Noncovalent Modification of Carbon Nanotubes with Ferrocene

Feb 26, 2008 - ... Richmond Street, London, ON, N6A 5B7 Canada, and DRDC Suffield, P.O. Box 4000, Station Main, Medicine Hat, AB, T1A 8K6 Canada...
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Anal. Chem. 2008, 80, 2574-2582

Noncovalent Modification of Carbon Nanotubes with Ferrocene-Amino Acid Conjugates for Electrochemical Sensing of Chemical Warfare Agent Mimics Mohammad A. K. Khan,† Kagan Kerman,‡ Michael Petryk,§ and Heinz-Bernhard Kraatz*,†,‡

Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK, S7N 5C9 Canada, Department of Chemistry, The University of Western Ontario, 1151 Richmond Street, London, ON, N6A 5B7 Canada, and DRDC Suffield, P.O. Box 4000, Station Main, Medicine Hat, AB, T1A 8K6 Canada

The electrochemical detection of chemical warfare agent (CWA) mimics was explored using multiwalled carbon nanotubes (MWCNTs) on indium tin oxide (ITO) surfaces in connection with ferrocene-amino acid conjugates. Various ferrocene-amino acid conjugates were synthesized and utilized as the recognition layer for the detection of CWA mimics. The ferrocene-amino acid conjugates were noncovalently attached to the pretreated MWCNTs on the ITO surface and reacted with CWA mimics, upon which the electrical properties of the MWCNTs and the Fc group were affected significantly. Alternating current voltammetry and capacitance-based detection offered large dynamic ranges for the detection of methylphosphonic acid, diethyl cyanophosphonate, ethylmethylphosphonate, and pinacolyl methylphosphonate in water. Electrochemical measurements showed dramatic changes upon the electrostatic interaction between the CWA mimics and the ferrocene-amino acid conjugates immobilized on MWCNTs on ITO surfaces. Electrochemical sensing in connection with MWCNTs is shown to be a promising analytical tool for the trace-level detection of CWA mimics in aqueous solutions. In the past decade, electrochemical sensors have become very popular due to their suitability to miniaturization, low-power consumption, and high sensitivity. Sensors generally use a transducer modified with a recognition layer that is sensitive to the analytes of interest. Carbon nanotubes (CNTs) have become extremely useful for this purpose due to their nanoscale diameter with promising electrical and electromechanical properties.1-5 Due to their high aspect ratio, simple adsorption of molecules causes significant changes in the electrical properties of CNTs. The * To whom correspondence should be addressed. E-mail: [email protected]. † University of Saskatchewan. ‡ The University of Western Ontario. § DRDC Suffield. (1) Ijima, S.; Ichihashi, T. Nature 1993, 363, 603. (2) Ajayan, P. M. Chem. Rev. 1999, 99, 1787. (3) Wong, S. S.; Harper, J. D.; Lansbury, J., P. T.; Lieber, C. M. J. Am. Chem. Soc. 1998, 120, 603. (4) Baughman, R. H.; Zakhidov, A. A.; de Heer, W. A. Science 2002, 297, 787. (5) Treacy, M. M. J.; Ebbesen, T. W.; Gibson, J. M. Nature 1996, 381, 678.

