AptamerAssisted Gold Nanoparticles/PEDOT Platform for ...

28 downloads 3718 Views 693KB Size Report
Jan 23, 2013 - Aptamer-Assisted Gold Nanoparticles/PEDOT Platform for. Ultrasensitive Detection of .... hanced the thermodynamic stability and the electron-.
Full Paper

Aptamer-Assisted Gold Nanoparticles/PEDOT Platform for Ultrasensitive Detection of LPS Wenqiong Su,a MiSuk Cho,a Jae-Do Nam,b Woo-Seok Choe,a Youngkwan Lee*a a

School of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea Department of Polymer Science and Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea *e-mail: [email protected] b

Received: August 20, 2012 Accepted: October 11, 2012 Published online: January 23, 2013 Abstract Neatly arranged gold nanoparticles (AuNPs) were directly electrodeposited on an electrochemically polymerized self-assembled monolayer (SAM) of thiol-functionalized 3,4-ethylenedioxythiophene (EDOT) derivative, EDTMSHA. A thiolated single-stranded DNA (ssDNA) aptamer with high specificity to LPS was immobilized on the AuNPs/conducting polymer composite film, serving as sensing platform for LPS detection. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), scanning electron microscope (SEM), and atomic force microscopy (AFM) were utilized to characterize the modification and detection processes. The electron transfer resistance was found to have a linear relationship with LPS concentration from 0.1 pg/mL to 1 ng/mL. Keywords: Conducting polymer, Gold nanoparticles, LPS, ssDNA aptamers, PEDOT, EDTMSHA

DOI: 10.1002/elan.201200453 Supporting Information for this article is available on the WWW under http://dx.doi.org/10.1002/elan.201200453.

1 Introduction Endotoxin, also referred to as lipopolysaccharide (LPS), is a structural molecule of the Gram-negative bacteria [1]. When bacteria die and dissolve, endotoxin is released to the surroundings. Even a small amount of endotoxin present in human blood can produce fever, decrease in blood pressure, and activate inflammation and coagulation. If the immune response to endotoxin is severely manifested, it can lead to septic shock [2]. FDA (U.S. Food and Drug Administration) recommends that the endotoxin concentration limit is 0.5 EU/mL (Endotoxin Unit, 1 EU = 0.1 ng) for medical devices, 0.06 EU/mL for devices in contact with cerebrospinal fluid, and 0.2 EU/ kg/hr for intrathecal drugs [3]. Therefore, detection and removal of traces of endotoxin is an important subject in the field of pharmaceutical production, medical therapy and any in vivo applications. Nowadays, Limulus Amoebocyte Lysate (LAL) assay is the most popular FDA- approved method for endotoxin detection [4]. As an enzymatic reaction based on clotting of Limuluspolyphemus and endotoxin, LAL assay usually takes several hours for a round of test and has susceptibility to other LAL-reactive materials, such as b-(1,3)-dglucan. Therefore, a rapid, facile, and sensitive method to detect LPS has always been improved. Among all detection techniques, the electrochemical biosensors based on aptamers have attracted much attention due to their high sensitivity, ultra specificity, fast response, low cost, and 380

facile operation [5]. Aptamers are single-stranded DNA (ssDNA) or RNA molecules that not only have the comparable affinity and specificity to those of antibodies, but also have a wider range of targets from small ions to large proteins, even whole cells [6]. Since its development in 1990 [7], the aptamer has been considered as a promising candidate with great potential for the new generation biological recognition element. Previously, we identified 10 different single-stranded DNA aptamers showing specific affinity to LPS with dissociation constants in the nanomolar range using a NECEEM-based non-SELEX method [8] and immobilized one of these aptamers (Kd = 11.9 nM) on a gold electrode for LPS determination [9]. The aptasensor showed a linear detection range and high sensitivity. The detection limit achieved as low as 0.001 ng/mL in present of pDNA, RNA, BSA, glucose, sucrose and cholesterol. To further enhance the sensitivity of LPS sensor, the strategy of utilizing electrode materials possessing a nanostructure and/or a hybrid type of conducting polymer and metal nanoparticles is a vital candidate. Conducting polymer (CP), which combines the properties of organic polymers and electronic properties of semiconductors, have attracted much interests and appeared as a type of novel materials in the development of biosensors [10]. Conducting polymers have the abilities to transfer and/or amplify electric charge produced by biochemical reaction and to provide suitable environment for biomolecular immobilization [11]. The composite of con-

