Impedance Based Detection of Chemical Warfare Agent Mimics Using

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Anal. Chem. 2010, 82, 3191–3197

Impedance Based Detection of Chemical Warfare Agent Mimics Using Ferrocene-Lysine Modified Carbon Nanotubes Piotr M. Diakowski,† Yizhi Xiao,† Michael W. P. Petryk,‡ and Heinz-Bernhard Kraatz*,† Department of Chemistry, University of Western Ontario, London, Ontario, Canada N6A 5B7 and DRDC Suffield, P.O. Box 4000, Station Main, Medicine Hat, Alberta, Canada T1A 8K6 A recognition layer formed by multiwalled carbon nanotubes (MWCNTs) covalently modified with a ferrocenelysine conjugate deposited on the indium tin oxide (ITO) was investigated as a sensor for chemical warfare agent (CWA) mimics. Electrochemical impedance spectroscopy measurements showed that upon addition of CWA mimic dramatic changes occurred in the electrical properties of the recognition layer. These changes allowed the detection of nerve agent analogues at the micromolar level, and a limited sensitivity was observed toward a sulfur mustard mimic. Experimental parameters were optimized so as to allow the detection of CWAs at single frequency, thereby significantly reducing acquisition time and simplifying data treatment. A proposed method of detection represents a significant step toward the design of an affordable and “fieldable” electrochemical CWA sensor. Since the introduction of modern chemical warfare agents (CWAs) at the beginning of 20th century, there has been continuous interest in the development of robust and reliable methods of detection of such agents.1-3 Current methods for CWA detection can be grouped into point and standoff detectors.4 Point (or in situ) detectors respond to the presence of CWAs in the immediate vicinity of the detector. Common techniques which are used in point detectors include ion mobility spectrometry,5 flame photometry,6 mass spectrometry,7-9 photoacoustic infrared spectroscopy,10 surface acoustic wave techniques,11 electrochemistry,12,13 * To whom correspondence should be addressed. E-mail: [email protected]. Fax: +1 519 661 3022. † University of Western Ontario. ‡ DRDC Suffield. (1) Sanderson, H.; Fauser, P.; Thomsen, M.; Soerensen, P. B. J. Hazard. Mater. 2009, 162, 416–422. (2) Sidell, F. R. Clin. Toxicol. 1974, 7, 1–17. (3) Grob, D.; Harvey, A. M. Am. J. Med. 1953, 14, 52–63. (4) Xie, Y.; Popov, B. N.; White, R. E. J. Electroanal. Chem. 1999, 466, 169– 176. (5) McHugh, V. M.; Harden, C. S.; Shoff, D. B.; Ince, B. S.; Harper, S. E.; Blethen, G. E.; Schafer, R. J.; Arnold, P.; Pavitt, S.; Thomas, M.; Conner, T.; Terzic, E.; Espander, W. Int. J. Ion Mobility Spectrom. 2003, 6, 49–52. (6) Le Harle, J.-P.; Bellier, B. J. Chromatogr., A 2005, 1087, 124–130. (7) Dubey, D. K.; Pardasani, D.; Kanaujia, P. K.; Tak, V.; Gupta, H. K. Rapid Commun. Mass Spectrom. 2008, 22, 2526–2532. (8) Kubachka, K. M.; Richardson, D. D.; Heitkemper, D. T.; Caruso, J. A. J. Chromatogr., A 2008, 1202, 124–131. (9) Owens, J.; Koester, C. J. Agric. Food Chem. 2009, 57, 8227–8235. (10) Webber, M. E.; Pushkarsky, M.; Patel, C. K. N. J. Appl. Phys. 2005, 97, 113101/113101–113101/113111. 10.1021/ac902694d  2010 American Chemical Society Published on Web 03/23/2010