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modification of CNTs has been carried out using covalent and noncovalent bonds. For example, sodium dodecyl sulfate can be adsorbed with noncovalent forces on the multiwalled CNT (MWCNT) surface and form rolled-up half-cylinders with the alkyl chains pointing toward the MWCNT.6 As a result, the surfactant molecules are loosely packed around the MWCNTs. Nonspecific hydrophobic interactions are believed to be involved in this phenomenon. Molecules containing aromatic groups or electronrich environments have been reported to modify nanotubes via π-π stacking interactions with the graphite surface. For example, sodium dodecylbenzenesulfonate was reported to interact with single-walled carbon nanotubes (SWCNTs) via π-π stacking interactions.7 Biological molecules have also been reported to interact noncovalently with the surface and interior of SWCNTs without changing their native reactivity.8-14 The electronic properties of CNTs along with the specific recognition properties of the immobilized biosystems can form a robust sensor platform for different analytes. Oligonucleotides,9,10 small proteins such as streptavidin,11 metallothionein,12-14 and DNA9-11 have been reported to adsorb to the surface and interior of the MWCNTs due to nonspecific interactions (e.g., van der Waals interaction). CNTs have been utilized for the detection of organic vapor as chemiresistors, where resistance changes in the CNTs were recorded in real time upon exposure to vapors.15,16 These studies (6) O’Connell, M. J.; Bachilo, S. M.; Huffman, C. B.; Moore, V. C.; Strano, M. S.; Haroz, E. H.; Rialon, K. L.; Boul, P. J.; Noon, W. H.; Kittrell, C.; Ma, J.; Hauge, R. H.; Weisman, R. B.; Smalley, R. E. Science 2002, 297, 593. (7) Islam, M. F.; Rojas, E.; Bergey, D. M.; Johnson, A. T.; Yodh, A. G. Nano Lett. 2003, 3, 269. (8) Davis, J. J.; Green, M. L. H.; Hill, O. A. H.; Leung, Y. C.; Sadler, P. J.; Sloan, J.; Xavier, A. V.; Tsang, S. C. Inorg. Chim. Acta 1998, 272, 261. (9) Tsang, S. C.; Guo, Z.; Chen, Y. K.; Green, M. L. H.; Hill, O. A. H.; Hambley, T. W.; Sadler, P. J. Angew. Chem. Int. Ed. Engl. 1997, 36, 2198. (10) Guo, Z.; Sadler, P. J.; Tsang, S. C. Adv. Mater. 1998, 10, 701. (11) Balavoine, F.; Schultz, P.; Richard, C.; Mallouh, V.; Ebbesen, T. W.; Mioskowski, C. Angew. Chem., Int. Ed. Engl. 1999, 38, 1912. (12) Chen, R. J.; Zhang, Y.; Wang, D.; Dai, H. J. Am. Chem. Soc. 2001, 123, 3838. (13) Tsang, S. C.; Davis, J. J.; Green, M. L. H.; Hill, H. A. O.; Leung, Y. C.; Sadler, P. J. J. Chem. Soc., Chem. Commun. 1995, 2579. (14) Lago, R. M.; Tsang, S. C.; Lu, K. L.; Chen, Y. K.; Green, M. L. H. J. Chem. Soc., Chem. Commun. 1995, 1355. (15) Collins, P. G.; Bradley, K.; Ishigami, M.; Zettl, A. Science 2000, 287, 1801. (16) Kong, J.; Franklin, N. R.; Zhou, C.; Chapline, M. G.; Peng, S.; Cho, K.; Dai, H. Science 2000, 287, 622. 10.1021/ac7022876 CCC: $40.75

