Aptasensor platform based on carbon nanofibers enriched screen printed electrodes for impedimetric detection of thrombin

Journal of Electroanalytical Chemistry 758 (2015) 12–19

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Aptasensor platform based on carbon nanofibers enriched screen printed electrodes for impedimetric detection of thrombin Arzum Erdem a,⁎, Gulsah Congur a, Günter Mayer b,⁎ a b

Ege University, Faculty of Pharmacy, Analytical Chemistry Department, 35100 Bornova, Izmir, Turkey LIMES Institute, Program Unit Chemical Biology and Medicinal Chemistry, University of Bonn, 53121 Bonn, Germany

a r t i c l e

i n f o

Article history: Received 10 April 2015 Received in revised form 4 October 2015 Accepted 5 October 2015 Available online 9 October 2015 Keywords: Carbon nanofibers Screen printed electrodes Electrochemical impedance spectroscopy Aptamer Thrombin Fetal bovine serum

a b s t r a c t Herein, impedimetric detection of THR could be achieved using an aptasensing platform based on carbon nanofibers enriched screen-printed electrodes (CNF-SPE). The resistance to charge transfer (Rct) was recorded using electrochemical impedance spectroscopy (EIS) technique before/after the immobilization of amino-modified DNA aptamer (APT) selective to thrombin (THR) onto the surface of CNF-SPEs and the specific interaction between APT and THR. The selectivity of the aptasensor was also tested in the presence of a random DNA oligonucleotide and a DNA aptamer that were different from THR specific APT. The impedimetric aptasensing of target protein was also explored in the fetal bovine serum (FBS) medium at different concentration levels of THR. Additionally, the selectivity of the aptasensor was tested against bovine serum albumin (BSA) and protein C (PC) in FBS medium. This CNF-SPE based aptasensor platform allows a reliable, sensitive and selective impedimetric monitoring of THR. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Aptamers are known as a new class of nucleic acids which are used to design sensitive, selective and robust biodetection platforms. The electrochemical biosensing technology can be combined with aptamers for recognition of nucleic acids, proteins, drugs, and toxins. Thus, there have been many reports in the literature about development of electrochemical aptamer based sensors (aptasensors) [1–22]. Elshafey et al. [12] reported the selection of a high affinity DNA aptamer and development of an impedimetric aptasensor by using gold electrode for sensitive and selective detection of Anatoxin-a (ATX) that is cyanobacterial toxin known as the smallest potent neurotoxin. The selectivity of the impedimetric aptasensor was also tested against cylindrospermopsin, Microcystin-LR and their mixture. In the study of Gao et al. [14], an electrochemical aptasensor for detection of hemin which was a well-known natural porphyrin to iron complex and applied in pharmacy, environmental and food industry was developed. Glassy carbon electrode (GCE) was modified with the nanocomposite comprised of hemin binding aptamer and carboxylated graphene, then, the interaction of hemin and its aptamer was monitored. Thrombin (THR) is the last serine protease involved in the coagulation cascade and its concentration in blood is of relevance in many

⁎ Corresponding authors. E-mail addresses: [email protected], [email protected] (A. Erdem), [email protected] (G. Mayer).

http://dx.doi.org/10.1016/j.jelechem.2015.10.002 1572-6657/© 2015 Elsevier B.V. All rights reserved.