and “wet chemistry” detection kits.14 Point detectors often offer good sensitivity at an affordable price, but some techniques (such as “wet chemistry” methods) may not be suitable for continuous monitoring. Furthermore, some very powerful analytical techniques, e.g., chromatographic methods, flame photometry, and mass spectrometry, may not be suitable for field applications due to the lack of portability, power requirements, and the need for resupply of consumables. In contrast, many standoff detectors use infrared remote sensing to detect the presence of CWAs at a distance from the threat, typically, in a range of 1-5 km.15,16 However, many standoff detectors may generate false readings due to the variations in humidity, temperature, and composition of air4 and suffer from problems such as heterogeneous backgrounds in urban environments and low sensitivities. Immunoassays and inhibition studies such as monitoring of acetylcholinesterase activity are promising alternatives, but owing to the irreversible nature of the reaction and the need for water, they are presently not suitable for real-time monitoring.17,18 Electrochemical detectors offer advantages in terms of high sensitivity, ease of miniaturization and integration, and low cost and power requirements. Since CWAs and their degradation products are electrochemically inactive, modified electrodes that use specific recognition elements need to be employed to enable CWA detection.19,20 In particular, aminoferrocene and ferroceneamino acids derivatives have been shown to exhibit interesting donor properties which were exploited for the detection of electrophilic species which include nerve agents and their degradation products.19-22 Arguably, one of the most powerful (11) Nieuwenhuizen, M. S.; Harteveld, J. L. N. Sens. Actuators, B 1994, 19, 502–505. (12) Nishiyama, K.; Yamada, H.; Kishi, S.; Sato, K.; Matsuura, H.; Nakano, N.; Seto, Y.; Taniguchi, I. Chem. Sens. 2009, 25, 79–81. (13) Oh, I.; Masel, R. I. Electrochem. Solid-State Lett. 2007, 10, J19–J22. (14) Eisenkraft, A.; Markel, G.; Simovich, S.; Layish, I.; Hoffman, A.; Finkelstein, A.; Rotman, E.; Dushnitsky, T.; Krivoy, A. Mil. Med. 2007, 172, 997–1001. (15) Gottfried, J. L.; De Lucia, F. C., Jr.; Munson, C. A.; Miziolek, A. W. Appl. Spectrosc. 2008, 62, 353–363. (16) Lavoie, H.; Puckrin, E.; ThEriault, J.-M. Int. J. High Speed Electron. Syst. 2008, 18, 457–468. (17) Liu, G.; Lin, Y. Anal. Chem. 2006, 78, 835–843. (18) Joshi, K. A.; Prouza, M.; Kum, M.; Wang, J.; Tang, J.; Haddon, R.; Chen, W.; Mulchandani, A. Anal. Chem. 2006, 78, 331–336. (19) Khan, M. A. K.; Kerman, K.; Petryk, M.; Kraatz, H.-B. Anal. Chem. 2008, 80, 2574–2582. (20) Khan, M. A. K.; Long, Y.-T.; Schatte, G.; Kraatz, H.-B. Anal. Chem. 2007, 79, 2877–2884. (21) Khan, M. A. K.; Thomas, D. S.; Kraatz, H.-B. Inorg. Chim. Acta 2006, 359, 3339–3344.

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Scheme 1. General Procedure for the Preparation of the Recognition Layer: Acid Treatment of Commercially Available MWCNTs Results in Shortening of the CNTs and Carboxy-Functionalization, Which Are Exploited to Covalently Attach the Ferrocene-Lysine Conjugate, Fc-CO-Lys-OMe, through the ε-amino Group of the Lys Residuea

a The resulting MWCNT conjugates are supported onto an indium tin oxide (ITO) surface and used for the detection of CWA mimics by electrochemical impedance spectroscopy.

electrochemical methods for the investigation of surface processes is electrochemical impedance spectroscopy (EIS).23 Unfortunately, the application of impedance based methods in chemical analysis is less common than “classical” voltammetric methods despite the fact that the former are inherently capable of providing more accurate data,24 perhaps due to less intuitive interpretation of experimental results often accompanied by development of appropriate mathematical models. Carbon nanotubes (CNTs) are interesting from the electrochemical sensor design perspective, because they display extreme sensitivity of the current flow to a local chemical environment.25 Importantly, CNT based transducers are known to give stable instrumental response,26,27 and they have been shown to effectively improve the redox currents and reduce the peak separations, as they contribute to charge transfer and mass transfer processes mostly through the increase of porosity and interface surface area.28-30 Furthermore, CNTs are easy to handle and functionalize.31,32 In this report, we demonstrate the use of covalently modified multiwalled carbon nanotubes (MWCNTs) for the detection of (22) Duffy, N. W.; Harper, J.; Ramani, P.; Ranatunge-Bandarage, R.; Robinson, B. H.; Simpson, J. J. Organomet. Chem. 2005, 564, 125–131. (23) Barsoukov, E.; MacDonald, R. Impedance Spectroscopy: Theory, Experiment, and Applications, 2nd ed.; John Wiley & Sons, Inc.: Hoboken,NJ, 2005. (24) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications, 2nd ed.; John Wiley & Sons, Inc.: Hoboken,NJ, 2001. (25) Wang, J.; Yang, S.; Guo, D.; Yu, P.; Li, D.; Ye, J.; Mao, L. Electrochem. Commun. 2009, 11, 1892–1895. (26) Xu, Z.; Chen, X.; Qu, X.; Dong, S. Electroanalysis 2004, 16, 684–687. (27) Dumitrescu, I.; Unwin, P. R.; MacPherson, J. V. Electrochem. Commun. 2009, 11, 2081–2084. (28) Hu, C.; Yuan, S.; Hu, S. Electrochim. Acta 2006, 51, 3013–3021.