© 2008 American Chemical Society Published on Web 02/26/2008

have recently been extended to the detection of chemical warfare agents (CWAs) and their mimics.17-23 Chemiresistor-based vapor sensors made from network films of SWCNT bundles on flexible plastic substrates were used to detect CWA mimics.24 Robinson et al.25 has recently reported improved chemical detection of CWA mimics using SWCNT-based capacitance measurements. In this paper, we are reporting a highly sensitive electrochemical sensor for CWA mimics based on the noncovalent modification of COOH group functionalized MWCNT using ferrocene-conjugated amino acids. We used several different reagents (diethyl cyanophosphonate (DECP), methylphosphonic acid (MPA), pinacolyl methylphosphonate (PMP), and ethylmethylphosphonate (EMP)), which are reasonable mimics for the nerve agent, tabun and its degradation products.26 The selectivity of the sensor was investigated by exposure to diethyl ethylsulfide (DEES), which is as a mustard gas mimic. Initially, MWCNTs were treated with concentrated H2SO4 and HNO3 in a 3:1 ratio to shorten them and increase surface area.27-29 Treatment with acid makes the interior of the nanotubes more accessible by opening the tubes and creating sidewall defects with COOH groups. MWCNTs were then immobilized by placing a drop of their suspension in DMF on the indium-tin oxide (ITO) surface. After drying, the surface was covered by the ferrocene-amino acid conjugate solution. Amino acids adsorbed on the MWCNT surface changed the electrical properties of the MWCNT by noncovalent modifications. Atomic force microscopy (AFM), transmission electron microscopy (TEM), and Raman spectroscopy were used to study the morphology and the functional groups on the MWCNTs. Alternating current voltammetry (ACV) and capacitance measurements were taken upon the exposure of the ferrocene-amino acid conjugate-modified MWCNTs to CWA mimics. EXPERIMENTAL SECTION General Remarks. All reagents were obtained from SigmaAldrich and used without further purification. BocHN-Fc-COOMe was prepared according to a literature procedure.30 MWCNTs with ∼5 µm in length and ∼2 nm in diameter were purchased from Cheaptubes Inc. A Bruker Avance 500-MHz NMR spectrometer (1H 500.3 MHz, 13C 125.8 MHz, and 31P 202.5 MHz) equipped with (17) Goldoni, A.; Larciprete, R.; Petaccia, L.; Lizzit, S. J. Am. Chem. Soc. 2003, 125, 11329. (18) Li, J.; Lu, Y.; Ye, Q.; Cinke, M.; Han, J.; Meyyappan, M. Nano Lett. 2003, 3, 929. (19) Novak, J. P.; Snow, E. S.; Houser, E. J.; Park, D.; Stepnowski, J. L.; McGill, R. A. Appl. Phys. Lett. 2003, 83, 4026. (20) Smeya, T.; Small, J.; Kim, P.; Nuckolls, C.; Yardley, J. T. Nano Lett. 2003, 3, 877. (21) Wei, C.; Dai, L.; Roy, A.; Tolle, T. B. J. Am. Chem. Soc. 2006, 128, 1412. (22) Snow, E. S.; Perkins, F. K. Nano Lett. 2005, 5, 2414. (23) Snow, E. S.; Perkins, F. K.; Houser, E. J.; Badescu, S. C.; Reinecke, T. L. Science 2005, 307, 1942. (24) Cattanach, K.; Kulkarni, R. D.; Kozlov, M.; Manohar, S. K. Nanotechnology 2006, 17, 4123. (25) Robinson, J. A.; Snow, E. S.; Perkins, F. K. Sens. Actuators, A 2007, 135, 309. (26) Munro, N. B.; Talmage, S. S.; Griffin, G. D.; Waters, L. C.; Watson, A. P.; King, J. F.; Hauschild, V. Environ. Health Perspect. 1999, 107, 933-974. (27) Liu, J.; Rinzler, A. G.; Dai, H.; Hafner, J. H.; Bradley, R. K.; Boul, P. J.; Lu, A.; Iverson, T.; Shelimov, K.; Huffman, C. B.; Rodriguez-Macias, F.; Shon, Y.-S.; Lee, T. R.; Colbert, D. T.; Smalley, R. E. Science 1998, 280, 1253. (28) Liu, Z.; Shen, Z.; Zhu, T.; Hou, S.; Ying, L.; Shi, Z.; Gu, Z. Langmuir 2000, 16, 3569. (29) 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. (30) Barisˇic´, L.; Rapic´, V.; Kovacˇ, V. Croatia Chim. Acta 2002, 75, 199.

