Turkey In the present work, an electrochemical nucleic acid biosensor has been designed for the purpose of detection of miR-122 in real samples, i.e cell lysates. The fabrication of the biosensor has been done first by immobilization of complemantary anti-miR-122 to the surfaces of the graphene (GRP) modified pencil graphite (PGEs) electrodes then solid phase hybridization with either synthetic miR-122 or miR-122 included in total RNA isolated from HUH-7 cell line. The characterization of GRP modification onto the PGE surface has been proved via Raman spectroscopy analysis, Electrochemical Impedance Spectroscopy (EIS) and Differential Pulse Voltammetry (DPV). The intrinsic guanine oxidation signal measured via DPV and the charge transfer resistance, R ct values recorded via electrochemical circle fit option of EIS have been used for hybridization detection. The proposed biosensor with the limit of detection (LOD) 1 pmol is applicable for analysis of certain miRNAs as well as miR-122 from total RNAs isolated from cell lysates. © 2016 The Electrochemical Society. [DOI: 10.1149/2.0481606jes] All rights reserved. Manuscript submitted October 28, 2015; revised manuscript received February 24, 2016. Published March 10, 2016. Current understanding of “central dogma” experiencing a paradigm shift by the discovery of 18–25 nucleotide long, non-coding, post-transcriptional gene regulators, naturally occuring RNAs; mi- croRNAs (miRNAs). 1 Growing evidence suggests that miRNAs not only have a pivotal role in various cell processes like differentiation, apoptosis, homeostasis and development but also responsible for vari- ous pathologies including cancer. 2 The biogenesis of miRNAs consists of three steps; i) transcription of primary miRNA (pri-miRNA) from miRNA coding genes ii) formation of pre-miRNA iii) maturation of pre-miRNA to miRNA. 3 Previous studies have paved the way for miRNA mediated can- cer occurences and miRNAs are classified into two categories as; oncogenic miRNAs which are up-regulated in cancer tissues or tumor suppressor miRNAs whose expression level is decreased compared to healthy tissue. 4 Such a specific relation between dyseregulation of certain miRNAs with specific types of cancers have taken great atten- tion in academia and excited researchers for the idea of designing new tools for early cancer diagnosis based on miRNA detection and/or classification. 5,6 miR-122 is a liver-specific microRNA that is down regulated in liver cancers and known to be a tumor suppressor. Additionaly, there are several functions of miR-122 in biological processes like anti apoptosis, lipid metabolism and hepatit-C occurence. 7 Current detection methods of miRNAs are based on conven- tional RNA analysis techniques like Northern blot, 8 microarray 9,10 and qRT-PCR. 11 Although, these methods have some advantages such as enabling multiplexed detection, quantification and classification of miRNAs, they are time and sample-consuming and often require expensive equipments and toxic chemicals like some dye labels. Oligonucleotide microarray-based detection methods have the ca- pability of simultaneous detection of large numbers of miRNAs in biological systems. 12 Although the locked nucleic acid 13 probes have been shown to detect miRNAs in a much more sensitive manner than traditional DNA probes in Northern blotting of miRNA, the cost in chip manufacturing and availability of specialized read-out instruments can be complex for the studies wherein the concentra- tion of a specific target miRNA is measured. Electrochemical biosen- sors based on nucleic acid detection are one of the most fascinating techniques for miRNA analysis since they are simple, suitable for point-of-care testing and cost effective. 14 Recently, those biosensors have been combined with nanomaterials for miRNA detection like z E-mail: mehmet.ozsoz@gediz.edu.tr; arzum.erdem@ege.edu.tr carbon nanotubes, 15,16 gold nanoparticles, 17 magnetic beads, 18,19 sili- con nanowires 20 quantum dots 21 or graphene (GRP). 22,23 Additionally, electrochemical biosensors depend on hybridization techniques were evolved as label free 23,24 or label based techniques like the ones using enzymes 25 and virus encoded silencing proteins. 26 The latest form of nanocarbon, graphene is an inspiring material and exhibits great electrical, thermal, mechanical, optic and magnetic properties due to its unique, anatomically thin honeycomb structure of sp 2 hybridized carbon atoms. 27 Graphene has single, bi-, tri- or few layers of thickness. It is easier to adsorb DNA/RNA onto GRP surfaces due to stronger π−π stacking, hydrophobic interaction, hy- drogen bonding, and van der Waals forces exist between the surface of GRP electrode and DNA/RNA rather than graphene Oxide (GO) which is an oxidized form of GRP that has negative surface charge. 28 Owing to those superior advantages compared to other nanomaterials, graphene subjected to many studies across different disciplines. Like- wise, various electrochemical nucleic acid based research conducted 29 based on graphene with the purpose of miRNA detection, 30 drug-DNA interaction, 31 and glucose 32 detection. The aim of our study is to develop novel, rapid, inexpensive, label- free, sensitive and selective method to detect miR-122 in total RNA extracted from HUH-7 cell line based on guanine oxidation signal and the charge transfer resistance measured by electrochemical impedance spectroscopy. After graphene modification onto electrode surface, the enhancement at the guanine signal was obtained due to good electri- cal conductivity properties of graphene. Total RNA sample is used directly without using any purification, or amplification step. To the best of our knowledge, this is the first study that presents the detection of miR-122 in cell lysates by EIS and DPV techniques by graphene modified pencil graphite electrodes GRP/PGEs. The characterization of electrode surface area was done by raman spectroscopy, EIS and DPV. The designed biosensor exhibits 1 pmol detection limit with a good selectivity that makes it applicable for detection of other miR- NAs as well as miR-122 in real total RNA samples. Experimental Apparatus and chemicals.—AUTOLAB PGSTAT204 Compact and modular potentiostat/galvanostat system was used DPV and EIS measurements. 33 The conventional three electrode system, consist- ing of the pencil graphite electrode (PGE) as the working electrode, (Ag/AgCl) as a reference electrode and a platinum wire as an auxiliary electrode were engaged in connection with the Nova 1.10 software. ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.179.180.181 Downloaded on 2016-03-15 to IP