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Multi channel screen printed array of electrodes for enzyme-linked voltammetric detection of MicroRNAs
Sensors and Actuators B 188 (2013) 1089–1095
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Multi channel screen printed array of electrodes for enzyme-linked voltammetric detection of MicroRNAs Arzum Erdem a,∗ , Gulsah Congur a,b , Ece Eksin a,b a b
Ege University, Faculty of Pharmacy, Analytical Chemistry Department, 35100 Bornova, Izmir, Turkey Ege University, The Institute of Natural and Applied Sciences, Biotechnology Department, 35100 Bornova, Izmir, Turkey
a r t i c l e
i n f o
Article history: Received 2 June 2013 Received in revised form 21 July 2013 Accepted 30 July 2013 Available online 8 August 2013 Keywords: Multi channel screen-printed array of electrodes Streptavidin-alkaline phosphatase Pencil graphite electrode miRNA Magnetic beads MUX-SPE
a b s t r a c t In the present work, a sensitive and selective enzyme-linked electrochemical sensor technology using magnetic beads assay was demonstrated for voltammetric detection of microRNAs (miRNAs) as the first time herein by using the multi-channel screen-printed array of electrodes (MUX-SPE16s). The streptavidin coated magnetic beads were used as a solid phase to immobilize biotinylated DNA capture probe, and then the complementary target RNA sequence (miRNA-15a) was recognized with the capture DNA probe. After attachment of the streptavidin–alkaline phosphatase enzyme (SALP) to biotinylated hybrid, the electroactive product ␣-naphthol was measured +0.188 V by linear sweep voltammetry (LSV) technique in combination with a single-use pencil graphite electrode (PGE) compared to MUX-SPE16. The oxidation signal of ␣-naphthol indicates the hybridization occurred between DNA probe and its RNA target in the sample. The selectivity of our assay was tested in the presence of non-complementary miRNA sequence, a very good discrimination was achieved compared to the results obtained with the full match hybridization of probe with miRNA-15a target. The detection limit of miRNA-15a target sequence was also calculated as 0.992 g/mL (98.60 pmole in 1 mL sample) at PGE, and 0.114 g/mL (34.20 fmole in 3 L sample) with MUX-SPE16 system. © 2013 Elsevier B.V. All rights reserved.
1. Introduction MicroRNAs (miRNAs) are a class of non-coding RNA gene whose final product is a ∼22 nt functional RNA molecule. They play important roles in the regulation of target genes by binding to complementary regions of messenger transcripts to repress their translation or regulate degradation [1–3]. Since the discovery of the founding members of the miRNA class, lin-4 and let-7 in Caenorhabditis elegans [4], over 2000 miRNA sequences have been described in vertebrates, flies, worms and plants, and even in viruses. However, the functions of only a handful of these miRNAs have been experimentally determined. In parallel with novel gene identification efforts, the miRNA community is therefore focused on predicting and validating miRNA gene targets [5]. The great interest in miRNAs reflects their central role in gene-expression regulation and the implication of miRNA-specific aberrant expression in the pathogenesis of cancer, cardiac, immune-related and other diseases. Another avenue of current research is the study of circulating miRNAs in serum, plasma, and other body fluids—miRNAs may act not only within cells, but also at
∗ Corresponding author. Tel.: +90 232 311 5131; fax: +90 232 388 5258. E-mail addresses: [email protected], [email protected] (A. Erdem). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.07.114
other sites within the body. The presence of miRNAs in body fluids may represent a gold mine of noninvasive biomarkers in cancer. Since deregulated miRNA expression is an early event in tumorigenesis, measuring circulating miRNA levels may also be useful for early cancer detection, which can contribute greatly to the success of treatment [6]. Mitchell and co-workers have recently showed that tumor cells shed tumor derived microRNAs into the blood stream and that microRNAs are present in the blood and serum in remarkably stable forms [7]. Serum circulating miRNAs are promising novel biomarkes for early cancer detection and for improved cancer screening, even if the detection protocol is required labeling of miRNAs with any different groups; such as, biotin [8,9], or any metal complexes [3]. At the present time, miRNAs are detected by using numerous techniques; Northern blot [10], in situ hybridization [11], RT-PCR [12], and microarrays [13] using various types of commercial diagnostic kit, even after the biotinylation of miRNAs. Nevertheless, they are expensive, time-consuming for performing the assay, and need an equipped laboratory with specialized and well-trained people. Because of these reason, they are not feasible for routine detection of serum-based microRNAs [14]. Magnetic beads (MBs) have received a great attention due to their potential use in various biotechnological applications, including protein and enzyme immobilization, separation and sorting proteins and cells, drug delivery system, hyperthermia treatment for cancerous tumors and immunoassays [15,16]. A major
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advantage of MBs is the easy and rapid recovery of target molecules from the reaction mixture by the efficient magnetic separation. The performance of MBs could be significantly affected by surface modifications. Various molecules could be easily immobilized onto the surfaces of MBs by surface chemistry [17]. The double surface technique based on magnetic beads sepration are well-known for electrochemical biosensing of nucleic acids (NAs) [18–22]. Under the principle of magnetic beads assay, a biotinylated DNA probe was firstly immobilized in our study onto the streptavidin coated magnetic particles (MB) and then, the hybridization of probe with its complementary RNA sequence was performed at MB surface. To the best of our knowledge, the multi-channel screen-printed array of electrodes (MUX-SPE16s) has been used as the first time herein for enzyme-linked detection of miRNA-15a hybridization related to Alzheimer disease. After labeling the biotinylated hybrid with a streptavidin–alkaline phosphatase (SALP) conjugate, the enzymatic reaction was performed. Consequently, the electroactive product, ␣-naphthol occurred, and it was then measured by using LSV technique in combination with MUX-SPE16s. The same experimental procedure was followed for detection of miRNA using the single use pencil grahite electrode (PGE). The features of both sensor technologies in combination with magnetic beads for the electrochemical detection of miRNA-15a were then discussed concerning to their detection limit with reproducibility, and also the selectivity of each sensor by comparing the results of earlier reports on the electrochemical miRNA detection related to different miRNA sequences. 