a 5-mm broadband probe and dedicated 1H, 13C, and 2H probes for locking were employed in all experiments. Peak positions in both 1H NMR and 13C NMR spectra are reported in ppm relative to TMS and coupling constants J in hertz. The 1H NMR spectra are referenced to residue CHCl3 at δ 7.27. 13C{1H} NMR spectra are referenced to the CDCl3 signal at δ 77.23. Mass spectrometry was carried out on a VG Analytical 70/20 VSE instrument. Infrared spectra were obtained on a Perkin-Elmer 1605 FT-IR at a resolution of 4 cm-1. UV-visible spectra were recorded on a Varian Cary 500 spectrometer. Synthesis of Ferrocene-Amino Acids Conjugates. To a solution of BocHN-Fc-COOMe (500 mg, 1.39 mmol) in DCM, 40% TFA in DCM is added and the resultant mixture stirred for 30 min, and then Et3N was added to neutralize the extra TFA. Then, amino acids (1.0 equiv) in DCM, HOBt (1.5 equiv), and EDC (1.5 equiv) were added. The mixture was stirred for 1 h. Then the product obtained from the BocHN-Fc-COOMe was added to the reaction mixture and the resultant mixture stirred for 10 h at room temperature. The yellow reaction mixture was then subjected to an aqueous workup by washing with saturated NaHCO3 and 10% citric acid and again with saturated NaHCO3. After drying of the organic phase over anhydrous Na2SO4, the solvent was evaporated to dryness and the residue was chromatographed on silica (nhexane/ EtOAc 4:1; Rf ) 0.30), giving the desired orange-red product. [Boc-Gln-Fc-COOMe] (1). 52% yield (350 mg). UV-vis (CH3CN, λmax in nm,  in L mol-1 cm-1): 445 (537). IR (KBr, cm-1): 3321 (NH), 2976 (Fc), 2928 (CH3), 1719 (CdO), 1696 (amide I), 1544 (amide II). 1H NMR (500 MHz, CDCl3, 293 K): 6.59 (s, FcNH), 6.00 (s, Boc-NH), 5.06 (s, 2H, Fc-H), 4.80 (s, 2H, Fc-H), 4.67 (s, 2H, Fc-H), 4.50 (s, CONH2), 4.36 (s, 2H, Fc-H), 4.14 (s, R-H), 3.79 (s, COOMe), 3.76 (q, 2H, CH2, J ) 29 Hz), 1.48 (s, 9H, Boc), 1.23 (m, 2H, CH2, J ) 30 Hz). 13C{1H} NMR (500 MHz, CDCl3, 293 K): δ 168.9 (ester CdO), 165.8 (amide CdO), 143.5 (Boc CdO), 120.4 (NH2CdO), 108.7 (Boc-tert-C), 100.0 (ipso-C, FcC(O)), 74.3 (ipso-C, Fc-NH), 72.2, 71.7, 71.0, 66.5, 65.9, 62.2 (FcC), 61.9 (ester CH3), 51.7 (R-C), 28.4 (Boc-CH3), 28.0 (CH2CONH2), 22.1 (CH2-R-C). ESI-MS: exact mass m/z calcd. for C22H29FeN3O6 487.1405, found 486.0878. Anal. Calcd for C22H29FeN3O6: C, 54.22; H, 6.00; N, 8.62. Found: C, 56.38; H, 5.09; N, 8.23%. [Boc-Asn-Fc-COOMe] (2). 46% yield (300 mg). UV-vis (CH3CN, λmax in nm,  in L mol-1 cm-1): 446 (301). IR (KBr, cm-1): 3309 (NH), 2949 (Fc), 2918 (CH3), 1710 (CdO), 1697 (amide I), 1563 (amide II). 1H NMR (500 MHz, CDCl3, 293 K): 7.88 (d, FcNH, J ) 7 Hz), 5.88 (d, 1H, Boc-NH, J ) 7 Hz), 4.77 (d, 2H, Fc-H, J ) 7 Hz), 4.60 (d, 2H, Fc-H, J ) 7 Hz), 4.50 (s, 2H, CONH2), 4.42 (s, 2H, Fc-H), 4.05 (s, 2H, Fc-H), 3.81(s, COOMe), 3.20 (s, 1H, R-H), 1.51 (s, 9H, Boc), 1.33 (t, 2H, CH2, J ) 15 Hz). 13C NMR (500 MHz, CDCl3, 293 K): δ 171.7 (ester CdO), 167.1 (amide CdO), 140.2 (Boc CdO), 117.0 (NH2CdO), 94.2 (Boc-tert-C), 81.5 (ipso-C, Fc-C(O)), 72.6 (ipso-C, Fc-NH), 72.6, 71.5, 71.2, 66.6, 66.4, 63.6 (Fc-C), 62.9 (ester CH3), 51.9 (R-C), 28.3 (Boc-CH3), 20.8 (CH2-R-C). ESI-MS: exact mass m/z calcd for C21H27FeN3O6 473.1249, found 473.1529. Anal. Calcd for C21H27FeN3O6: C, 53.29; H, 5.75; N, 8.88. Found: C, 53.65; H, 5.31; N, 8.87%. Analytical Chemistry, Vol. 80, No. 7, April 1, 2008