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nerve agent analogues by combining the use of recognition with impedance sensing. Scheme 1 outlines the preparation of the recognition layer and subsequent detection of CWAs. First, MWCNTs are activated, which creates sidewall defects, adds carboxylate groups, and increases the solubility of CNTs in aqueous media.19,33 Next, the carboxylic groups on shortened MWCNTs are covalently modified with ferrocene-lysine conjugate to give Fc-CO-Lys-OMe-MWCNT, which is subsequently deposited onto indium tin oxide (ITO) to form the recognition layer. This new covalent modification method differs considerably from the noncovalent one reported earlier by our group;19 it also offers significant advantages (i.e., more reproducible surface preparation and improved surface stability). Interactions between the recognition layer and the organophosphonate result in a change in the impedance characteristics of the system (as compared to the system in the absence of phosphonate), forming the basis for impedance based detection of organophosphate simulants. As the system has shown promise toward the detection of organophosphate nerve agents, its selectivity was also tested by evaluating its efficiency at detecting sulfur mustard. In the current investigation, 2-chloroethyl ethyl sulfide (2-CLEES) and diethyl cyanophosphonate (DECP) were used as reasonable mimics of sulfur mustard (agent HD) and tabun (agent GA), (29) Siqueira, J. R., Jr.; Gasparotto, L. H. S.; Oliveira, O. N., Jr.; Zucolotto, V. J. Phys. Chem. C 2008, 112, 9050–9055. (30) Obradovic, M. D.; Vukovic, G. D.; Stevanovic, S. I.; Panic, V. V.; Uskokovic, P. S.; Kowal, A.; Gojkovic, S. L. J. Electroanal. Chem. 2009, 634, 22–30. (31) Singh, P.; Campidelli, S.; Giordani, S.; Bonifazi, D.; Bianco, A.; Prato, M. Chem. Soc. Rev. 2009, 38, 2214–2230. (32) Sgobba, V.; Guldi, D. M. Chem. Soc. Rev. 2009, 38, 165–184. (33) Gooding, J. J. Electrochim. Acta 2005, 50, 3049–3060.

Scheme 2. Chemical Structures of Pinacolyl Methyl Phosphonate (PMP), Which Is a Hydrolysis Product of Soman (GD), Methylphosphonic Acid (MPA), Which Is the Final Hydrolysis Product of a Series of Nerve Agents (GB, GS, and VX), and the Two Mimics Diethyl Cyanophosphonate (DECP) and 2-Chloroethyl Ethyl Sulfide (2-CLEES)

respectively (see Scheme 2). In addition, the responses of the system to two nerve agent decomposition products were evaluated. Pinacoyl methylphosphonate, PMP (hydrolysis product of soman, agent GD), and methyl phosphonic acid, MPA (ultimate hydrolysis product of GB, GD, and VX agents), were chosen for this purpose (see Scheme 2). EXPERIMENTAL SECTION Chemicals and Materials. All reagents were used as received, unless otherwise indicated. All aqueous solutions were freshly prepared using deionized water (Milli-Q, 18.2 MΩ). Pinacolyl methylphosphonate (PMP), ethyl methylphosphonate (EMP), methylphosphonic acid (MPA), diethyl cyanophosphonate (DECP), 2-chloroethyl ethyl sulfide (2-CLEES), and sodium perchlorate (99.99%) were purchased from Sigma-Aldrich Corp. St. Louis, MO. H-Lys(Boc)-OMe · HCl was purchased from EMD Chemicals Inc., Gibbstown, New Jersey. Dichloromethane (DCM), dimethylforamide (DMF), and trifluoroacetic acid (TFA) were obtained from Caledon Laboratories Ltd., Georgetown, Ontario, Canada. O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate (HBTU) was from Advanced ChemTech, Louisville, KY. Multiwalled carbon nanotubes (MWCNTs) with 8-15 nm diameter and 3-8 µm length were obtained from NanoNB Corp., Fredricton, NB, Canada. ITO substrates were obtained from SPI Supplies, West Chester, PA. Polytetrafluoroethylene (PTFE) membranes were purchased from Sartorius AG, Go¨ttingen, Germany. Measurements and Instrumentation. A custom-made electrochemical cell was used to house the modified ITO substrates (Figure S1, Supporting Information) and a conventional three electrode arrangement was used. The surface area of the substrate exposed to the supporting electrolyte solution was 2.01 cm2. A coiled platinum wire, 5 cm long, (0.2 mm diameter, Alfa Aesar, Ward Hill, MA) served as an auxiliary electrode, while Ag/ AgCl reference electrode was purchased from CH Instruments (Austin, TX), and it was connected to the electrochemical cell via a homemade agar salt bridge (1 M KNO3). The cell was filled with 5 mL of 2.0 M sodium perchlorate solution. All electrochemical measurements were carried out under ambient conditions (without removal of dissolved oxygen) using a CompactStat-Electrochemical Interface and Impedance analyzer (Ivium Technologies B.V., Eindhoven, The Netherlands). All electrochemical measurements were carried out in a grounded Faraday cage. In the measurements involving chemical agents,