2. Experimental 2.1. Apparatus The oxidation signal of ␣-naphthol was measured by AUTOLAB PGSTAT electrochemical analysis system and GPES 4.9 software package (Eco Chemie, The Netherlands) using linear sweep voltammetry (LSV) technique. The raw data were also treated using the Savitzky and Golay filter (level 2) of the GPES software, followed by the moving average baseline correction with a “peak width” of 0.03. MUXSCNR16-multichannel system was used to perform LSV measurements with MUX-SPE16s. The three-electrode system consisted of the PGE, an Ag/AgCl/KCl reference electrode and a platinum wire as the auxiliary electrode was used. The experiments were also performed by using multi-channel screen-printed array of electrodes (MUX-SPE16). The preparation of magnetic beads and hybridization process were performed on the magnetic separators, MCB 1200 (Sigris, USA). 2.2. Chemicals Streptavidin–alkaline phosphatase (SALP), ␣-naphthyl phosphate, bovine serum albumin (BSA) were obtained from Sigma–Aldrich. The DNA probes and complementary RNA or DNA oligonucleotides (RNA or DNA ODN), were purchased (as lyophilized powder) from TIB Molbiol (Germany). The base sequences of ODNs are listed below (X: Uracil): miRNA-15a DNA probe 5 -TCA AAA TCC ACA AAC CAT TAT GTG CTG CTA CTT TAC TCC AAG G–Biotin-3 Complementary miRNA-15a (DNA) 5 -TAG CAG CAC ATA ATG GTT TGT GGA TTT TGA–Biotin-3 Complementary miRNA-15a (RNA) 5 -XAG CAG CAC AXA AXG GXX XGX GGA XXX XGA–Biotin-3 Non-Complementary miRNA ODN (NC-miRNA) 5 -AGX GXX XCG GCC CAA XCA CXA XCX CCC GXC–Biotin-3
All ODN stock solutions (500 g/mL) were prepared with TrisEDTA buffer solution (10 mM Tris–HCl, 1 mM EDTA, pH: 8.00; TE) and kept frozen. More dilute solutions of ODNs were prepared with either TTL buffer (100 mM Tris–HCl, pH 8.00, and 0.1% Tween 20 and 1 M LiCl) or 50 mM phosphate buffer solution (PBS, pH 7.40) according to the hybridization protocol. SALP stock solution (1000 g/mL) was prepared with 0.1 M Tris–HCl (pH 9.20) containing 1 mM MgCl2. More diluted solutions of SALP were prepared in 50 mM phosphate buffer solution (PBS, pH 7.40) containing 2% BSA. Required amount of ␣-naphthyl phosphate was prepared in carbonate buffer (CBS, pH 9.50) containing 0.50 M Na2 CO3 and 0.50 M KHCO3 . Streptavidin coated magnetic particles (magnetic beads) in 0.94 mm diameter size were purchased from Estapor, Merck (France). Other chemicals were in analytical reagent grade and they were supplied from Sigma and Merck. 2.3. Electrode preparation 2.3.1. Pencil graphite electrodes (PGEs) A Tombo® pencil was used as a holder for the graphite lead. Electrical contact with the lead was obtained by soldering a metallic wire to the metallic part. The pencil was held vertically with 14 mm of the lead extruded outside (10 mm of which was immersed in the solution). PGEs were prepared according to the protocol previously reported in the literature [19,23]. The PGEs were electrochemically pretreated by applying +1.40 V for 30 s in 0.50 M acetate buffer solution containing 20 mM NaCl (pH 4.80; ABS). 2.3.2. Screen-printed array of electrodes (MUX-SPE16s) The MUXSCNR-16 compatible MUX-SPE16 was developed and characterized by Dropsens Company (Spain) containing one main electrochemical cell with sixteen working carbon electrodes combined with one reference electrode and one auxiliary electrode. Each working electrode at MUX-SPE16s was pretreated one by one by applying +0.90 V for 60 s in ABS. 2.4. Preparation of probe-coated magnetic particles and DNA hybridization at the surface of magnetic particles The preparation of ODN-coated microspheres (magnetic beads, MBs) was performed by following the reported procedure [19,20]. 5 L of streptavidin-coated MBs was transferred into a 1.5 mL centrifuge tube. The microspheres were then washed twice with 90 L TTL buffer and resuspended in 30 L TTL buffer containing 60 g/mL biotinylated probe and incubated for 15 min at room temperature with gentle mixing. The immobilized probe was then separated and washed once with 90 L TT buffer (250 mM Tris–HCl, pH 8.0, and 0.1% Tween 20), resuspended in 20 L PBS containing desired concentration of biotin capped complementary RNA/DNA (target) of probe, or non-complementary miRNA sequences. The hybridization reaction was carried out at room temperature during 10 min. The hybridized microsphere conjugates were then washed twice with 90 L PBS. After hybridization and washing steps, MBs were incubated with a solution containing required amount of SALP for 15 min. MBs were then washed with 90 L of PBS-T buffer (PBS containing 0.05% Tween20) and washed again with 90 L PBS buffer. SALP conjugated MBs resuspended in 100 L CBS buffer solution containing required amount of ␣-naphthyl phosphate and incubated for 15 min at room temperature with gentle mixing. As a result of enzymatic reaction, electroactive product ␣-naphthol was occurred. 100 L supernatant was transferred into the vial, which contains 900 L of CBS. The mixed sample containing ␣-naphthol was transferred into the 1 mL electrochemical cell, and the voltammetric transduction was performed.
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For miRNA detection at MUX-SPE16s, 3 L of sample was only dropped onto the surface of consecutively each working electrode of MUX-SPE16s, and kept for passive adsorption step during 15 min. Then, the MUX-SPE16s was washed with CBS three times. The MUX-SPE16s was connected with multichannel system integrated to electrochemical analyzer. 850 L CBS was then dropped onto the full surface of MUX-SPE16s to perform consecutively the LSV measurements. 2.5. Voltammetric transduction The oxidation signal of ␣-naphthol was measured by using LSV by scanning from −0.3 V to +1.2 V at a pulse amplitude of 4.8 mV and a scan rate of 1 V/s. 3. Results and discussion As seen in Scheme 1 the miRNA detection protocol required firstly hybridization step performed at MB surface modified with biotinylated miRNA-15a DNA probe and with its complementary sequence labeled with biotin. The streptavidin labeled enzyme, alkaline phosphatase (SALP) was then introduced to this sample for performing the enzymatic reaction binding of enzyme to the biotin linked target sequence. After the interaction between the substrate of SALP; ␣-naphthyl phosphate and SALP, the electroactive product, ␣-naphthol was occurred in the media and consequently, the oxidation signal of product was measured by using LSV technique. Fig. 1 shows the oxidation signal of ␣-naphthol measured before and after hybridization between 60 g/mL miRNA-15a DNA probe and 60 g/mL miRNA-15a DNA target. The control experiments were firstly done by using MB alone, and full hybridization in the absence of substrate, and no signal was observed (Fig. 1a and b). In the presence of full hybridization case, 11 times higher ␣-naphthol
Fig. 1. Histograms and voltammograms (inset) representing the ␣-naphthol oxidation signals measured by using PGEs at control experiment of (a) MB alone, in the case of the full hybridization, (b) in the absence of ␣-naphthyl phosphate, and (c) before hybridization between 60 g/mL miRNA-15a DNA probe and 60 g/mL miRNA-15a DNA target, and (d) after hybridization in the presence of 50 g/mL SALP and 1 mM ␣-naphthyl phosphate.