pathological situations [23–26]. Fibrinogen is converted into insoluble fibrin in the presence of THR. Moreover, THR is involved in thrombosis and platelet activation. Whereas THR is nearly absent in blood under normal conditions, its presence or absence under pathological conditions can be indicative of coagulation abnormalities [25]. Therefore, sensitive detection of THR has a vital importance not only for diagnosis of cardiovascular disease, but also of different types of cancer. The thrombin-binding aptamer (HD1) was the first aptamer selected in vitro, specific for a protein without nucleic acid-binding properties [26]. The interaction between THR and the HD1 has been taken as a model system by many authors. G-quartet structure of HD1 and the binding site of THR with HD1 has been characterized thoroughly [27, 28]. Due to the importance of THR, many sensors for thrombin detection, using different techniques and approaches have been developed [1–6]. Label-free electrochemical detection of human α-THR in human blood serum was explored by Kwon et al. [1] by using thiolated aptamers immobilized gold electrodes. It was reported that positively charged ferrocene-coated gold nanoparticles were electrostatically bound to the negatively charged aptamers and accordingly, the electrochemical response was recorded by cyclic voltammetry and differential pulse voltammetry (DPV). Li et al. [4] developed a novel electrochemical aptasensor for the voltammetric detection of THR based on multiwalled carbon nanotubes modified GCE surface. Evtugyn and co-workers [20] introduced an aptasensor platform for impedimetric detection of thrombin by using neutral red attached and thiacalix[4]arene modified GCE.

A. Erdem et al. / Journal of Electroanalytical Chemistry 758 (2015) 12–19

Carbon nanofibers (CNFs) are hollow cylinders, which have different diameters varying from 50 to 500 nm and a few tens of microns-size lengths. Due to the fact that they have high aspect ratios (length/ diameter N 100), they can align homogeneously at the surface, on which they were immobilized [29,30]. In addition, it was found that they can provide a stable surface for robust biosensor applications [31–38]. Detection of dopamine and serotonin in the presence of excess ascorbic acid, a biosensor based on CNF modified GCE in combination with DPV technique was described by Rand et al. [32]. The detection limits (DLs) were found to be as low as 50 nM and 250 nM respectively for dopamine and serotonin. Periyakaruppan and co-workers developed a selective and sensitive assay based on CNF modified nanoelectrode in combination with EIS to analyze a toxic protein, ricin [33]. An amperometric lactate biosensor was also developed by using screen printed carbon electrodes modified with platinum nanoparticles supported on graphitized carbon nanofibers (PtNps/GCNF) [37]. In our study, the impedimetric detection of THR using an aptasensor platform was comprehensively investigated. To the best of our knowledge, no report describing an impedimetric detection of aptamer–THR interaction at the surface of carbon nanofibers enriched screen printed electrodes (CNF-SPEs) is available as of yet. A sensitive and selective THR detection was performed herein at the surface of CNF-SPEs using EIS technique. The surface morphologies of CNF-SPE were explored using scanning electron microscopy (SEM) technique. The effect of different experimental conditions upon the sensor response was examined; e.g. the concentration of DNA aptamer and THR. The selectivity of the aptasensor was also investigated using random DNA sequence, or random DNA aptamer. The impedimetric detection of interaction between DNA aptamer and its cognate protein was also investigated even in the presence of a complex medium, fetal bovine serum (FBS). Moreover, the selectivity of the aptasensor was tested against bovine serum albumin (BSA) and protein C (PC) in FBS medium. 2. Experimental 2.1. Apparatus Electrochemical measurements were performed by using AUTOLABPGSTAT 302 electrochemical system supplied with a FRA 2.0 module for impedance measurements and NOVA software package (Eco Chemie, The Netherlands). Electrochemical impedance spectroscopy (EIS) measurements were performed in the Faraday cage (Eco Chemie, The Netherlands). 2.2. Chemicals The amino-linked DNA aptamers (APT) and random DNA (DNA ODN) oligonucleotides were synthesized from Ella Biotech (Germany). The DNA APTs were designed according to the information given in the literature [39]. Anti-thrombin DNA aptamer-1 (APT-1): 5′-NH2-C6-GGT TGG TGT GGT TGG – 3′ Anti-thrombin DNA aptamer-2 (APT-2): 5′-NH2-C6-GGT TGG TGT GGT TGG AAA AAA AAA AAA AAA AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3′ Non-binding point mutant of DNA aptamer (APT-MM): 5′-NH2-C6-GGT AGG TGT GGT TGG-3′ Random DNA oligonucleotide (DNA ODN): 5′-NH2-TCA-AAT-CAG-GTT-GCT-TA-3′ The stock solutions of the DNA APTs and random DNA ODN were prepared using fresh ultrapure triple distilled water and kept frozen. The diluted solutions of the APTs and DNA ODN were prepared with 5 mM Tris–HCl buffer containing 20 mM NaCl (TBS, pH 7.00).