the desired concentrations of the chemical agent was obtained by a method of subsequent additions. After an aliquot of chemical agent was added, the system was allowed to equilibrate for 15 min before a measurement was taken. Replicate data were obtained on multiple electrodes. NMR spectra were recorded using a Varian MERCURY 400 MHz spectrometer operating at 400.1 MHz (1H) and 100.62 MHz (13C). Mass spectrometry was carried out on a Finnigan MAT 8200 instrument. Scanning electron microscopy (SEM) was performed on a Leo 1540 FIB/SEM CrossBeam microscope. IR experiments were carried out on Bruker FT-IR spectrometer as a KBr disk. Raman spectra were recorded on a Renishaw 2000 Raman spectrometer equipped with a LN2 cooled CCD detector. Modification of MWCNTs with Fc-CO-Lys(Boc)-OMe. The ferrocene-amino acid conjugate (Fc-CO-Lys(Boc)-OMe) was synthesized and characterized as described in the Supporting Information. The following steps were then required to obtain MWCNTs covalently modified with Fc-Lys-conjugate (also, see Scheme S1 in the Supporting Information). First, the MWCNTs were shortened according to the procedures described elsewhere.34,35 Briefly, MWCNTs were heated (70 °C) and sonicated in an acidic mixture (conc. H2SO4 and conc. HNO3, 3:1) for 12 h. This procedure is known to produce MWCNTs that can be easily suspended, sorted, and modified with other molecules. As a result, shortened carbon nanotubes functionalized with COOH groups were obtained. Next, the shortened MWCNTs (1 mg) in N-N′-dimethylformamide, DMF, (100 µL) were sonicated for 1 h, followed by addition of the peptide coupling reagent O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluorophosphate, HBTU (1 mg), and triethylamine (TEA) and sonicated for another 10 min. Then, Fc-CO-Lys(Boc)-OMe (5 mg) in DMF was treated with trifluoroacetic acid, TFA, neutralized and added to the CNT suspension. The reaction mixture was allowed to stir overnight, and resulting precipitate was thoroughly washed with methanol and centrifuged (12 000 rpm for 20 min) to remove any unreacted Fc-Lys. The final product was dried under vacuum at 40 °C for 1 h. The modified MWCNTs showed lower dispersion in ethanol or water than unmodified MWCNTs. This can be explained by the reduced number of carboxylic groups on the MWCNTs and introduction of alkyl chains from the Fc-amino acid conjugates. The shortened MWCNTs were examined by FT-IR spectroscopy before and after modification with Fc-CO-Lys-OMe (see Supporting Information, Figure S3). Additional examination of the Fc-CO-Lys-OMe was also performed by means of Raman spectroscopy (see Figure S4 in Supporting Information). Modification and Characterization of the ITO Substrates. First, ITO substrates were cleaned with a wet soft cotton cloth. Next, the substrates were thoroughly rinsed with Milli-Q water, followed by cleaning in an ultrasonic bath (30 min each cycle; first in Milli-Q water/Triton X-100, then in Milli-Q water, and finally in freshly distilled ethanol). The ITO substrates were dried in a stream of pure nitrogen prior to use. Then, a suspension of (34) Liu, Z.; Shen, Z.; Zhu, T.; Hou, S.; Ying, L.; Shi, Z.; Gu, Z. Langmuir 2000, 16, 3569–3573. (35) Gooding, J. J.; Wibowo, R.; Liu, J.; Yang, W.; Losic, D.; Orbons, S.; Mearns, F. J.; Shapter, J. G.; Hibbert, D. B. J. Am. Chem. Soc. 2003, 125, 9006– 9007.