signal (Fig. 1d) was measured at +0.188 V comparison to the one recorded before hybridization (Fig. 1c). It indicated that the spesific miRNA-15a detection was successfully achieved by using magnetic beads assay combined with PGE. Next, the effect of enzyme concentration based on ␣-naphthol signal was investigated in the presence of full hybridization at PGE surface (Fig. S1). The highest ␣-naphthol signal was obtained in the presence of enzymatic reaction with 50 g/mL of SALP (Fig. S1B-b). In the case of full hybridization between 60 g/mL DNA probe and 60 g/mL miRNA-15a DNA target, the average ␣-naphthol signal was measured as 314.88 ± 35.09 A with relative standart deviation (RSD%) 11.14% (n = 3), that was 11 times higher than one obtained in the absence of hybridization (probe alone). Therefore,
Scheme 1. Schematic representation of enzymatic detection protocol in combination with magnetic beads assay using disposable graphite sensor technologies; (A) PGE and (B) MUX-SPE16s.
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Fig. 2. Voltammograms (I), histograms (II) representing ␣-naphthol signals measured at PGE in the presence of enzymatic reaction between 50 g/mL SALP and 2 mM ␣-naphthyl phosphate. Before (a) and after hybridization between 60 g/mL miRNA-15a DNA probe and miRNA-15a DNA target in different concentrations: 2.5 g/mL (b), 5 g/mL (c), 7.5 g/mL (d), 10 g/mL (e), 12.5 g/mL (f), 15 g/mL (g).
50 g/mL of SALP concentration was chosen as optimum for further studies in our study. The effect of substrate concentration was also investigated at 50 g/mL SALP concentration (Fig. S2). ␣-naphthol signal gradually increased till 2 mM substrate concentration (Fig. S2C-b), then it leveled off. A series of three repetitive LSV measurements at 2 mM concentration level of ␣-naphthyl phosphate using PGE, the average signal was measured as 408.46 ± 21.25 A with the RSD% of 5.20% (n = 4). The further experiments were performed in 2 mM concentration of ␣-naphthyl phosphate. Upon the changes at the ␣-naphthol signal, the effect of RNA target concentration was also studied in the case of full hybridization between probe DNA and miRNA-15 target in its different concentration from 2.5 to 15 g/mL (Fig. 2). The response gradually increased till 10 g/mL target RNA concentration (Fig. 2-I and II-e), and then it almost leveled off. The RSD % was found as 9.97% for three successive determinations at 10 g/mL target RNA concentration by using PGE. The linear graph based on the sensor response upon various target RNA concentrations was shown in Fig. 3. The limit of detection (DL) was calculated with the aid of the section of the calibration plot close to the origin, which is linear, utilizing both the regression equation and the definition y = yB + 3sB (yB is the signal of the blank solutions and sB is the standard deviation of the blank solution) as outlined in reference [24]. Thus, the DL was estimated as 0.992 g/mL (98.60 pmole in 1 mL sample) at PGE. The selectivity of this biosensor system was analyzed based on ␣-naphthol signal, that was obtained in the presence of hybridization between DNA probe and miRNA-15a RNA target, or non-complementray miRNA sequences by using PGE (Fig. 4). In the case of hybridization with NC-miRNA sequence, the negligible small ␣-naphthol signal was measured compared to the signals of full match hybridization between DNA and 10 g/mL miRNA15a (Fig. 4A-I and II-b to c), and 60 g/mL (Fig. 4B-I and II-b to
Fig. 3. The calibration plot based on the ␣-naphthol signals measured at PGE in the presence of enzymatic reaction in the case of full hybridization between 60 g/mL miRNA-15a DNA probe and miRNA-15a RNA target concentrations varying from 0 to 10 g/mL by using 50 g/mL SALP and 2 mM ␣-naphthyl phosphate.
c) miRNA-15a. For further selectivity study, it was tested in the mixture samples containing RNA target and NC-miRNA sequences at both concentrations; 10 g/mL (Fig. 4A-I and II-d), and 60 g/mL (Fig. 4B-I and II-d). However there was an excess of NC-miRNA sequence including target RNA in the medium (1:1), it was found that a selective voltammetric detection of miRNA-15a was explored by the advantage of efficient magnetic separation even in the mixture samples. Compared to the first part of our study performed by using PGE, more sensitive and selective enzyme-linked electrochemical sensor technology using magnetic beads was demonstrated for voltammetric detection of miRNAs as the first time herein by using MUX-SPE16s, which have also brought the numerous opportunities herein; such as, easy-to-use, disposable, cost effective, sensitive and
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Fig. 4. Voltammograms (I), histograms (II) representing the ␣-napthol signals measured at PGE after hybridization between 60 g/mL miRNA-15a DNA probe and (A) 10, (B) 60 g/mL miRNA-15a RNA target, or NC-RNA, or miRNA-15a RNA target in the mixture sample with NC-RNA (1:1) by using PGE. ␣-naphthol signals (a) before and after hybridization with miRNA-15a DNA probe and (b) miRNA-15a RNA target, (c) NC-RNA, or (d) the mixture sample of RNA target:NC-RNA (1:1).
signal in various target concentration from 0.5 to 3 g/mL (Fig. 5). Although the highest ␣-naphthol signal was measured at 3 g/mL concentration level of RNA target (Fig. 5-A and B-f) with a RSD% as 19.20% (n = 3), the better reproducible result was recorded in the case of 2 g/mL concentration level of target (38.09 ± 2.90 A with the RSD of 7.63%, n = 3). The DL was calculated by following the equation [24] as explained above, and it was found as 0.114 g/mL (34.20 fmole in 3 L sample) at MUX-SPE16s. The selectivity studies were firstly performed at 2 g/mL concentration level of miRNA-15a target, however the selective results could not be obtained at this concentration level (not shown). Afterwards, the selectivity of our assay was then tested at a higher RNA concentration as 10 g/mL (Fig. 6). In the case of full hybridization, 2.5 times higher ␣-naphthol signal (Fig. 6-b) was measured compared to one obtained in the absence of DNA-RNA hybridization (probe alone) (Fig. 6-a). After probe hybridization with NC-miRNA, there was a negligible change observed at ␣-naphthol signal (Fig. 6c) due to the efficient magnetic separation using magnetic beads assay. Even in a mixture media containing excess amount of NCmiRNA besides target RNA, the ␣-naphthol signal was measured almost in the same level (Fig. 6-d) of full hybridization signal.