 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Electroanalysis 2013, 25, No. 2, 380 – 386

Ultrasensitive Detection of LPS

ducting polymer and gold nanoparticles (AuNPs) enhanced the thermodynamic stability and the electrontransfer ability [12], and promoted the chemical functionality to immobilize the biological recognition elements [13], providing an ideal matrix for electrochemical biosensors [14]. Shim et al. developed a chronoamperometric sensor to detect inducible nitric oxide synthase in neuronal cell culture based on CP-encapsulating AuNPs [15]. The conducting polymer-covered AuNPs, obtained from electropolymerization of a thiophene derivative, which is self-assembled on AuNP surface, were demonstrated to provide not only the active sites to immobilize antibodies, but also to increase surface conductivity. Recently, a series of new thiol-substituted monomers or thiophene derivants were synthesized and incorporated with AuNPs to form CP/AuNPs composites applied in the fields of electrochemical and optical sensors [16]. In this paper, an impedance biosensor using ssDNA aptamer as the biological recognition element was designed and developed to detect LPS. A monomer, named 3-((2,3-dihydrothieno[3,4b][1,4]dioxin-2-yl)methoxy)propane-1-thiol (EDTMSHA), was synthesized and its selfassembled monolayer (SAM) on gold surface was electrochemically polymerized, giving rise to a thin layer of conducting polymer named PEDTMSHA SAM [17]. A composite film of gold nanoparticles and the conducting polymer (i.e. PEDTMSHA SAM) was included to develop an ordered matrix for aptamer immobilization. The hybrid synergy of PEDTMSHA SAM and gold nanoparticles was able to transfer and amplify electric charge produced in the detection process, which accordingly increased the sensitivity of the developed biosensor. The developed biosensor was applied to detect LPS from 0.1 pg/mL to 1 ng/mL. The performance of the CP/AuNPs-based biosensor was compared with that of other LPS detection reports published recently.

2 Experimental 2.1 Materials and Apparatus LPS from Escherichia coli 055:B5 (L4524) was purchased from Sigma and stored at 20 8C before use. The ssDNA aptamer with high affinity to LPS [9], 5’-CTT CTG CCC GCC TCC TTC C- TAG CCG GAT CGC GCT GGC CAG ATG ATA TAA AGG GTC AGC CCC CCA -GGA GAC GAG ATA GGC GGA CAC T-3’, was purchased from Integrated DNA Technologies (IDT, Coralville, USA) and stored at 20 8C before use. All solutions were prepared using Millipore MilliQ water (resistivity: 18.2 MW/cm) produced by Satorious Arium 61316 RO system (Sartorius Stedim Biotech, Aubagne Cedex, France). HAuCl4, K3Fe(CN)6, K4Fe(CN)6, LiClO4, anhydrous acetonitrile (ACN), 1,4-dithiothreitol (DTT) and 6-mercapto-1-hexanol (MCH), were purchased from Sigma-Aldrich and used without further purification. The materials for EDTMSHA synthesis are described in [17]. Electroanalysis 2013, 25, No. 2, 380 – 386