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Thrombin (THR), bovine serum albumin (BSA) and protein C (PC) were purchased as lyophilisates from Sigma-Aldrich (Germany). The stock solutions of proteins were prepared by dissolving the lyophilisates in fresh ultrapure triple-solutions. Diluted solutions of proteins were prepared in 50 mM phosphate buffer solution containing 20 mM NaCl (PBS, pH 7.40). Fetal bovine serum (FBS) was purchased from SigmaAldrich (Germany). Other chemicals were supplied from Sigma (USA) and Merck (Germany) in analytical reagent grade. 2.3. Carbon nanofibers enriched screen printed electrodes (CNF-SPEs) Graphitized carbon nanofibers enriched screen printed electrodes (CNF-SPEs) were purchased from DropSens (Oviedo-Asturias, Spain). CNF-SPEs were designed for the development of (bio)sensors with an enhanced electrochemical active area. The planar screen-printed electrode 3.3 × 1.0 × 0.05 cm (length × width × height) consists of three main parts, which are graphitized carbon nanofiber modified graphite working electrode, a graphite counter electrode and a silver pseudoreference electrode. The graphite working screen printed electrode surface is 4 mm in diameter. A specific DropSens connector (ref. DSC) allows the connection of CNF-SPE to the potentiostat. The electrodes were pretreated by applying + 0.9 V for 60 s with 40 μL droplet of acetate buffer solution (ABS, pH 4.8). 2.4. Microscopic characterization of bare (unmodified) SPE and CNF-SPE by scanning electron microscopy (SEM) The microscopic characterizations of bare SPE and CNF-SPE were obtained by Quanta 400 FEI, field emission scanning electron microscope (FE-SEM) (Tokyo, Japan) with 10.0 kV acceleration voltage with the resolution in various magnitudes; 500 nm, 1 μm, 2 μm and 4 μm. 2.5. DNA APT/DNA ODN immobilization onto the surface of CNF-SPE, THR-APT interaction at CNF-SPE surface and impedance measurements EIS measurements were performed in the redox probe solution containing 2.5 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) prepared in 0.1 M KCl. The impedance was measured in the frequency range from 100 mHz to 100 KHz in a potential of +0.23 V versus silver reference with a sinusoidal signal of 10 mV. The frequency interval was divided into 98 logarithmically equidistant measure points. The respective semicircle diameter corresponds to the charge-transfer resistance, Rct, the values of which are calculated using the fitting program AUTOLAB 302 (FRA, version 4.9 Eco Chemie, The Netherlands). EIS measurements were performed by placing a 40 μL drop of the corresponding solution onto the working area at the surface of pretreated CNF-SPE. Before and after the immobilization of various types of aptamers, or control oligonucleotides onto the surface of CNFSPE, EIS measurements were performed by dropping 40 μL of redox probe onto the electrode surface. For DNA APT/DNA ODN immobilization onto the electrode, the surface of CNF-SPE was covered by 40 μL of the indicated amount of aptamer, or control oligonucleotides and it was kept for 30 min. Thereby the formation of peptide bounds between carboxylic groups of the nanofibers and amine groups of aptamer, or DNA ODN was occurred at the electrode surface without using any chemicals (EDC, NHS) used for covalent attachment. Similar immobilization process for amino linked DNA APT specific for lysozyme was applied for its immobilization onto the surface of carboxylated multiwalled carbon nanotube modified SPE in earlier works [40,41]. Each electrode was then rinsed with TBS (pH 7.0) for 5 s to remove unbound aptamer/DNA ODN from the surface. Then, EIS measurements were performed as described above. 40 μL of the indicated concentration of THR or BSA was added on the surface of aptamer or oligonucleotide-modified electrode and incubated for 30 min. Subsequently, each electrode was rinsed with PBS (pH 7.4)