Fig. 5. (A) Histograms representing the ␣-naphthol signals measured at MUX-SPE16 system before (a) and after hybridization between 60 g/mL miRNA-15a DNA probe and 2.5 g/mL (b), 5 g/mL (c), 7.5 g/mL (d), 10 g/mL (e), 12.5 g/mL (f), 15 g/mL (g) miRNA-15a RNA target concentrations. (B) The calibration plot obtained from ␣naphthol signals after hybridization with DNA probe and miRNA-15a RNA target concentrations varying from 0.5 to 3 g/mL.
selective detection compared to the results of earlier electrochemical assays for detection of miRNAs [25–29]. The effect of target miRNA-15a concentration was firstly investigated using MUX-SPE16s based on the changes of the ␣-naphthol
Fig. 6. Histograms representing the ␣-naphthol signals measured at MUX-SPE16s before (a) and after hybridization between 60 g/mL miRNA-15a DNA probe and (b) 10 g/mL miRNA-15a RNA target, (c) 10 g/mL NC-miRNA and (d) the mixture sample of miRNA-15a:NC-miRNA(1:1).
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4. Conclusion The detection and monitoring of miRNAs has recently become a frontline topic due to the fact that they are well-known biomarkers for different types of cancer [30–35], Alzheimer disease [36–39] and myocardial disease [40,41]. On the purpose of introducing a selective and sensitive approach for miRNA detection, an enzyme-linked electrochemical single-use sensors; PGE and multi-channel screenprinted array of electrodes (MUX-SPE16s) combined with magnetic beads assay was tested herein as the first time for detection of miRNA-15a, which is a biomarker for Alzheimer disease [38,42–44]. The DL of miRNA-15a target sequence was found as 0.992 g/mL (98.60 pmole in 1 mL sample) at PGE, and 0.114 g/mL (34.20 fmole in 3 L sample) at MUX-SPE16s system. A lower DL was obtained in the less sample volume as 3 L by using MUX-SPE16s array system in combination with LSV method, that is quite lower comparison to the DL obtained by using differential pulse voltammetry (DPV) technique performed by Kilic et al. [28]. The LSV technique brings the important features, such as sensitive detection by preventing the product degradation in our assay due to the rapid measurement could be performed using this electrochemical technique. As a conclusion, MUX-SPE16s system tested herein as the first time for voltammetric miRNA detection brings several advantages; such as, disposable, easy-to-use, robust, time-saving, ability of sensitive and selective recognition of target RNA contrary to other chip technologies developed for electrochemical monitoring of different miRNA targets [21,25–28,45]. It was also shown that the multi channel screen-printed array of electrodes could be used not only for detection of nucleic acids, but also for gene and protein monitoring in future toward to chip technology. Acknowledgements A.E acknowledges the financial support from Turkish Scientific and Technological Council (TUBITAK; Project No 110S146) and she also would like to express her gratitude to the Turkish Academy of Sciences (TUBA) as an Associate member for its partial support. E.E acknowledges a master project scholarship through by TUBITAK (Project No 110S146). The authors also acknowledge to Prof. Gorsev Yener and Prof. Sermin Genc due to their valuable scientific comments during this study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2013.07.114. References [1] D.P. Bartel, Micro RNAs Target recognition and regulatory functions, Cell 136 (2009) 215–233. [2] E. Palecek, M. Bartosik, Electrochemistry of nucleic acids, Chem. Rev. 112 (2012) 3427–3481. [3] M. Bartosik, M. Trefulka, R. Hrstka, B. Vojtesek, E. Palecek, Os(VI)bipy-based electrochemical assay for detection of specific microRNAs as potential cancer biomarkers, Electrochem. Commun. 33 (2013) 55–58. [4] A.E. Pasquinelli, G. Ruvkun, Control of developmental timing by microRNAs and their targets, Annu. Rev. Cell Dev. Biol. 18 (2002) 495–513. [5] S. Griffiths-Jones, R.J. Grocock, S. Dongen, A. Bateman, A.J. Enright, miRBase: microRNA sequences, targets and gene nomenclature, Nucleic Acids Res. 34 (2006) D140–D144. [6] M.A. Cortez, MicroRNAs in body fluids—the mix of hormones and biomarkers, Nat. Rev. Clin. Oncol. 8 (2011) 467–477. [7] P.S. Mitchell, R.K. Parkin, E.M. Kroh, B.R. Fritz, S.K. Wyman, E.L. PogosovaAgadjanyan, A. Peterson, J. Noteboom, K.C. O’Briant, A. Allen, D.W. Lin, N. Urban, C.W. Drescher, B.S. Knudsen, D.L. Stirewalt, R. Gentleman, R.L. Vessella, R.S. Nelson, D.B. Martin, M. Tewari, Circulating microRNAs as stable bloodbased markers for cancer detection, Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 10513–10518. [8] U.A. Ørom, A.H. Lund, Isolation of microRNA targets using biotinylated synthetic microRNAs, Methods 43 (2007) 162–165.
