Indicator-free electrochemical biosensor for microRNA detection based on carbon nanofibers modified screen printed electrodes Arzum Erdem ⁎, Ece Eksin, Gulsah Congur Faculty of Pharmacy, Analytical Chemistry department, Ege University, Bornova, 35100, Izmir, Turkey The Institute of Natural and Applied Sciences, Biotechnology department, Ege University, Bornova, 35100, Izmir, Turkey abstract article info Article history: Received 18 January 2015 Received in revised form 2 July 2015 Accepted 21 July 2015 Available online 30 July 2015 Keywords: Carbon nanofibers screen printed electrode miRNA differential pulse voltammetry electrochemical impedance spectroscopy Carbon nanofibers (CNFs) are fiber-structured nano-sized carbon materials, which have excellent mechanical, thermal and electrical properties. CNFs possess a wide range of application especially in biorecognition area. In this study, we developed an indicator-free voltammetric biosensor for detection of miRNA-34a target RNA by using carbon nanofiber enriched disposable screen printed electrodes (CNF-SPEs). The sensitive and selective recognition of miRNAs has been an attractive topic due to they have been defined as small RNAs and play major roles in regulating the translation and degradation of messenger RNAs. The characterization of CNF-SPEs in contrast to unmodified ones was firstly performed by scanning electron microscopy (SEM) and electrochem- ical impedance spectroscopy (EIS) techniques. The amino linked DNA probe, that was the complementary se- quence of miRNA-34a, was immobilized onto the surface of CNF-SPEs. After measuring the guanine oxidation signal measured by differential pulse voltammetry (DPV), the detection of sequence-selective hybridization was then investigated between probe and miRNA-34a RNA target, or other miRNA sequences; miRNA-15a, or miRNA-660, which were chosen as noncomplementary sequences (NC). The detection limit was corresponded to 10.98 μg/mL (54 pmol in 35 μL sample) in the linear concentration range of target miRNA-34a from 25 to 100 μg/mL by using CNF-SPEs. The features of this present study was comprehensively discussed by means of the unique characteristics of single-use CNF-SPEs and importance of miRNA monitoring. © 2015 Elsevier B.V. All rights reserved. 1. Introduction Development of biosensing strategies based on nanomaterials has received a great attention in the last decade due to unique physical, chemical and mechanical properties of nanomaterials. Carbon based nanomaterials have been synthesized in different forms as nanoparticle, nanotube, nanowire and nanofiber. Each of them have been utilized to fabricate biosensors in order to detect drugs [1–4], glucose [5–7], nucleic acids [8,9] and other biomolecules [10,11]. Carbon nanofibers are fiber- structured nano-sized carbon materials which have many edge sites on the outer wall without any hollow core. Also, they possess excel- lent mechanical, thermal and electrical characteristics [12]. They are promising nanomaterials and have a potential to be used in a wide range of application, especially in biosensing area for recognition of drugs, proteins, toxins, nucleic acids or other (bio)molecules [1,5,6, 10,11]. Nucleic acids are a class of biomarkers and they have a wide range of application for fabrication of biosensing platforms due to they provide to develop sensitive and selective recognition systems. Although DNA and RNA are the well-known nucleic acids, new generation of nucleic acids have been discovered in diagnostic area such as microRNAs. MicroRNAs (miRNAs) are defined as small RNAs which are length of 18–25 nucleotides and play major roles in regulating the translation and degradation of messenger RNAs [13]. They have been used as bio- markers for diagnosis of fatal diseases [14–19] by using different detec- tion techniques. At this point, electrochemical techniques offer to obtain fast and reliable response, to achieve sensitive and selective detection and to miniaturize fabricated biosensors. Tran and coworkers fabricated a label free electrochemical biosensor platform by glassy carbon (GC) electrodes modi fied with multi walled carbon nanotubes (MWCNT) to detect miRNA-141, which was a biomarker for diagnosis of prostate cancer [16]. In order to test the selectivity of the biosensor, the hybridization was also performed in the presence of different miRNAs, miRNA-103 or miRNA-29b-1. The re- sults of cyclic voltammetry (CV) and square wave voltammetry (SWV) measurements were evaluated by means of detection based on hybrid- ization protocol. In another report [20], a label free miRNA biosensor developed by using thin insulating polymer film modified gold electrodes in order to enhance the analytical signal. After full-match hybridization and Journal of Electroanalytical Chemistry 755 (2015) 167–173 ⁎ Corresponding author at: Faculty of Pharmacy, Analytical Chemistry department, Ege University, Bornova, 35100, Izmir, Turkey. E-mail addresses: arzum.erdem@ege.edu.tr, arzume@hotmail.com (A. Erdem). http://dx.doi.org/10.1016/j.jelechem.2015.07.031 1572-6657/© 2015 Elsevier B.V. All rights reserved. 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