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Table 1. Optical Properties of Compounds 1 and 2

cm-1)

IR (KBr, NMR (δ/ppm)

1H

13C

MS

NMR (δ/ppm)

1

2

3321 (N-H), 1719 (CdO) 6.59 (s, 1H, Fc-NH), 6.00 (s, Boc-NH), 4.50 (s, 2H, CONH2), 4.14 (s, 1H, R-H) 3.79 (s, 3H, COOMe) 168.9 (amide CdO), 165.8 (Boc CdO), 28.4 (ester CH3) calcd 487.1405; expt 486.0878

3309 (N-H), 1710 (CdO) 7.88 (d, 1H, Fc-NH), 5.88 (d, 1H, Boc-NH), 4.50 (s, 2H, CONH2), 3.81(s, 3H, COOMe), 3.20 (s, 1H, R-H) 171.7 (amide CdO), 167.0 (Boc CdO), 28.3 (ester CH3) calcd 473.1249; expt 473.1529

Figure 1. Tapping mode AFM image showing aggregates of MWCNTs on bare mica surface.

Scheme 1. Scheme for Synthesizing Compounds 1 and 2 (R ) Boc-Gln (1), Boc-Asn (2))

Pretreatment of MWCNTs. MWCNTs were shortened using the standard literature procedure.26-28 Briefly, 8 mg of MWCNTs in a solution mixture of H2SO4 and HNO3 (3:1) was ultrasonicated at 70 °C for 84 h. The reaction mixture was diluted with MilliQ water and cooled to room temperature in an ice bath. The mixture was then filtered through poly(tetrafluoroethylene) membrane filter with a 200-nm pore size. The MWCNT-acid solution was transferred to a separation funnel with a small amount of hexane. Next, ethanol was added and the mixture shaken until the nanotubes were extracted into the hexane layer. After the initial separation, more Milli-Q water was added to the funnel. The twophase mixture was allowed to settle for 30 min, and the organic layer was separated and washed 4-5 times until the pH became neutral. The solvent was evaporated in a rotary evaporator at 40 °C. Film Preparation. The suspension of MWCNT in DMF was prepared by ultrasonicating the pretreated MWCNTs in DMF for 1 h. An aliquot (10 µL) of the suspension was placed on the ITO surface. The substrates were dried at room temperature in a 2576

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laminar-flow chamber, and then, one drop (5 µL) of ethanolic solution of ferrocene-amino acid conjugates (1 mM) was placed on the top of the MWCNT layer. All the substrates were dried in the air before used. Raman Spectroscopy. Raman spectra were recorded by Renishaw system 2000. The laser excitation was provided by an air-cooled HeNe source operating at 17 mW of 514-nm output. The laser power at the sample was ∼1 mW and was focused to ∼1 µm. Frequency calibration was carried out using the 520.5cm-1 line of the silicon wafer. Transmission Electron Microscopy. MWCNTs were suspended in Milli-Q water by ultrasonication for 1 h, and an aliquot (10 µL) of the suspension was placed on the carbon grid surface. Then, the carbon grids were dried at room temperature in the air in a laminar-flow chamber. TEM was carried out using a Philips 410LS electron microscope at various acceleration potentials. Electrochemical Measurements. MWCNT-modified ITO substrate was placed into the homemade electrochemical cell (Supporting Information Figure 1). Electrolyte solution (5 mL of 2 M NaClO4) was put into the cell slowly. Then, the MWCNTmodified ITO was incubated in the blank buffer for 15 min. ACV measurement from 0 to 0.8 V was performed at an amplitude of 25 mV and a frequency of 100 Hz. For the measurements with the CWA mimics, a desired concentration of the CWA mimic solution was added into the electrolyte and the resultant mixture incubated for 15 min. Serial dilutions of CWA mimic solution were prepared using Milli-Q

Chart 1. Chemical Drawings of the Compounds 1 and 2

Figure 2. TEM image showing MWCNTs on carbon grid surface. Resolution 110000×.