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Biographies Arzum Erdem is a Professor at the Analytical Chemistry Department in the Faculty of Pharmacy of Ege University in Turkey. She received her PhD in Analytical Chemistry from Ege University in 2000. Dr. Erdem was awarded by the Turkish Academy of Sciences (TUBA) as the one of highly skilled young scientists selected in 2001, and she also received Juniour Science Award 2006 given by The Scientific and Technological Research Council of Turkey (TUBITAK). She has initiated many international collaborative research on development and applications of electrochemical (bio)sensors based on drug, enzyme and nucleic acids. Her recent research is centered on the development of novel transducers and chemical and biological recognition systems by using different nanomaterials (e.g., magnetic nanoparticles, carbon nanotubes, gold and silver nanoparticles, nanowires etc.) designed for electrochemical sensing of nucleic acid (DNA, RNA) hybridization, and also the specific interactions between drug and DNA, or protein and DNA, aptamer-protein and also the development of integrated analytical systems for environmental, industry and biomedical monitoring. Gulsah Congur has a B.Sc. in bioengineering from Faculty of Engineering, Ege University (Izmir, Turkey), and a M.Sc. in biotechnology from the Institute of Natural and Applied Sciences at Ege University. She’s still continued her Ph.D. in biotechnology from Natural and Applied Sciences, Ege University. Her current research is on the development of electrochemical biosensors for the purpose of monitoring of (bio)molecule–DNA interaction, detection of genetic disease by nucleic acid hybridization, investigation of protein-aptamer interaction. Ece Eksin received her B.Sc. degree in Biochemistry Majored Biotechnology Program in the Faculty of Science from Ege University (Izmir, Turkey) in 2011. Currently, she is studying for MSc degree in Biotechnology from the Institute of Natural and Applied Sciences at Ege University. Her field of interest is electrochemical biosensors for biomedical applications.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Multi channel screen printed array of electrodes for enzyme-linked voltammetric detection of MicroRNAs Arzum Erdem a,∗ , Gulsah Congur a,b , Ece Eksin a,b a b
Ege University, Faculty of Pharmacy, Analytical Chemistry Department, 35100 Bornova, Izmir, Turkey Ege University, The Institute of Natural and Applied Sciences, Biotechnology Department, 35100 Bornova, Izmir, Turkey
a r t i c l e
i n f o
Article history: Received 2 June 2013 Received in revised form 21 July 2013 Accepted 30 July 2013 Available online 8 August 2013 Keywords: Multi channel screen-printed array of electrodes Streptavidin-alkaline phosphatase Pencil graphite electrode miRNA Magnetic beads MUX-SPE
a b s t r a c t In the present work, a sensitive and selective enzyme-linked electrochemical sensor technology using magnetic beads assay was demonstrated for voltammetric detection of microRNAs (miRNAs) as the first time herein by using the multi-channel screen-printed array of electrodes (MUX-SPE16s). The streptavidin coated magnetic beads were used as a solid phase to immobilize biotinylated DNA capture probe, and then the complementary target RNA sequence (miRNA-15a) was recognized with the capture DNA probe. After attachment of the streptavidin–alkaline phosphatase enzyme (SALP) to biotinylated hybrid, the electroactive product ␣-naphthol was measured +0.188 V by linear sweep voltammetry (LSV) technique in combination with a single-use pencil graphite electrode (PGE) compared to MUX-SPE16. The oxidation signal of ␣-naphthol indicates the hybridization occurred between DNA probe and its RNA target in the sample. The selectivity of our assay was tested in the presence of non-complementary miRNA sequence, a very good discrimination was achieved compared to the results obtained with the full match hybridization of probe with miRNA-15a target. The detection limit of miRNA-15a target sequence was also calculated as 0.992 g/mL (98.60 pmole in 1 mL sample) at PGE, and 0.114 g/mL (34.20 fmole in 3 L sample) with MUX-SPE16 system. © 2013 Elsevier B.V. All rights reserved.
1. Introduction MicroRNAs (miRNAs) are a class of non-coding RNA gene whose final product is a ∼22 nt functional RNA molecule. They play important roles in the regulation of target genes by binding to complementary regions of messenger transcripts to repress their translation or regulate degradation [1–3]. Since the discovery of the founding members of the miRNA class, lin-4 and let-7 in Caenorhabditis elegans [4], over 2000 miRNA sequences have been described in vertebrates, flies, worms and plants, and even in viruses. However, the functions of only a handful of these miRNAs have been experimentally determined. In parallel with novel gene identification efforts, the miRNA community is therefore focused on predicting and validating miRNA gene targets [5]. The great interest in miRNAs reflects their central role in gene-expression regulation and the implication of miRNA-specific aberrant expression in the pathogenesis of cancer, cardiac, immune-related and other diseases. Another avenue of current research is the study of circulating miRNAs in serum, plasma, and other body fluids—miRNAs may act not only within cells, but also at
∗ Corresponding author. Tel.: +90 232 311 5131; fax: +90 232 388 5258. E-mail addresses: [email protected], [email protected] (A. Erdem). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.07.114
other sites within the body. The presence of miRNAs in body fluids may represent a gold mine of noninvasive biomarkers in cancer. Since deregulated miRNA expression is an early event in tumorigenesis, measuring circulating miRNA levels may also be useful for early cancer detection, which can contribute greatly to the success of treatment [6]. Mitchell and co-workers have recently showed that tumor cells shed tumor derived microRNAs into the blood stream and that microRNAs are present in the blood and serum in remarkably stable forms [7]. Serum circulating miRNAs are promising novel biomarkes for early cancer detection and for improved cancer screening, even if the detection protocol is required labeling of miRNAs with any different groups; such as, biotin [8,9], or any metal complexes [3]. At the present time, miRNAs are detected by using numerous techniques; Northern blot [10], in situ hybridization [11], RT-PCR [12], and microarrays [13] using various types of commercial diagnostic kit, even after the biotinylation of miRNAs. Nevertheless, they are expensive, time-consuming for performing the assay, and need an equipped laboratory with specialized and well-trained people. Because of these reason, they are not feasible for routine detection of serum-based microRNAs [14]. Magnetic beads (MBs) have received a great attention due to their potential use in various biotechnological applications, including protein and enzyme immobilization, separation and sorting proteins and cells, drug delivery system, hyperthermia treatment for cancerous tumors and immunoassays [15,16]. A major