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for 5 s and EIS measurements were performed. The impedimetric detection of THR by using APT-1 modified CNF-SPE was shown representatively in Scheme 1. 3. Results and discussion First, the surface morphologies of CNF-SPE were explored using scanning SEM in the magnitudes varying from 500 nm to 4 μm (Fig. 1). The aligned-fiber structures could be clearly seen at the surface of CNF-SPE in contrast to the bare SPE. Electrochemical impedance spectroscopy (EIS) is a useful technique, which provides information on the step-by-step immobilization and interaction that occurs at the electrode surface. Thus, it is an effective electrochemical characterization technique based on the change of the charge transfer resistance (Rct) to understand biomolecular interaction processes. Figs. 2–6 show the changes at the impedance of the CNFSPEs associated with the stepwise of DNA immobilization/interaction. Real (Z′) and imaginary (− Z″) components consisted of the complex impedance (Z). The resistance related to the dielectric and insulating characteristics at the electrode/electrolyte interface is defined as the Rct [42,43]. It was reported [42,43] that an equivalent circuit model (Randles circuit, inset in each figure) was utilized to fit the impedimetric results. In addition, the electron transfer was limited at higher frequencies; the linear section seen at lower frequencies may be attributed to the diffusion [42,43].

The Nyquist diagrams related to unmodified and CNF modified SPE were also given in Fig. S1. The Rct obtained by CNF-SPE was quite smaller than the one obtained by unmodified SPE (i.e., 129 Ω). This result may be attributed to the conductive structure of CNF [31,44,45]. The electron transfer became easier at the surface of CNF-SPE resulting with the decrease at repulsive forces occurred between electrode/electrolyte interface. The effect of aptamer in different concentrations varying from 0 to 200 μg mL−1 upon the impedimetric response of aptasensor was then studied (shown in Fig. 2). It was found that the Rct was gradually increased after the immobilization of APT-1 varying between 0 and 150 μg mL−1 concentration, then it decreased when its concentration was also increased to 200 μg mL−1. This increase at the Rct value could be attributed the repulsive forces occurred between the negatively charged phosphate groups of DNA APT and the anionic [Fe(CN)6]3−/4− ions similarly to the results of earlier reports [7–9,12,14,17,20,40, 46–48]. The average Rct value was found as 192.2 ± 26.4 Ω with a relative standard deviation (RSD) % = 13.7% (n = 3) after the modification of 150 μg mL−1 APT-1 onto the surface of CNF-SPE (Fig. 2B, b), which is 50 times higher than the one obtained by unmodified CNF-SPE (i.e., 3 Ω). This result is a strong verification that APT-1 had been immobilized onto the CNF-SPE. Next, the effect of THR concentration on the aptasensor response was explored in the presence of interaction between APT-1 and protein at different concentrations of THR from 25 to 150 μg mL−1. Fig. S2 shows

Scheme 1. Impedimetric THR detection using APT-1 modified CNF-SPE.

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Fig. 1. SEM images of bare SPE (a), CNF-SPE (b). Identical acceleration voltage 10.0 kV with resolution in various magnitudes; (A) 500 nm, (B) 1 μm, (C) 2 μm and (D) 4 μm.

the impedance changes observed at the CNF-SPEs associated with the stepwise interaction process. The Rct value increased linearly 65.97%, 71.17%, 104.63% and 58.16% after interaction between 150 μg mL− 1 APT-1 and THR in different protein concentrations from 25 to 150 μg mL−1 respectively. The increase at the Rct value can be explained that the repulsive interaction between ferricyanide ions and negatively charged THR ions [4,6,17,20,40,46–48]. The negativity of the electrode

surface increased after specific interaction of THR with its DNA aptamer, then, the Rct increased till 100 μg mL− 1 THR concentration. The net negative charges of the interaction between 100 μg mL−1 THR and APT-1 in turn lead to 125.80% increase (369.33 Ω) at the Rct value. Moreover, the Rct value was measured as 86 Ω in the control experiment (Fig. S2-inset,b) after 100 μg/mL THR immobilization onto the electrode surface in the absence of its APT (APT-1).