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Scheme 3. Proposed Interactions of the Fc-CO-Lys-OMe to the ITO Surface via H-Bonding Interactions between Functionalized Groups of the MWCNTs and -OH Groups Present at the ITO Surface

Figure 1. Nyquist plot (Z′ vs -Z′′) obtained for a typical stable FcCO-Lys(MWCNT-ITO)-OMe surface in supporting electrolyte. The data points (]) represent experimental data, and the solid line corresponds to the theoretical spectrum. Experimental conditions: Edc ) 0.43 V, fmin ) 10 Hz, fmax ) 10 kHz, and Vamp ) 20 mV. The inset is an equivalent circuit model: Rs is the solution resistance, Cdl is the double layer capacitance, Rct is the charge transfer resistance, CPE is the constant phase element, Cf is the film capacitance, and Rf is the film resistance.

modified MWCNTs in DMF was sonicated for 1 h, and 5 µL of the suspension was placed on a clean ITO surface. The ITO substrates were dried at room temperature in the air in a laminarflow chamber. Photographs of the resulting Fc-CO-Lys(MWCNTITO)-OMe substrate are included in the Supporting Information. The effect of MWCNTs on electrochemical characteristics of the ferrocene-conjugate system was evaluated by means of cyclic voltammetry (see Figure S6 in the Supporting Information). We believe that interaction of the Fc-CO-Lys-OMe with the ITO surface occurs via H-bonding interactions between the functionalized groups (carboxylate residues) of the shortened acid-treated nanotubes, and the -OH groups are present at the ITO surface, as depicted in Scheme 3. Cyclic voltammetry (CV) measurements were performed at different time intervals after the substrate was immersed in the electrolyte solution (see Figure S7a, Supporting Information). A decrease in the Faradaic response of the system occurred immediately after immersion in the supporting electrolyte solution, presumably due to dissociation of weakly bound material. However, after approximately 24 h, a stable electrochemical behavior was observed. After this stabilization of the film, variable sweep rate CV measurements (see Figure S7b, Supporting Information) resulted in a linear dependence of the peak current on the sweep rate, which is characteristic of electrochemically active, surface bound species.36 In addition, the overall number of Fc-Lysine conjugates was determined from the cyclic voltammetry measurements. For a “fresh” surface, the number of Fc groups was determined to be 2.0 ± 0.7 · 1012, and 3.5 ± 0.6 · 1011 redox centers were present after stable system response was obtained (24 h). It is worth emphasizing that a typical Fc-CO-Lys(MWCNTITO)-OMe surface remains stable for days under ambient conditions and that all further experiments were carried out

RESULTS AND DISCUSSION Impedance Characteristics of the Sensing Interface. A thorough evaluation of the modified ITO surface was carried out using a series of experiments. Figure 1 shows a typical Nyquist plot for a stable Fc-CO-Lys(MWCNT-ITO)-OMe substrate in the supporting electrolyte solution (2.0 M NaClO4). The EIS (Figure 1) was interpreted with the help of an equivalent circuit. Because of the familiar semicircular shape with a diffusion-like tail, a modified Randles circuit was employed to model the experimental data. A proposed equivalent circuit is shown as the inset in Figure 1. Rs represents the resistance of the electrolyte solution and is determined by supporting electrolyte concentration. Cdl is the double layer capacitance of the external Fc-CO-Lys-OMe-MWCNT/electrolyte interface, and Rct represents the charge-transfer resistance related to the process of Fc group oxidation/reduction. CPE is the constant phase element, which is used to account for the frequency dispersion of the pseudocapacitance resulting from the surface inhomogenity.23,37 The impedance of the constant phase element is given by the following relationship ZCPE ) 1/(Q(j · ω)n, where Q is the frequency-independent constant relating to the redox properties of the surface, j ) -11/2, ω is the angular frequency, and the exponent n arises from the slope of log Z vs log f (-1

(36) Bard, A. J.; Faulkner, L. R., 2nd ed.;John Wiley & Sons, Inc.: Hoboken, NJ, 2001, pp 591.

(37) Brug, G. J.; Van den Eeden, A. L. G.; Sluyters-Reehbach, M.; Sluyters, J. H. J. Electroanal. Chem. 1984, 176, 275–295.

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using surfaces that were stabilized for at least 24 h in the supporting electrolyte solution. Also, SEM images were obtained for freshly prepared Fc-CO-Lys(MWCNT-ITO)-OMe substrates and after a 24 h stabilization period (Figure S5a,b, Supporting Information). Comparison of SEM included in the Supporting Information supports the earlier assumption that part of the weakly adsorbed material is lost during substrate stabilization.

Figure 2. AC voltammograms (AC current vs E) obtained using FcCO-Lys(MWCNT-ITO)-OMe (a) and MWCNT-ITO (b). Curves (Ia) and (Ib), DECP absent; curve (IIa), 1 µM DECP; curves (IIIa) and (IIb), 50 µM DECP; curve (IIIb), 100 µM DECP; and curve (IVb), 500 µM DECP. Experimental conditions: f ) 10 Hz, Vamp ) 20 mV.