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advantage of MBs is the easy and rapid recovery of target molecules from the reaction mixture by the efficient magnetic separation. The performance of MBs could be significantly affected by surface modifications. Various molecules could be easily immobilized onto the surfaces of MBs by surface chemistry [17]. The double surface technique based on magnetic beads sepration are well-known for electrochemical biosensing of nucleic acids (NAs) [18–22]. Under the principle of magnetic beads assay, a biotinylated DNA probe was firstly immobilized in our study onto the streptavidin coated magnetic particles (MB) and then, the hybridization of probe with its complementary RNA sequence was performed at MB surface. To the best of our knowledge, the multi-channel screen-printed array of electrodes (MUX-SPE16s) has been used as the first time herein for enzyme-linked detection of miRNA-15a hybridization related to Alzheimer disease. After labeling the biotinylated hybrid with a streptavidin–alkaline phosphatase (SALP) conjugate, the enzymatic reaction was performed. Consequently, the electroactive product, ␣-naphthol occurred, and it was then measured by using LSV technique in combination with MUX-SPE16s. The same experimental procedure was followed for detection of miRNA using the single use pencil grahite electrode (PGE). The features of both sensor technologies in combination with magnetic beads for the electrochemical detection of miRNA-15a were then discussed concerning to their detection limit with reproducibility, and also the selectivity of each sensor by comparing the results of earlier reports on the electrochemical miRNA detection related to different miRNA sequences. 2. Experimental 2.1. Apparatus The oxidation signal of ␣-naphthol was measured by AUTOLAB PGSTAT electrochemical analysis system and GPES 4.9 software package (Eco Chemie, The Netherlands) using linear sweep voltammetry (LSV) technique. The raw data were also treated using the Savitzky and Golay filter (level 2) of the GPES software, followed by the moving average baseline correction with a “peak width” of 0.03. MUXSCNR16-multichannel system was used to perform LSV measurements with MUX-SPE16s. The three-electrode system consisted of the PGE, an Ag/AgCl/KCl reference electrode and a platinum wire as the auxiliary electrode was used. The experiments were also performed by using multi-channel screen-printed array of electrodes (MUX-SPE16). The preparation of magnetic beads and hybridization process were performed on the magnetic separators, MCB 1200 (Sigris, USA). 2.2. Chemicals Streptavidin–alkaline phosphatase (SALP), ␣-naphthyl phosphate, bovine serum albumin (BSA) were obtained from Sigma–Aldrich. The DNA probes and complementary RNA or DNA oligonucleotides (RNA or DNA ODN), were purchased (as lyophilized powder) from TIB Molbiol (Germany). The base sequences of ODNs are listed below (X: Uracil): miRNA-15a DNA probe 5 -TCA AAA TCC ACA AAC CAT TAT GTG CTG CTA CTT TAC TCC AAG G–Biotin-3 Complementary miRNA-15a (DNA) 5 -TAG CAG CAC ATA ATG GTT TGT GGA TTT TGA–Biotin-3 Complementary miRNA-15a (RNA) 5 -XAG CAG CAC AXA AXG GXX XGX GGA XXX XGA–Biotin-3 Non-Complementary miRNA ODN (NC-miRNA) 5 -AGX GXX XCG GCC CAA XCA CXA XCX CCC GXC–Biotin-3
All ODN stock solutions (500 g/mL) were prepared with TrisEDTA buffer solution (10 mM Tris–HCl, 1 mM EDTA, pH: 8.00; TE) and kept frozen. More dilute solutions of ODNs were prepared with either TTL buffer (100 mM Tris–HCl, pH 8.00, and 0.1% Tween 20 and 1 M LiCl) or 50 mM phosphate buffer solution (PBS, pH 7.40) according to the hybridization protocol. SALP stock solution (1000 g/mL) was prepared with 0.1 M Tris–HCl (pH 9.20) containing 1 mM MgCl2. More diluted solutions of SALP were prepared in 50 mM phosphate buffer solution (PBS, pH 7.40) containing 2% BSA. Required amount of ␣-naphthyl phosphate was prepared in carbonate buffer (CBS, pH 9.50) containing 0.50 M Na2 CO3 and 0.50 M KHCO3 . Streptavidin coated magnetic particles (magnetic beads) in 0.94 mm diameter size were purchased from Estapor, Merck (France). Other chemicals were in analytical reagent grade and they were supplied from Sigma and Merck. 2.3. Electrode preparation 2.3.1. Pencil graphite electrodes (PGEs) A Tombo® pencil was used as a holder for the graphite lead. Electrical contact with the lead was obtained by soldering a metallic wire to the metallic part. The pencil was held vertically with 14 mm of the lead extruded outside (10 mm of which was immersed in the solution). PGEs were prepared according to the protocol previously reported in the literature [19,23]. The PGEs were electrochemically pretreated by applying +1.40 V for 30 s in 0.50 M acetate buffer solution containing 20 mM NaCl (pH 4.80; ABS). 2.3.2. Screen-printed array of electrodes (MUX-SPE16s) The MUXSCNR-16 compatible MUX-SPE16 was developed and characterized by Dropsens Company (Spain) containing one main electrochemical cell with sixteen working carbon electrodes combined with one reference electrode and one auxiliary electrode. Each working electrode at MUX-SPE16s was pretreated one by one by applying +0.90 V for 60 s in ABS. 2.4. Preparation of probe-coated magnetic particles and DNA hybridization at the surface of magnetic particles The preparation of ODN-coated microspheres (magnetic beads, MBs) was performed by following the reported procedure [19,20]. 5 L of streptavidin-coated MBs was transferred into a 1.5 mL centrifuge tube. The microspheres were then washed twice with 90 L TTL buffer and resuspended in 30 L TTL buffer containing 60 g/mL biotinylated probe and incubated for 15 min at room temperature with gentle mixing. The immobilized probe was then separated and washed once with 90 L TT buffer (250 mM Tris–HCl, pH 8.0, and 0.1% Tween 20), resuspended in 20 L PBS containing desired concentration of biotin capped complementary RNA/DNA (target) of probe, or non-complementary miRNA sequences. The hybridization reaction was carried out at room temperature during 10 min. The hybridized microsphere conjugates were then washed twice with 90 L PBS. After hybridization and washing steps, MBs were incubated with a solution containing required amount of SALP for 15 min. MBs were then washed with 90 L of PBS-T buffer (PBS containing 0.05% Tween20) and washed again with 90 L PBS buffer. SALP conjugated MBs resuspended in 100 L CBS buffer solution containing required amount of ␣-naphthyl phosphate and incubated for 15 min at room temperature with gentle mixing. As a result of enzymatic reaction, electroactive product ␣-naphthol was occurred. 100 L supernatant was transferred into the vial, which contains 900 L of CBS. The mixed sample containing ␣-naphthol was transferred into the 1 mL electrochemical cell, and the voltammetric transduction was performed.
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For miRNA detection at MUX-SPE16s, 3 L of sample was only dropped onto the surface of consecutively each working electrode of MUX-SPE16s, and kept for passive adsorption step during 15 min. Then, the MUX-SPE16s was washed with CBS three times. The MUX-SPE16s was connected with multichannel system integrated to electrochemical analyzer. 850 L CBS was then dropped onto the full surface of MUX-SPE16s to perform consecutively the LSV measurements. 2.5. Voltammetric transduction The oxidation signal of ␣-naphthol was measured by using LSV by scanning from −0.3 V to +1.2 V at a pulse amplitude of 4.8 mV and a scan rate of 1 V/s. 3. Results and discussion As seen in Scheme 1 the miRNA detection protocol required firstly hybridization step performed at MB surface modified with biotinylated miRNA-15a DNA probe and with its complementary sequence labeled with biotin. The streptavidin labeled enzyme, alkaline phosphatase (SALP) was then introduced to this sample for performing the enzymatic reaction binding of enzyme to the biotin linked target sequence. After the interaction between the substrate of SALP; ␣-naphthyl phosphate and SALP, the electroactive product, ␣-naphthol was occurred in the media and consequently, the oxidation signal of product was measured by using LSV technique. Fig. 1 shows the oxidation signal of ␣-naphthol measured before and after hybridization between 60 g/mL miRNA-15a DNA probe and 60 g/mL miRNA-15a DNA target. The control experiments were firstly done by using MB alone, and full hybridization in the absence of substrate, and no signal was observed (Fig. 1a and b). In the presence of full hybridization case, 11 times higher ␣-naphthol
Fig. 1. Histograms and voltammograms (inset) representing the ␣-naphthol oxidation signals measured by using PGEs at control experiment of (a) MB alone, in the case of the full hybridization, (b) in the absence of ␣-naphthyl phosphate, and (c) before hybridization between 60 g/mL miRNA-15a DNA probe and 60 g/mL miRNA-15a DNA target, and (d) after hybridization in the presence of 50 g/mL SALP and 1 mM ␣-naphthyl phosphate.