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Fig. 3. Nyquist diagrams (A), histograms (B) representing the Rct values obtained before/ after interaction at CNF-SPE surface between 100 μg mL−1 THR and 150 μg mL−1 APT-1 or DNA ODN: unmodified CNF-SPE (a), APT-1 immobilized CNF-SPE (b), interaction between APT-1 and THR (c), DNA ODN modified CNF-SPE (d), interaction between DNA ODN and THR (e).

Fig. 2. (A) Nyquist diagrams recorded in the redox probe solution containing 2.5 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) prepared in 0.1 M KCl by using CNF-SPE immobilized with APT-1 at different concentrations in TBS (pH 7.0). The Randles circuit was present as inset which was used to fit the impedance data, the parameters of which are listed in the text; Rs is the solution resistance. The constant phase element Cd is then related to the space charge capacitance at the DNA/electrolyte interface. Rct is related to the charge transfer resistance at the DNA/electrolyte interface. The constant phase element W is the Warburg impedance due to mass transfer to the electrode surface. (B) Nyquist diagrams given as inset were obtained before (a) and after (b) 150 μg mL−1 APT-1 immobilization onto CNF-SPE surface.

Impedimetric detection of a possible interaction between THR and a random DNA oligonucleotide (DNA ODN) was also tested (shown in Fig. 3). The average Rct value (Fig. 3A-d) was measured as 389.5 ± 33.2 Ω with a RSD % = 8.5% (n = 3) after the immobilization of DNA ODN onto the CNF-SPE surface, which is 130 times higher than the one measured by unmodified CNF-SPE. However, there was a small decrease obtained at the average Rct (18.9% decrease) (Fig. 3B, d to e) after the interaction between 100 μg/mL THR and 150 μg/mL DNA ODN at electrode surface whereas 104.63% increase at the Rct after the interaction between 150 μg/mL APT-1 and 100 μg/mL THR (Fig. 3B, b to c). This result claimed that no specific binding of THR to random DNA occurred at the surface of CNF-SPE. The response of impedimetric aptasensor was also investigated in the presence of different aptamers, APT-2 and APT-MM (shown in Fig. 4). The average R ct value was calculated as 136.35 ± 34.86 Ω (RSD % = 25.6%; n = 3), and 193.5 ± 44.54 Ω (RSD % = 23.02%, n = 3), after APT-2 and APT-MM modification onto the surface of CNFSPE, respectively. In the presence of interaction between THR and each aptamer, the Rct increased due to the increase at the negativity occurred at the electrode surface through a negatively charged structure of protein [4,6,17,20,40,46–48]. The increase ratio % at Rct value was calculated, and

found to be 104.63%, 63% and 60% respectively for interaction between THR and APT-1, APT-2 and APT-MM. These results indicate that (i) THR binding capacity of APT-1 was more different than the one of APT-2 and (ii) it could selectively recognize its target protein, THR even if an aptamer with single-base mismatch has been tested. In the next step, the impedimetric detection of THR at low protein concentrations varying from 5 to 20 μg mL− 1 was investigated by

Fig. 4. Nyquist diagrams obtained by CNF-SPE. (a) unmodified CNF-SPE, (b) 150 μg mL−1 APT-1 modified CNF-SPE, (c) interaction between 100 μg mL−1 THR and APT-1, (d) 150 μg mL−1 APT-2 modified CNF-SPE, (e) interaction between 100 μg mL−1 THR and APT-2, (f) 150 μg mL−1 APT-MM modified CNF-SPE, (g) interaction between 150 μg mL−1 THR and APT-MM.