All electrochemical experiments were performed using VSP Potentiostat (Princeton applied research, USA) and the resulting curves were recorded with VSP EC-Lab software. A three-electrode system with a gold disc working electrode (CH Instruments, Inc., USA; diameter: 2 mm), an Ag/AgCl (Saturated KCl) reference electrode and a platinum plate counter electrode was used to carry out CV and EIS measurements. In the EIS measurement, a 10 mV amplitude sine wave was applied in the frequency range of 0.1 Hz–100 kHz, and 2 mM Fe(CN)63 /4 (1 : 1) in phosphate buffer saline (PBS, 10 mM, pH 7.4, 50 mM NaCl) was used as the electrolyte. The ZSimpWin EIS DATA analysis software (Perkin-Elmer, Version 2.00) was used to analyze the obtained EIS data and fit an equivalent circuit. SEM (JSM-7600F, JEOL, Japan) and AFM (SPA300HV, SIINT, Japan) studies of bare and modified gold electrodes were employed to confirm the morphology of conducting polymer and gold nanoparticles. Stainless steel plates coated by a layer of gold with the thickness of 40 nm were used to carry out these studies. AFM images were taken in air at room temperature (25 8C). Silicon nitride cantilevers with a typical force constant of 40 N/m were used for AFM in the tapping mode. Scan areas with a resolution of 512  512 pixels were obtained ranging from 5.0 mm  5.0 mm. Images were obtained from at least three different sites in each given samples. 2.2 Pretreatment of Aptamer The ssDNA aptamer molecules were modified by Integrated DNA Technologies to introduce a disulfide modifier at their 5’ terminus. In order to enable self-assembly on the gold surface, the disulfide group was reduced by DTT for 1 h at room temperature to give thiolated aptamer. Following the reaction, the thiolated aptamer was purified by centrifugal filter (UFC900324, Millipore) at 10000 g for 30 min three times. The purified aptamer was diluted by PBS (10 mM, pH 7.4) to give an ssDNA aptamer concentration of 14 nM based on UV absorption at 260 nm. 2.3 Preparation of Gold Electrodes The gold disc electrodes were polished with 0.05 mm alumina slurry, and then ultrasonically cleaned in distilled water and absolute ethanol, respectively. After that, the gold electrode was immersed in Piranha solution [30 % H2O2/98 % H2SO4 (1/3, v/v)] and washed with distilled water. Subsequently, the gold electrode was electrochemically activated by CV in 0.5 M H2SO4 aqueous solution from 0 to 1.5 V at a scan rate of 100 mV/s for 20 cycles [5]. 2.4 Preparation of PEDTMSHA SAM The synthesis of EDTMSHA is described in [17]. Following the procedure, the final product (EDTMSHA) was

 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.electroanalysis.wiley-vch.de

381

Full Paper

W. Su et al.

Fig. 1. A schematic of fabricating aptasensor to detect LPS.

analyzed by 1H NMR and 13C NMR to confirm its structure. The fresh EDTMSHA was purified and dissolved in ACN to get a 0.1 M solution. EDTMSHA SAM was performed on a cleaned gold disc electrode for 24 h incubation and then electrochemically polymerized in 0.1 M LiClO4/ACN solution under the potential between 0.4 V and 1.2 V (vs. Ag/AgCl) at a scan rate of 50 mV/ s for 20 redox cycles.

2.5 Biosensor Preparation The gold nanoparticles were directly electrodeposited on the PEDTMSHA SAM-coated gold surface in 0.5 M H2SO4 aqueous solution containing 10 mM HAuCl4 at 0.2 V for 30 s [18]. The thiolated aptamers were assembled on the AuNPs/ PEDTMSH SAM-covered gold electrode for 3 h at room temperature. The aptamer-modified gold electrode was further blocked in 1 mM MCH aqueous solution for 1 h at room temperature [19]. The modification process was illustrated in Figure 1. For control, in absence of conducting polymer layer, an AuNP-coated electrode was fabricated under the same process. The performance of the developed biosensors was evaluated in a series of LPS/PBS solutions with varying concentrations.

2.6 Electrochemical Measurements Electrochemical impedance spectroscopy (EIS) is a useful electrochemical technique to track the changes of the electrode/electrolyte interface by applying an oscillating potential as a function of frequency [20]. Based on the charge transfer kinetics of the redox probe (i.e. Fe(CN)63 /4 ), the physiochemical process in the electrochemical system was modeled by a suitable equivalent circuit. In this study, the modified Randle model consisting of solution resistance, electron transfer resistance, content phase element (CPE) and Warburg impedance [21] was used to describe the obtained EIS data (Figure 5A Inset). EIS measurement in 2 mM Fe(CN)63 /4 (1 : 1)/PBS was conducted to electrochemically assess interaction of the aptamer-modified gold electrode with LPS after incubation in a series of PBS solutions containing different con382

www.electroanalysis.wiley-vch.de

centration of LPS (0.0001, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 ng/mL) for 15 min each.