Figure 3. Raman spectra showing (a) MWCNT and (b) MWCNT and compound 1 on ITO surface.

water. ACV and capacitance measurements were recorded using the same parameters described above, but in the absence of agitation. RESULTS AND DISCUSSION The ferrocene-amino acid conjugates, [(RNH)Fc(COOMe)], were synthesized using the EDC/HOBt protocol31 starting from the Boc-protected aminoferrocenecarboxylate ester, BocHN-FcCOOMe (1) and amino acids as shown in Scheme 1. Bocdeprotection of 1 was carried out by adding trifluoroacetic acid in dichloromethane solution under inert conditions and results in the formation of 1-amino-n′-ferrocenemethylcarboxylate, 1,n′H2N-Fc-COOMe (2). This species was then reacted with amino (31) Liu, C.-M.; Cao, H.-B.; Li, Y.-P.; Xu, H.-B.; Zhang, Y. Carbon 2006, 44, 2919.

acid solutions, Boc-Gln, Boc-Asn, Boc-Cys, Boc-Glu, and Boc-Asp to form 3-7. Purification of these materials was achieved by column chromatography, giving orange-red solids, which were further analyzed spectroscopically. All of the compounds exhibited a weak absorption band in the visible region with a λmax G 444 nm, typical of the d-d transition in ferrocene derivatives. The IR spectrum of these compounds show a typical CdO band close to 1700 cm-1, with all N-H stretching bands found below 3400 cm-1 (Table 1). The 1H and 13C{1H} NMR spectra of compounds 3-7 were recorded in CDCl3. In the 1H NMR spectrum, 3-7 exhibited peaks at around δ 5.43 and δ 4.58, assigned to the NH attached to the Cp ring and the protected NH group in the amino acid functional groups, respectively. These two groups also gave distinct signals in the 13C NMR at around δ 171.9 for the amide CdO group and at around δ 167.0 for the Boc CdO group. The chemical shifts of the inequivalent Cp protons of the Fc group were comparable with reported shifts and are observed as four distinct singlets in the region of δ 4.72-4.05. These chemical shifts were comparable to those of other disubstituted Fc-systems, like [Fe(η-C5H4CH(CH2)4NMe)2],32 [Fe(η-C5H4(CH2)2NMe2)2],32 and the Fc-urea dimethyl-1′,1′-ureylenedi(1-ferrocenecarboxylate).33 MWCNTs were shortened by heating and ultrasonication in an acidic mixture (concentrated H2SO4 and concentrated HNO3, 3:1) according to the literature procedure (Supporting Information Figure 2).27-29 This method was chosen as the acidic mixture is known to intercalate and exfoliate graphite. Also, it helps ensure a smooth cut by separating the cut tube pieces from the underlying tubes. At this condition, the average cut nanotube shortened at a rate of roughly 130 nm/h.27 Thus, this oxidation procedure created short MWCNTs, which can be easily suspended, sorted, and modified with other molecules. An AFM investigation was carried out in order to ascertain the effectiveness of the cutting process and immobilization on mica from hexane solution. This investigation showed islands of nanotubes on the mica surface (Figure 1). The diameter of the spots was found to be significantly greater (tens of nanometers) than the diameter of a single nanotube. Since the MWCNTs are functionalized with COOH groups, it is possible that more than one carboxylic group could participate in the surface condensation (32) Bradley, S.; McGowan, P. C.; Oughton, K. A.; Camm, K. D.; Liu, X.; Mumtaz, R.; Podesta, T. J.; Thornton-Pett, M. Inorg. Chem. 2002, 41, 715. (33) Mahmoud, K.; Long, Y. T.; Schatte, G.; Kraatz, H. B. J. Organomet. Chem. 2004, 689, 2250.