signal (Fig. 1d) was measured at +0.188 V comparison to the one recorded before hybridization (Fig. 1c). It indicated that the spesific miRNA-15a detection was successfully achieved by using magnetic beads assay combined with PGE. Next, the effect of enzyme concentration based on ␣-naphthol signal was investigated in the presence of full hybridization at PGE surface (Fig. S1). The highest ␣-naphthol signal was obtained in the presence of enzymatic reaction with 50 g/mL of SALP (Fig. S1B-b). In the case of full hybridization between 60 g/mL DNA probe and 60 g/mL miRNA-15a DNA target, the average ␣-naphthol signal was measured as 314.88 ± 35.09 A with relative standart deviation (RSD%) 11.14% (n = 3), that was 11 times higher than one obtained in the absence of hybridization (probe alone). Therefore,
Scheme 1. Schematic representation of enzymatic detection protocol in combination with magnetic beads assay using disposable graphite sensor technologies; (A) PGE and (B) MUX-SPE16s.
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Fig. 2. Voltammograms (I), histograms (II) representing ␣-naphthol signals measured at PGE in the presence of enzymatic reaction between 50 g/mL SALP and 2 mM ␣-naphthyl phosphate. Before (a) and after hybridization between 60 g/mL miRNA-15a DNA probe and miRNA-15a DNA target in different concentrations: 2.5 g/mL (b), 5 g/mL (c), 7.5 g/mL (d), 10 g/mL (e), 12.5 g/mL (f), 15 g/mL (g).
50 g/mL of SALP concentration was chosen as optimum for further studies in our study. The effect of substrate concentration was also investigated at 50 g/mL SALP concentration (Fig. S2). ␣-naphthol signal gradually increased till 2 mM substrate concentration (Fig. S2C-b), then it leveled off. A series of three repetitive LSV measurements at 2 mM concentration level of ␣-naphthyl phosphate using PGE, the average signal was measured as 408.46 ± 21.25 A with the RSD% of 5.20% (n = 4). The further experiments were performed in 2 mM concentration of ␣-naphthyl phosphate. Upon the changes at the ␣-naphthol signal, the effect of RNA target concentration was also studied in the case of full hybridization between probe DNA and miRNA-15 target in its different concentration from 2.5 to 15 g/mL (Fig. 2). The response gradually increased till 10 g/mL target RNA concentration (Fig. 2-I and II-e), and then it almost leveled off. The RSD % was found as 9.97% for three successive determinations at 10 g/mL target RNA concentration by using PGE. The linear graph based on the sensor response upon various target RNA concentrations was shown in Fig. 3. The limit of detection (DL) was calculated with the aid of the section of the calibration plot close to the origin, which is linear, utilizing both the regression equation and the definition y = yB + 3sB (yB is the signal of the blank solutions and sB is the standard deviation of the blank solution) as outlined in reference [24]. Thus, the DL was estimated as 0.992 g/mL (98.60 pmole in 1 mL sample) at PGE. The selectivity of this biosensor system was analyzed based on ␣-naphthol signal, that was obtained in the presence of hybridization between DNA probe and miRNA-15a RNA target, or non-complementray miRNA sequences by using PGE (Fig. 4). In the case of hybridization with NC-miRNA sequence, the negligible small ␣-naphthol signal was measured compared to the signals of full match hybridization between DNA and 10 g/mL miRNA15a (Fig. 4A-I and II-b to c), and 60 g/mL (Fig. 4B-I and II-b to
Fig. 3. The calibration plot based on the ␣-naphthol signals measured at PGE in the presence of enzymatic reaction in the case of full hybridization between 60 g/mL miRNA-15a DNA probe and miRNA-15a RNA target concentrations varying from 0 to 10 g/mL by using 50 g/mL SALP and 2 mM ␣-naphthyl phosphate.
c) miRNA-15a. For further selectivity study, it was tested in the mixture samples containing RNA target and NC-miRNA sequences at both concentrations; 10 g/mL (Fig. 4A-I and II-d), and 60 g/mL (Fig. 4B-I and II-d). However there was an excess of NC-miRNA sequence including target RNA in the medium (1:1), it was found that a selective voltammetric detection of miRNA-15a was explored by the advantage of efficient magnetic separation even in the mixture samples. Compared to the first part of our study performed by using PGE, more sensitive and selective enzyme-linked electrochemical sensor technology using magnetic beads was demonstrated for voltammetric detection of miRNAs as the first time herein by using MUX-SPE16s, which have also brought the numerous opportunities herein; such as, easy-to-use, disposable, cost effective, sensitive and
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Fig. 4. Voltammograms (I), histograms (II) representing the ␣-napthol signals measured at PGE after hybridization between 60 g/mL miRNA-15a DNA probe and (A) 10, (B) 60 g/mL miRNA-15a RNA target, or NC-RNA, or miRNA-15a RNA target in the mixture sample with NC-RNA (1:1) by using PGE. ␣-naphthol signals (a) before and after hybridization with miRNA-15a DNA probe and (b) miRNA-15a RNA target, (c) NC-RNA, or (d) the mixture sample of RNA target:NC-RNA (1:1).
signal in various target concentration from 0.5 to 3 g/mL (Fig. 5). Although the highest ␣-naphthol signal was measured at 3 g/mL concentration level of RNA target (Fig. 5-A and B-f) with a RSD% as 19.20% (n = 3), the better reproducible result was recorded in the case of 2 g/mL concentration level of target (38.09 ± 2.90 A with the RSD of 7.63%, n = 3). The DL was calculated by following the equation [24] as explained above, and it was found as 0.114 g/mL (34.20 fmole in 3 L sample) at MUX-SPE16s. The selectivity studies were firstly performed at 2 g/mL concentration level of miRNA-15a target, however the selective results could not be obtained at this concentration level (not shown). Afterwards, the selectivity of our assay was then tested at a higher RNA concentration as 10 g/mL (Fig. 6). In the case of full hybridization, 2.5 times higher ␣-naphthol signal (Fig. 6-b) was measured compared to one obtained in the absence of DNA-RNA hybridization (probe alone) (Fig. 6-a). After probe hybridization with NC-miRNA, there was a negligible change observed at ␣-naphthol signal (Fig. 6c) due to the efficient magnetic separation using magnetic beads assay. Even in a mixture media containing excess amount of NCmiRNA besides target RNA, the ␣-naphthol signal was measured almost in the same level (Fig. 6-d) of full hybridization signal.