3 Result and Discussion 3.1 The Characterization of AuNPs/PEDTMSHA SAM Composite Film As it is shown in Figure 2A, the redox peaks of 2 mM K3Fe(CN)6 in 0.1 M KCl/H2O provided an indirect measurement of the EDTMSHA self-assembly process. After self-assembly by EDTMSHA, the CV current (Figure 2 A2) was almost flat, compared to that of bare gold electrode (Figure 2 A1), which clearly showed a pair of redox peaks of K3Fe(CN)6. The tightly packed EDTMSHA SAM posed a high steric and electrostatic hindrance to the redox probes (i.e. K3Fe(CN)6) in accessing the electrode surface [22], so the CV current was decreased dramatically. The cyclic voltammogram of EDTMSHA SAM electropolymerization is shown in Figure 2B. The application of repetitive potential cycles led to the progressive development of a reversible redox system, which is indicative of the formation of longer conjugated chains. After ca.20 recurrent scans, the shape and intensity of the cyclic voltammogram waves stabilized, which indicated that all of the assembling monomers had been oxidized to format thin conjugated film on the gold disc electrode surface [17]. Theoretically, when self-assembled EDTMSHA monomers fasten on gold surface via their thiol groups, the sulfur atoms of their thiophene groups should expose outwards [23]. The electrochemical polymerization of EDTMSHA monomers produced ordered conjugated chains firmly attached on the gold surface through the Au-S bond. Thus the exposed sulfur atoms of the thiophene groups were arranged in order and offered a regular and compatible surface for further modification. The morphology of the bare gold electrode was presented by SEM, as shown in Figure 3A. Figure 3B shows the morphology of gold nanoparticles, which were electrochemically deposited on the PEDTMSHA SAM-covered gold surface. In the presence of PEDTMSHA SAM, Au NPs grew in order and uniformly, spreading out

 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Electroanalysis 2013, 25, No. 2, 380 – 386

Ultrasensitive Detection of LPS

Fig. 2. Cyclic voltammogram of EDTMSHA SAM modified gold electrode in (A) 2 mM K3Fe(CN)6 + 0.1 M KCl/H2O and (B) 0.1 M LiClO4/ACN solution producing PEDTMSHA SAM (A1: bare gold; A2: EDTMSHA SAM-covered gold electrode; B inner: the last cycle).

a monolayer along the straight grain of the conducting polymer. The uniform particles completely covered the electrode surface with an average diameter of 160nm. The AuNPs were successfully arranged in order, probably due to the exposed sulfur atoms of thiophene groups produced by PEDTMSHA SAM. The HAuCl4 molecules might have been partially reduced on the conducting polymer-covered surface, and the generated gold nanoparticles were instantly adsorbed by the sulfur atoms placed on the electrode surface. AFM images gave more information. The root-mean-square (RMS) roughness was 11 nm of bare gold (Figure 3C). After self-assembled of EDTMSHA (Figure 3D), the RMS roughness of gold surElectroanalysis 2013, 25, No. 2, 380 – 386

face was increased to 61 nm, indicating the successful deposition of EDTMSHA SAM. The RMS roughness (59 nm) was not changed too much after electrochemical polymerization of that SAM (Figure 3E). Finally, the RMS roughness (184 nm) was increased largely after eletrodeposition of Au NPs (Figure 3F), indicating the successful deposition of gold nanoparticles. 3.2 The Fabrication of Aptasensor The modification process of a gold electrode was characterized by EIS. The typical Nyquist plots in 2 mM Fe(CN)63 /4 (1 : 1)/PBS (10 mM, pH 7.4), including a semi-

 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.electroanalysis.wiley-vch.de

383

Full Paper

W. Su et al.

Fig. 3. SEM (A, B) and AFM (C–F) images of bare (A, C), EDTMSHA SAM-covered (D), PEDTMSHA SAM-covered (E) and Au NPs/PEDTMSHA SAM-covered (B, F) electrodes.