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Fig. 5. (I) Nyquist plots obtained by (a) CNF-SPE, (b) 150 μg/mL APT-1 immobilized SPE, (c) after interaction of 150 μg/mL APT-1 with THR in the concentration level of THR as (A) 5, (B) 10, (C) 15 and (D) 20 μg/mL at CNF-SPE surface. Inset was the equivalent electrical model used to fit the impedance data. (II) Calibration plot representing the changes at the ΔRct value measured in the presence of interaction between 150 μg mL−1 APT-1 and THR in various protein concentrations from 5 to 20 μg mL−1.

using 150 μg mL−1 APT-1 modified CNF-SPE. When the THR concentration increased to 20 μg mL−1, the Rct linearly increased. A calibration graph (shown in Fig. 5) was obtained based on the ΔRct calculated using Eq. (1). ΔRct ¼ Rct2 −Rct1

The apparent fractional coverage (Q RIS) values obtained after interaction of APT-1 with THR in various concentration level of THR at the surface of CNF-SPE were calculated by using the Eq. (2) described by Janek et al. [50].

ð1Þ

Rct1 and Rct2 represent the Rct values obtained after interaction of THR and APT and after APT immobilization, respectively. Based on the ΔRct values, the detection limit (DL) was calculated according to Miller and Miller method [49] and it was found to be 1.8 μg mL−1 (equals to 17.84 nM).

Q RIS ¼ 1‐

APT=CNF‐SPE Rct THRþAPT=CNF‐SPE Rct

ð2Þ

The QRIS values were calculated for the purpose of the surface coverage of CNF-SPE after interaction of THR and DNA APT-1 (see Table S2)

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4. Conclusion

Fig. 6. (A) Nyquist diagrams, (B) histograms representing the Rct values recorded using (a) CNF-SPE, (b) 100 μg/mL APT-1 immobilized CNF-SPE, after interaction with 15 μg/mL THR (c), (d) BSA and (e) PC in diluted solution of FBS:PBS (1:1000). Inset was the equivalent electrical model used to fit the impedance data.

and accordingly, the highest surface coverage was obtained in the presence of 15 μg mL−1 THR. Next, the impedimetric detection of THR using CNF-SPE based aptasensor platform was studied in the artificial serum medium; i.e., FBS, that contains numerous biomolecules. Firstly, the effect of THR concentration upon to the Rct was investigated in diluted solution of FBS:PBS (1:1000) (Fig. S2). The interaction of aptamer with protein at its various concentration from 5 to 20 μg/mL THR prepared in 1:1000 FBS:PBS diluted solutions was performed at the surface of CNFSPEs, then the impedimetric measurements were performed. Based on the changes at ΔRct values (Fig. S3), the DL was calculated according to Miller and Miller method [49] and found to be 1.9 μg mL − 1 (equals to 18.83 nM), which was quite closer to the one obtained in PBS (i.e., 1.8 μg mL− 1). The average ΔRct obtained after interaction of 150 μg/mL APT-1 and 15 μg/mL THR was calculated as 192.3 ± 21.1 Ω with the RSD % as 10.9% (n = 3). Both DLs for THR obtained in buffer and in the artificial serum medium are found lower than those reported in earlier studies [6,22,51,52]. The selectivity of the aptasensor was then tested against BSA and PC in 1:1000 FBS:PBS diluted solutions (Fig. 6). There was 100.50%, 48.29%, 20.45% increase obtained at the average Rct value after interaction between 15 μg/mL THR, BSA and PC with 150 μg/mL APT-1 in diluted solution of FBS:PBS (1:1000) respectively (Fig. 6A,B b to c,d and e). The average Rct values were recorded respectively as 385.30 ± 26.40 Ω, 285.00 ± 5.70 Ω and 231.50 ± 20.50 Ω with the RSDs % as 3.50%, 2.00% and 8.90% (n = 3) after 15 μg/mL THR, BSA and PC with 150 μg/mL APT-1 in diluted solution of FBS:PBS (1:1000). These results indicated that our impedimetric aptasensing protocol could selectively detect THR, however it was available in a complex medium; such as an artificial serum.