e n e 1). For n ) 0, the CPE behaves as a pure resistance; for n ) 1, CPE behaves as a pure capacitor, and for n ) -1, CPE behaves as an inductor. When n ) 0.5, CPE corresponds to a Warburg impedance which is associated with the mass transport due to the diffusion of ions at the electrode/solution interface. Cf is the capacitance of the internal MWCNTs/ solution interface in the film micropores, and Rf is the resistance that develops between the MWCNTs and the ITO substrate. Similar equivalent circuit models were proposed in two separate reports that described electrochemical behavior of CNT modified surfaces.38,39 Consequently, an equivalent circuit shown in Figure 1 was employed to model experimental impedance spectra, and the following values of equivalent circuit elements were obtained for a typical stable substrate: Rs ) 17 ± 2 Ω, Cdl ) 5.0 ± 0.7 µF, Rct ) 280 ± 16 Ω, Q ) 23 ± 4 µS · sn, n ) 0.92 ± 0.04, Cf ) 1.8 ± 0.3 µF, and Rf ) 3.1 ± 0.4 Ω. A typical example of the calculated impedance spectrum is potted in Figure 1 (solid line) and is in good agreement with the experimental data points. Detection of CWA Mimics and CWA Degradation Products. The Fc-CO-Lys-OMe-OMe-MWCNT modified ITO surface was employed to perform detection of CWA mimics and their degradation products. Figure 2a shows alternating current voltammograms (ACV) obtained in the absence and presence of DECP (GA agent mimic). The first curve (Ia) corresponds to the measurement performed in the supporting electrolyte alone, while curves (IIa) and (IIIa) were obtained in the presence of 1 µM and 50 µM DECP, respectively. Curve (Ia) displays a single AC (38) Hsieh, C.-T.; Chou, Y.-W.; Chen, W.-Y. J. Solid State Electrochem. 2008, 12, 663–669. (39) Grodzka, E.; Pieta, P.; Dluzewski, P.; Kutner, W.; Winkler, K. Electrochim. Acta 2009, 54, 5621–5628.

Figure 3. Effects of DECP addition on the impedance response of the Fc-CO-Lys(MWCNT-ITO)-OMe surface. Spectra from right to left were obtained for the following concentrations of DECP, CDECP ) 0, 1 · 10-12, 1 · 10-9, 1 · 10-6, 1 · 10-5, 5 · 10-5, 1 · 10-4, 5 · 10-4, and 1 · 10-3 M. Data points represent experimental results and solid lines correspond to spectra calculated for an equivalent circuit shown in Figure 1. Experimental conditions for EIS measurements: Edc ) 0.43 V, fmin ) 10 Hz, fmax ) 10 kHz, and Vamp ) 20 mV.

current peak at approximately 0.43 V, which corresponds to the oxidation of the Fc moiety and low value of charge transfer resistance, Rct. The presence of 1 µM of DECP causes a decrease in the AC peak current (curve IIa). However, a 50 µM concentration of DECP, besides the further decrease in the first peak current, causes appearance of the second peak at about -20 mV (curve IIIa). Also, a similar growth of the cathodic current peak occurred in the case of MPA and PMP. It has to be stressed that the additional peak is not present if concentration of CWA mimic is lower than 50 µM and that equivalent circuit model shown in Figure 1 is only valid for Edc ) 0.43 V (vs Ag/AgCl). The change in the anodic peak (about 0.43 V) is most likely due to the interactions between chemical agent and Fc-Lys based recognition layer. These interactions are presumably H-bonding based, which affects the chemical environment of the Fc and, thus, modifies its electrochemical characteristics. However, to gain a better insight into the origin of the additional cathodic peak, further ACV measurements were performed. Figure 2b shows AC voltammograms of the MWCNT-ITO substrate without the ferrocene-amino acid conjugate obtained at various DECP concentrations. Curve (Ib) was recorded in the absence of DECP and curves (IIb), (IIIb), and (IVb) were acquired in the presence of 50, 100, and 500 µM DECP, respectively. Again, the appearance of the AC current peak was only observed at concentrations of CDECP g 50 µM. The increase in the DECP concentration caused an increase in the peak current accompanied by a slight anodic shift. Since this cathodic peak was observed using both FcCO-Lys(MWCNT-ITO)-OMe and MWCNT-ITO substrates, its appearance is presumably due to the direct interaction between DECP and the carbon nanotubes, indicative of possibly a nonspecific adsorption process. In addition, control experiments preformed with bare ITO did not show any concentration dependent behavior, as were observed for systems containing Analytical Chemistry, Vol. 82, No. 8, April 15, 2010

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Table 1. Values of the Equivalent Circuit Elements for the Fc-CO-Lys(MWCNT-ITO)-OMe Surface Modeled for Different Concentrations of DECP CDECP