circle portion at higher frequencies corresponding to the electron-transfer-limited process and a linear part at lower frequency range representing the diffusion-limited process [24], are shown in Figure 4. The bare gold corresponds to an almost straight line (Figure 4 curve 1), which is characteristic of a diffusion-controlled process over most frequency range [25]. The Nyquist plots of the PEDTMSHA SAM-modified gold surface (Figure 4 curve 2) contained a large semicircle without straight line, indicating an electron-transfer limited process over the whole frequency range [20]. After electrochemical deposition of gold nanoparticles, the semicircle was decreased

(Figure 4 curve 3). The presence of gold nanoparticles reduced the surface resistance for redox probes, and the network of gold nanoparticles and conducting polymer offered a good system for electron transfer [26]. The EIS data (Figure 4 curve 4 and 5) indicated that the electron transfer resistance (Ret) remarkably increased due to the formation of a mixed self-assembled monolayer of the aptamer and the spacer thiol [27]. The electronegative phosphate skeleton of the aptamer and a barrier of the blocking molecules prevented Fe(CN)63 /4 ions from reaching the electrode surface for electron transfer and mass transfer during redox reaction [25]. The post-treatment with MCH not only covered the exposed gold surface, but also replaced the nonspecific adsorbed aptamers to a great extent. The dilution of small thiol molecules provided space among aptamers and facilitated precise orientation of ssDNA molecules to reach their targets easily [19]. 3.3 The Performance of Aptasensor

Fig. 4. Nyquist plots for modification process. (1: bare gold electrode; 2: PEDTMSHA SAM-covered gold electrode; 3: gold nanoparticles-deposited gold electrode; 4: aptamer-immobilized gold electrode; 5: MCH-blocked gold electrode.)

384

www.electroanalysis.wiley-vch.de

The response of the aptamer/endotoxin interaction in supporting solution was directly probed and quantified by EIS. The biosensor was applied to a series of samples with different endotoxin concentrations. When the concentration of endotoxin was high, the semi-circles of Nyquist plots increased due to the inter-attraction between the aptamers and their targets (Figure 5A). The increase of Ret showed a good linear relationship with the endotoxin concentration from 0.1 pg/mL to 1 ng/mL, which was DRet = 23236.7 log ([LPS/ng·mL 1]) + 94457.5 and R2 was closed to 0.99 (Figure 5 B1). Compared to the results obtained from the biosensors based on a gold nanoparticle-coated electrode (Figure 5 B2), DRet from gold elec-

 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Electroanalysis 2013, 25, No. 2, 380 – 386

Ultrasensitive Detection of LPS

The interfering species, nucleic acids, proteins, sugars and lipids which frequently co-exist with LPS in biological liquors were selected and prepared into 50 mg/mL solutions in PBS (10 mM, pH 7.4) [8,9]. The developed LPS aptasensor based on AuNPs/PEDOT platform exhibited good selectivity, consistent with the previous report [9] [Figure S2 in Supporting Information]. When the target molecules are present, aptamers will change into their secondary structures in order to combine with their targets in specific sites [28], which is the way aptamers recognize their targets with high specificity. It is necessary to offer enough space for aptamers to complete the analyte-dependent conformational changes. The novel structure of PEDTMSHA SAM and gold nanoparticles network served as an ordered matrix that gave enough space among each aptamer molecules and also reduced the steric hindrance when aptamers changed into their secondary conformation [24]. The synergy between the gold nanoparticles and the conducting polymer that integrates the network not only provided a large surface area, but also facilitated electron transfer to the substrate [29]. For comparison purposes, we summarize some recent reports using different recognition elements and approaches for LPS sensing in Table 1.

4 Conclusions Fig. 5. Nyquist plots in Fe(CN)63 /4 /PBS solution for LPS detection (A, 1–9: LPS/PBS with the concentration 0, 0.0001, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5 and 1.0 ng/mL; inner: the equivalent circuit for modeling EIS data) and (B) linear relationship between DRet and LPS concentration with (curve 1) and without (curve 2) conducting polymer layer.

trode modified by both conducting polymer and gold nanoparticles was dramatically increased due to the ordered arrangement of aptamer molecules and the inherent electric properties [18,20]. The EIS data of the sensor performed in a concentration range more than 1 ng/mL were shown in Figure S1 (Supporting Information).