Aptasensor platform based on carbon nanofibers enriched screen printed electrodes (CNF-SPEs) was developed for the first time herein for impedimetric detection of thrombin. Firstly, CNF-SPE surface was characterized via SEM; then electrochemical characterization was performed by using EIS. The carbon nanofiber based sensor platform yielded a reliable and sensitive monitoring of THR through the interaction of protein with its specific DNA aptamer by EIS transduction of the resistance to charge transfer (Rct). The effect of DNA aptamer concentration and THR concentration upon the sensor response was evaluated. The selectivity of impedimetric aptasensor was also studied in the presence of other proteins; BSA and PC, or different aptamer, or random DNA. In this manner, impedimetric aptasensor platform based on carbon nanofibers enriched screen printed electrode has presented many advantages. Our aptasensor platform could be used as a single-use and implemented to a portable chip system. Moreover, they are easy to use without requirement of any complex process by using any chemical agents. Both DLs for THR obtained in buffer and in the artificial serum medium are found to be lower than those reported in earlier studies [6,22,51,52]. Sensitive and selective detection of THR at aptasensor platform could also be achieved even in the artificial serum medium without any separation, or centrifugation steps. Since there was no necessity for the usage of any time-consuming steps (such as, sonication, or chemical activation for preparation of the electrode surface), the whole detection protocol could be completed in 80 min, which was faster than the aptasensor platforms in the literature [1–5,17,20,21,53]. Carbon nanofibers enriched screen printed electrodes could provide an advanced analysis of biomolecules that was applied herein for impedimetric detection of THR through aptamer–protein interaction. Our approach could be possibly implemented further into a chip system, which enables concentration-dependent and selective detection of THR. Moreover, this sensor platform has a realistic potential for monitoring of nucleic acids, and other biomolecular recognitions. Acknowledgments A.E. would like to express her gratitude to the Turkish Academy of Sciences (TÜBA) as an Associate member for its partial support. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jelechem.2015.10.002. References [1] D. Kwon, H. Jeong, B.H. Chung, Label-free electrochemical detection of human αthrombin in blood serum using ferrocene-coated gold nanoparticles, Biosens. Bioelectron. 28 (2011) 454–458. [2] F. Yan, F. Wang, Z. Chen, Aptamer-based electrochemical biosensor for label-free voltammetric detection of thrombin and adenosine, Sensors Actuators B 160 (2011) 1380–1385. [3] C. Ding, Y. Ge, J.M. Lin, Aptamer based electrochemical assay for the determination of thrombin by using the amplification of the nanoparticles, Biosens. Bioelectron. 25 (2010) 1290–1294. [4] L.D. Li, H.T. Zhao, Z.B. Chen, X.J. Mu, L. Guo, Aptamer biosensor for label-free impedance spectroscopy detection of thrombin based on gold nanoparticles, Sensors Actuators B 157 (2011) 189–194. [5] N. Meini, C. Farre, C. Chaix, R. Kherrat, S. Dzyadevych, N. Jaffrezic-Renault, A sensitive and selective thrombin impedimetric aptasensor based on tailored aptamers obtained by solid-phase synthesis, Sensors Actuators B 166–167 (2012) 715–720. [6] A. Erdem, H. Karadeniz, G. Mayer, M. Famulok, A. Caliskan, Electrochemical sensing of aptamer–protein interactions using a magnetic particle assay and single-use sensor technology, Electroanalysis 21 (2009) 1278–1284. [7] A. Erdem, E. Eksin, M. Muti, Chitosan–graphene oxide based aptasensor for the impedimetric detection of lysozyme, Colloids Surf. B 115 (2014) 205–211. [8] A. Erdem, G. Congur, Dendrimer enriched single-use aptasensor for impedimetric detection of activated protein C, Colloids Surf. B 117 (2014) 338–345.

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