Rs/Ω

Cdl/µF

Rct/Ω

Q/µS · sn

n

Cf/µF

Rf/Ω

0 1 pM 1 nM 1 µM 10 µM 50 µM 100 µM 0.5 mM 1 mM

17 ± 2 18 ± 3 17 ± 2 18 ± 4 17 ± 3 16 ± 5 18 ± 2 18 ± 2 17 ± 2

5.0 ± 0.7 5.1 ± 0.4 5.0 ± 0.6 5.4 ± 1.2 5.7 ± 0.8 6.4 ± 1.5 6.8 ± 1.8 10 ± 2 12 ± 2

280 ± 16 278 ± 16 275 ± 20 271 ± 18 263 ± 16 243 ± 27 224 ± 12 184 ± 15 175 ± 14

23 ± 4 22 ± 3 20 ± 4 17 ± 3 16 ± 2 14 ± 3 12 ± 3 10 ± 3 10 ± 2

0.92 ± 0.04 0.90 ± 0.02 0.92 ± 0.03 0.92 ± 0.03 0.92 ± 0.06 0.90 ± 0.03 0.91 ± 0.04 0.86 ± 0.02 0.86 ± 0.02

1.8 ± 0.3 1.9 ± 0.2 1.9 ± 0.3 1.9 ± 0.2 1.8 ± 0.3 1.9 ± 0.2 2.1 ± 0.3 2.3 ± 0.2 2.5 ± 0.4

3.1 ± 0.4 2.7 ± 0.5 2.9 ± 0.3 3.2 ± 0.3 3.4 ± 0.2 4.0 ± 0.5 5.3 ± 0.4 6.8 ± 0.3 7.4 ± 0.4

carbon nanotubes (see Supporting Information Figure S9 and S10). Consequently, on the basis of the above observations, the impedance detection of CWA mimics and their degradation products was performed at constant potential corresponding to oxidation/reduction of the ferrocene group at Edc ) 0.43 V (vs Ag/AgCl). EIS measurements were carried out to further evaluate the effect of DECP on electrochemical characteristics of the system, and the effect of DECP concentration on the EIS of the Fc-CO-Lys(MWCNT-ITO)-OMe surface is shown in Figure 3. Clearly, the increase of the DECP concentration is reflected by the decrease of the overall system impedance (decrease in the semicircle radius, Figure 3). Also, a similar concentration dependence was observed for the Fc-CO-Lys(MWCNT-ITO)-OMe system in the presence of MPA and PMP (see Supporting Information). However, such a concentration dependence was not observed for 2-CLEES. In order to gain a better understanding of this concentration dependence, the system was evaluated in terms of the equivalent circuit shown in Figure 1, and the resulting numerical values of the individual circuit elements as a function of DECP concentrations are summarized in Table 1. A quick examination of the numerical values summarized in Table 1 reveals two important observations. First, the Faradic process (Rct) is affected over all of the DECP concentrations. Second, circuit elements associated with properties of the film display clear concentration dependence at concentrations higher than 50 µM. In addition, the nonspecific adsorption of DECP at 50 µM affects Fradaic process (Rct), which results in a significant increase of sensitivity. Interestingly, the decrease in n suggests a rise in the mass transfer contribution to the overall impedance with increasing DECP concentration. Therefore, it is reasonable to conclude that adsorption of the DECP on the transducer surface modulates the electrochemical characteristics of the system at a micromolar concentration level, thus in principle allowing for the impedance based detection of chemical warfare agent mimics and their degradation products. Put into a military context, U.S. Army Chemical Defense Equipment Process Action Team recommended an LD50 toxicity estimate for the percutaneous exposure to liquid tabun of a healthy 70 kg male of 1500 mg.40 This toxicity estimate (which must not be used for civilians) corresponds to a volume of 185 L at a solution strength of 50 µM, indicating that the detection limits achieved in this experiment are toxicologically relevant. (40) Committee on Toxicology, National Research Council. Review of Acute Human-Toxicity Estimates for Selected Chemical-Warfare Agents; National Academy Press: Washington, DC, 1997.

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Figure 4. Frequency dependence of the serial resistance (Rz, blue) and serial capacitance (Cz, red) obtained for Fc-CO-Lys(MWCNTITO)-OMe in supporting electrolyte (s) and in supporting electrolyte containing 0.5 mM DECP (---). Experimental conditions: Edc ) 0.43 V, fmin ) 10 Hz, fmax ) 10 kHz, and Vamp ) 20 mV.