In this study, an impedance biosensor based on a novel composite film, PEDTMSHA SAM and gold nanoparticles offered a simple, sensitive and rapid method to detect LPS. The developed aptasensor was demonstrated to detect endotoxin at a range of 0.1 pg/mL 1 ng/mL. Compared with the biosensors without the conducting polymer layer, the developed biosensor showed a high sensitivity and a low detection limit. The combination of conducting polymer and gold nanoparticles provides an appropriate microenvironment enhancing the stability and bioactivity of the immobilized aptamers by influencing their orientation and distribution. The network of conducting polymer and gold nanoparticles in favor of charge transfer has a tremendous prospect in the development of biosensors.

Table 1. Aanalysis results of various developed LPS sensors. Ref.

Technique

Probe

Sensitivity

Detection range

[30] [31] [32] [33] [34]

Uv-Vis Capacitance Chemiluminescence Cyclic voltammetric Fluorescent

Nonlinear 18 nF cm 2/log (M) Nonlinear Nonlinear 852 a.u./mM

0.011–10 EU/mL 1.0  10 13–1.0  10 0.01–10 ng/mL 0.1–1000 ng/mL 0.1–1.5 mM

[5b] [9] This study

EIS EIS EIS

LAL ENP Antibody ENP N,N-Dimethyl-N-(pyrenyl-1-methyl) dodecan-1-ammonium Polymyxin B LPS aptamer LPS aptamer

Nonlinear 597 W/log (ng mL 1) 23237 W/log (ng mL 1)

0.1 mg/mL–1000 mg/mL 1 pg/mL–1 ng/mL 0.1 pg/mL–1 ng/mL

Electroanalysis 2013, 25, No. 2, 380 – 386

 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.electroanalysis.wiley-vch.de

10

M

385

Full Paper

W. Su et al.

Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0083540) and by Ministry of Education, Science and Technology of Korea (Grant No. 2012-002285).

References [1] C. R. H. Raetz, Ann. Rev. Biochem. 1990, 59, 129. [2] S. Copeland, H. S. Warren, S. F. Lowry, S. E. Calvano, D. Remick, Clin. Vaccine Immunol. 2005, 12, 60. [3] a) Pyrogen and Endotoxins Testing: Questions and Answers, U.S. FDA, 2012; b) S. Poole, P. Dawson, R. E. Gaines Das, Innate Immunity 1997, 4, 21. [4] a) J. F. Cooperet, J. Levin, H. N. Wagner, J. Nucl. Med. 1970, 11, 310; b) A. Kakinuma, T. Asano, H. Torii, Y. Sugino, Biochem. Bioph. Res. Co. 1981, 101, 434. [5] a) S. Iijima, D. Kato, S. Yabuki, O. Niwa, F. Mizutani, Biosens. Bioelectron. 2011, 26, 2080; b) A. R. Mohd Syaifudin, S. C. Mukhopadhyay, P. L. Yu, I. R. Matias, J. Goicoechea, J. Kosel, C. P. Gooneratne, in 10th Sensors, IEEE, 2011, p. 588; c) M. D. L. Oliveira, C. A. S. Andrade, M. T. S. Correia, L. Coelho, P. R. Singh, X. Q. Zeng, J. Colloid Interf. Sci. 2011, 362, 194; d) M. H. Lan, J. S. Wu, W. M. Liu, W. J. Zhang, J. C. Ge, H. Y. Zhang, J. Y. Sun, W. W. Zhao, P. F. Wang, J. Am. Chem. Soc. 2012, 134, 6685; e) J. Sun, J. Ge, W. Liu, X. Wang, Z. Fan, W. Zhao, H. Zhang, P. Wang, S. T. Lee, Nano Res. 2012, 1, 1. [6] S. Song, L. Wang, J. Li, C. Fan, J. Zhao, TrAC – Trend Anal. Chem. 2008, 27, 108. [7] S. Tombelli, M. Minunni, M. Mascini, Biosens. Bioelectron. 2005, 20, 2424. [8] S. E. Kim, W. Su, M. Cho, Y. Lee, W. S. Choe, Anal. Biochem. 2012, 424, 12. [9] W. Su, M. Lin, H. Lee, M. Cho, W. S. Choe, Y. Lee, Biosens. Bioelectron. 2012, 32, 32. [10] a) A. C. Manju Gerard, B. D. Malhotra, Biosens. Bioelectron. 2002, 17, 345; b) L. Xia, Z. Wei, M. Wan, J. Colloid Interf. Sci. 2010, 341, 1. [11] a) F. R. R. Teles, L. P. Fonseca, Mater. Sci. Eng. C 2008, 28, 1530; b) W. Liao, B. Randall, N. Alba, X. Cui, Anal. Bioanal. Chem. 2008, 392, 861.