Fig. 5. (A) Histograms representing the ␣-naphthol signals measured at MUX-SPE16 system before (a) and after hybridization between 60 g/mL miRNA-15a DNA probe and 2.5 g/mL (b), 5 g/mL (c), 7.5 g/mL (d), 10 g/mL (e), 12.5 g/mL (f), 15 g/mL (g) miRNA-15a RNA target concentrations. (B) The calibration plot obtained from ␣naphthol signals after hybridization with DNA probe and miRNA-15a RNA target concentrations varying from 0.5 to 3 g/mL.
selective detection compared to the results of earlier electrochemical assays for detection of miRNAs [25–29]. The effect of target miRNA-15a concentration was firstly investigated using MUX-SPE16s based on the changes of the ␣-naphthol
Fig. 6. Histograms representing the ␣-naphthol signals measured at MUX-SPE16s before (a) and after hybridization between 60 g/mL miRNA-15a DNA probe and (b) 10 g/mL miRNA-15a RNA target, (c) 10 g/mL NC-miRNA and (d) the mixture sample of miRNA-15a:NC-miRNA(1:1).
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4. Conclusion The detection and monitoring of miRNAs has recently become a frontline topic due to the fact that they are well-known biomarkers for different types of cancer [30–35], Alzheimer disease [36–39] and myocardial disease [40,41]. On the purpose of introducing a selective and sensitive approach for miRNA detection, an enzyme-linked electrochemical single-use sensors; PGE and multi-channel screenprinted array of electrodes (MUX-SPE16s) combined with magnetic beads assay was tested herein as the first time for detection of miRNA-15a, which is a biomarker for Alzheimer disease [38,42–44]. The DL of miRNA-15a target sequence was found as 0.992 g/mL (98.60 pmole in 1 mL sample) at PGE, and 0.114 g/mL (34.20 fmole in 3 L sample) at MUX-SPE16s system. A lower DL was obtained in the less sample volume as 3 L by using MUX-SPE16s array system in combination with LSV method, that is quite lower comparison to the DL obtained by using differential pulse voltammetry (DPV) technique performed by Kilic et al. [28]. The LSV technique brings the important features, such as sensitive detection by preventing the product degradation in our assay due to the rapid measurement could be performed using this electrochemical technique. As a conclusion, MUX-SPE16s system tested herein as the first time for voltammetric miRNA detection brings several advantages; such as, disposable, easy-to-use, robust, time-saving, ability of sensitive and selective recognition of target RNA contrary to other chip technologies developed for electrochemical monitoring of different miRNA targets [21,25–28,45]. It was also shown that the multi channel screen-printed array of electrodes could be used not only for detection of nucleic acids, but also for gene and protein monitoring in future toward to chip technology. Acknowledgements A.E acknowledges the financial support from Turkish Scientific and Technological Council (TUBITAK; Project No 110S146) and she also would like to express her gratitude to the Turkish Academy of Sciences (TUBA) as an Associate member for its partial support. E.E acknowledges a master project scholarship through by TUBITAK (Project No 110S146). The authors also acknowledge to Prof. Gorsev Yener and Prof. Sermin Genc due to their valuable scientific comments during this study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2013.07.114. References [1] D.P. Bartel, Micro RNAs Target recognition and regulatory functions, Cell 136 (2009) 215–233. [2] E. Palecek, M. Bartosik, Electrochemistry of nucleic acids, Chem. Rev. 112 (2012) 3427–3481. [3] M. Bartosik, M. Trefulka, R. Hrstka, B. Vojtesek, E. Palecek, Os(VI)bipy-based electrochemical assay for detection of specific microRNAs as potential cancer biomarkers, Electrochem. Commun. 33 (2013) 55–58. [4] A.E. Pasquinelli, G. Ruvkun, Control of developmental timing by microRNAs and their targets, Annu. Rev. Cell Dev. Biol. 18 (2002) 495–513. [5] S. Griffiths-Jones, R.J. Grocock, S. Dongen, A. Bateman, A.J. Enright, miRBase: microRNA sequences, targets and gene nomenclature, Nucleic Acids Res. 34 (2006) D140–D144. [6] M.A. Cortez, MicroRNAs in body fluids—the mix of hormones and biomarkers, Nat. Rev. Clin. Oncol. 8 (2011) 467–477. [7] P.S. Mitchell, R.K. Parkin, E.M. Kroh, B.R. Fritz, S.K. Wyman, E.L. PogosovaAgadjanyan, A. Peterson, J. Noteboom, K.C. O’Briant, A. Allen, D.W. Lin, N. Urban, C.W. Drescher, B.S. Knudsen, D.L. Stirewalt, R. Gentleman, R.L. Vessella, R.S. Nelson, D.B. Martin, M. Tewari, Circulating microRNAs as stable bloodbased markers for cancer detection, Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 10513–10518. [8] U.A. Ørom, A.H. Lund, Isolation of microRNA targets using biotinylated synthetic microRNAs, Methods 43 (2007) 162–165.
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Biographies Arzum Erdem is a Professor at the Analytical Chemistry Department in the Faculty of Pharmacy of Ege University in Turkey. She received her PhD in Analytical Chemistry from Ege University in 2000. Dr. Erdem was awarded by the Turkish Academy of Sciences (TUBA) as the one of highly skilled young scientists selected in 2001, and she also received Juniour Science Award 2006 given by The Scientific and Technological Research Council of Turkey (TUBITAK). She has initiated many international collaborative research on development and applications of electrochemical (bio)sensors based on drug, enzyme and nucleic acids. Her recent research is centered on the development of novel transducers and chemical and biological recognition systems by using different nanomaterials (e.g., magnetic nanoparticles, carbon nanotubes, gold and silver nanoparticles, nanowires etc.) designed for electrochemical sensing of nucleic acid (DNA, RNA) hybridization, and also the specific interactions between drug and DNA, or protein and DNA, aptamer-protein and also the development of integrated analytical systems for environmental, industry and biomedical monitoring. Gulsah Congur has a B.Sc. in bioengineering from Faculty of Engineering, Ege University (Izmir, Turkey), and a M.Sc. in biotechnology from the Institute of Natural and Applied Sciences at Ege University. She’s still continued her Ph.D. in biotechnology from Natural and Applied Sciences, Ege University. Her current research is on the development of electrochemical biosensors for the purpose of monitoring of (bio)molecule–DNA interaction, detection of genetic disease by nucleic acid hybridization, investigation of protein-aptamer interaction. Ece Eksin received her B.Sc. degree in Biochemistry Majored Biotechnology Program in the Faculty of Science from Ege University (Izmir, Turkey) in 2011. Currently, she is studying for MSc degree in Biotechnology from the Institute of Natural and Applied Sciences at Ege University. Her field of interest is electrochemical biosensors for biomedical applications.