In order to simplify our considerations, we can describe our system in terms of a serial resistance, Rz, and a serial capacitance, Cz. The serial resistance is equivalent to the real component of the total impedance (Rz ) Z′), and the serial capacitance is determined directly from the imaginary component of the total impedance (Cz ) 1/(ω · Z′′), where angular frequency ω ) 2 · π · f). The effect of DECP on Rz and Cz of the Fc-COLys(MWCNT-ITO)-OMe surface plotted as function of AC frequency is shown in Figure 4. In Figure 4, we can see a decrease in resistive component, Rz, while the value of Cz rises in the presence of the DECP. Moreover, this change in Rs and Cs is most prominent in the low frequency regime (approximately less than 25 Hz) where the impedance of the capacitive elements is relatively high. On the basis of this observation, we conclude that the effective detection of chemical agents can be achieved by monitoring changes in Rz at a fixed frequency. This represents a significant simplification over classical EIS in terms of data interpretation and time-savings. The dependence of ∆Rz, measured at f ) 15 Hz, on concentration for DECP, PMP, MPA, and CLEES is presented in Figure 5. ∆Rz is a difference between the resistance Rz measured in the absence and presence of the chemical agent.

Figure 5. Dependence of ∆Rz on concentration of ∆, DECP; ], MPA; ×, PMP; and °, 2-CLEES observed at f ) 15 Hz and Edc ) 0.43 V. The inset is a magnified view of 10-12-10-6 M concentration range. Error bars represent the standard deviation for measurements at five different electrodes. Experimental conditions: Edc ) 0.43 V, f ) 15 Hz, and Vamp ) 20 mV.

From the data shown in Figure 5, it is clear that the Fc-Lys recognition layer shows a good sensitivity toward nerve agent mimics while it is much less sensitive to the sulfur mustard mimic 2-CLEES. This low sensitivity toward the sulfur mustard mimic can be attributed to the weak interactions between the recognition layer and 2-CLEES, which is the poor hydrogen bond acceptor. Possible interactions are most likely due to physisorption onto the transducer surface. The dramatic increase in the sensitivity of the surface toward the nerve agent mimics at 50 µm concentration essentially represents the onset of nonspecific adsorption at MWCNTs and its effect on the redox process. However, direct correlation of this phenomenon with the number of Fc-conjugates present at the surface was not observed, most likely due to the high aspect ratio of MWCNTs. Further studies were carried out to determine if the Fc-COLys(MWCNT-ITO)-OMe used surface is reusable. Figure 6 shows a set of typical EIS curves obtained using Fc-CO-Lys(MWCNTITO)-OMe sensor. (×) data points were recorded using a “fresh” surface in the supporting electrolyte solution, and (∆) spectrum was obtained in 100 µM DECP solution. Next, the sensor was rinsed three times with supporting electrolyte solution (5 mL each time), and EIS measurements were repeated, resulting in the spectrum represented by ()) for supporting electrolyte alone and (+) for 100 µM DECP. These results suggest that Fc-COLys(MWCNT-ITO)-OMe substrate senor can be reused, but washing does not completely restore the EIS characteristics of the surface to its prewash state. Overall, higher impedance is observed for the washed sensor ((×) and (4) in Figure 6) compared to before washing (()) and (+) in Figure, 6). This is presumably due to a minimal loss of material from the recognition layer due to the washing process. Nevertheless, there is some potential for the sensor to be reused. CONCLUSIONS In this report, we describe a novel impedance based method for the detection of chemical warfare agents. We have demon-

Figure 6. EIS spectra obtained for the sensor in supporting electrolyte (×) and in 100 µM DECP (∆) and after sensor washing in supporting electrolyte (]) and again after exposure of the sensor surface to 100 µM DECP (+). Solid lines represent spectra calculated for equivalent circuit shown in Figure 1. Experimental conditions: Edc ) 0.43 V, fmin ) 10 Hz, fmax ) 10 kHz, and Vamp ) 20 mV.

strated that electrochemical properties of the carbon nanotubes covalently modified with the ferrocene-lysine conjugate are sensitive to the presence of the nerve agent analogues. Our investigation of the Fc-CO-Lys(MWCNT-ITO)-OMe indicates that CWA mimics interact with the redox active centers (ferrocene groups) as well as with the carbon nanotubes. While interactions between the CWA and the Fc group affect the Faradaic signal, CWA-MWCNT interactions modify electrical properties (resistance and capacitance) of the recognition layer. These CWAinduced changes in system characteristics can be easily observed by employing impedance measurements and, consequently, are used to detect CW agents at micromolar level. Moreover, careful optimization of the experimental parameters allows for the detection of CWA presence by simple, single frequency measurements of the overall system resistance, which presents considerable simplification in comparison to “classical” EIS measurements. Our findings present a step forward toward the design of portable and affordable CWA sensors. ACKNOWLEDGMENT This work was carried out under Public Works Contract W7702-05R104/001/EDM. The authors would also like to thank NSERC for financial support and Dr. Todd Simpson from UWO Nanofabrication Facility for SEM images. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review November 25, 2009. Accepted March 7, 2010. AC902694D Analytical Chemistry, Vol. 82, No. 8, April 15, 2010

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