386

www.electroanalysis.wiley-vch.de

[12] M. C. Daniel, D. Astruc, Chem. Rev. 2003, 104, 293. [13] A. Balamurugan, K. C. Ho, S. M. Chen, T. Y. Huang, Colloid. Surf. A 2010, 362, 1. [14] H. B. Noh, K. S. Lee, B. S. Lim, S. J. Kim, Y.-B. Shim, Electrophoresis 2010, 31, 3053. [15] W. C. A. Koh, P. Chandra, D. M. Kim, Y. B. Shim, Anal. Chem. 2011, 83, 6177. [16] a) Z. Liu, J. Liu, G. Shen, R. Yu, Electroanalysis 2006, 18, 1572; b) Z. Zhang, F. Wang, F. Chen, G. Shi, Mater. Lett. 2006, 60, 1039; c) Y. Leroux, E. Eang, C. Fave, G. Trippe, J. C. Lacroix, Electrochem. Commun. 2007, 9, 1258; d) G. Zotti, B. Vercelli, A. Berlin, Chem. Mater. 2007, 20, 397; e) P. Kannan, S. A. John, Electrochim. Acta 2011, 56, 7029. [17] W. Su, H. T. Nguyen, M. Cho, Y. Son, Y. Lee, Synth. Met. 2010, 160, 2471. [18] K. A. Mahmound, S. Hrapovic, J. H. T. Luong, ACS Nano 2008, 2, 1051. [19] T. M. Herne, M. J. Tarlov, J. Am. Chem. Soc. 1997, 119, 8916. [20] M. I. Prodromidis, Electrochim. Acta 2010, 55, 4227. [21] C. Z. Li, Y. Liu, J. H. T. Luong, Anal. Chem. 2004, 77, 478. [22] M. I. Pividori, A. MerkoÅi, S. Alegret, Biosens. Bioelectron. 2000, 15, 291. [23] M. Ocafrain, T. K. Tran, P. Blanchard, S. Lenfant, S. Godey, D. Vuillaume, J. Roncali, Adv. Funct. Mater. 2008, 18, 2163. [24] L. V. Protsailo, W. R. Fawcett, Electrochim. Acta 2000, 45, 3497. [25] U. K. Sur, R. Subramanian, V. Lakshminarayanan, J. Colloid Interf. Sci. 2003, 266, 175. [26] E. Katz, I. Willner, Electroanalysis 2003, 15, 913. [27] M. K. Patel, P. R. Solanki, A. Kumar, S. Khare, S. Gupta, B. D. Malhotra, Biosens. Bioelectron. 2010, 25, 2586. [28] T. Hermann, D. J. Patel, Science 2000, 287, 820. [29] J. Heinze, B. A. Frontana-Uribe, S. Ludwigs, Chem. Rev. 2010, 110, 4724. [30] K. Ong, J. Leland, K. Zeng, G. Barrett, M. Zourob, C. Grimes, Biosens. Bioelectron. 2006, 21, 2270. [31] W. Limbut, M. Hedstrçm, P. Thavarungkul, P. Kanatharana, B. Mattiasson, Anal. Bioanal. Chem. 2007, 389, 517. [32] M. Yang, Y. Kostov, H. A. Bruck, A. Rasooly, Anal. Chem. 2008, 80, 8532. [33] D. Pallarola, F. Battaglini, Anal. Chem. 2009, 81, 3824. [34] L. Zeng, J. Wu, Q. Dai, W. Liu, P. Wang, C. S. Lee, Organic Lett. 2010, 12, 4014.

 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Electroanalysis 2013, 25, No. 2, 380 – 386