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Scheme 2. Surface Modification by MWCNT and Ferrocene-Amino Acid Conjugates and Possible Mechanism of Noncovalent Interaction (AA Represents the Conjugated Amino Acids)

Chart 2. Chemical Drawings of the Chemical Warfare Agent Mimics: Methylphosphonic Acid (MPA), Diethyl Cyanophosphonate (DECP), Ethylmethylphosphonate (EMP), and Pinacolyl Methylphosphonate (PMP)

reaction. We hypothesize that the MWCNTs were interconnected with hydrogen bonds. Moreover, the oxidation reaction of shortening MWCNTs could exfoliate them.27 Thus, the true width of the MWCNTs are likely to be smaller than the observed values. We assume that this discrepancy was due to the tip-induced broadening artifact and aggregation of CNTs in the assembly process.34-36 The functionalized MWCNTs were also observed using TEM (Figure 2). The MWCNT were observed to interweave each other in an irregular manner to form webs of MWCNTs and displayed a high degree of alignment at their free edges. Such kind of arrangement of nanotubes was also observed by Shaffer et al.37 Modification of ITO surfaces was characterized by Raman spectroscopy. For example, spectra a and b in Figure 3 show the (34) Wong, S. S.; Woolley, A. T.; Odom, T. W. H., J.-L.; Kim, P.; Vezenov, D. V.; Lieber, C. M. App. Phys. Lett. 1998, 73, 3465. (35) Diao, P.; Liu, Z.; Wu, B.; Nan, X.; Zhang, J.; Wei, Z. Chem. Phys. Chem. 2002, 3, 898. (36) Yu, X.; Chattopadhyay, D.; Galeska, I.; Papadimitrakopoulos, F.; Rusling, J. F. Electrochem. Commun. 2003, 5, 408. (37) Shaffer, M. S. P.; Fan, X.; Windle, A. H. Carbon 1998, 36, 1603.

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unmodified MWCNT and MWCNT modified with [Boc-Cys-FcCOOMe] on the ITO surface. The peaks at 1552 and 1579 cm-1 represent the tangential modes,38-40 and the peak at 1344 cm-1 represents the disorder mode.38-40 The disorder mode represents the conversion of sp2-hybridized carbon to a sp3hybridized carbon due to the acid treatment. As the disorder mode is the diagnostic in the hexagonal framework of the MWCNT, the fact that the relative intensity of this mode increased after the addition of ferrocene-amino acid conjugate solution provides direct evidence of the adsorption of amino acids onto the surface of MWCNTs.39,40 Moreover, it was noted that the amplitude of the tangential mode feature increased upon addition of ferrocene-amino acid conjugates. This observation indicates an extended electronic resonance due to the noncovalent modification of MWCNT surfaces. Moreover, the peaks shifted ∼5 cm-1 (38) Zhang, Y.; Li, J.; Shen, Y.; Wang, M.; Li, J. J. Phys. Chem. B 2004, 108, 15343. (39) Dyke, C. A.; Tour, J. M. Chem.-Eur. J. 2004, 10, 812. (40) Cioffi, C.; Campidelli, S.; Brunetti, F. G.; Meneghetti, M.; Prato, M. Chem. Commun. 2006, 2129.

compared to those in the unmodified and cut MWCNTs, further indicating noncovalent modification. Similar increases in both amplitude and frequency were observed by Zhang et al.,38 when they modified the SWCNTs with poly(L-lysine). Based on the observations, we envisaged the possible mechanism of noncovalent modification of MWCNTs by ferroceneamino acids conjugates as shown in Scheme 2. The carboxylic groups were distributed randomly on the MWCNT surfaces, which can form hydrogen-bonded complexes with the amide groups of ferrocene-amino acid conjugates. Both bond expansion and contraction are found to coexist in the multiple-functionalized MWCNTs. A distinct Raman shift was observed in the radial breathing mode and the G modes, depending not only on the tube diameter and chirality but also on carboxyl group coverage and adsorption configurations. With the carboxyl groups increasing, interestingly, a nonmonotonic upshift and downshift was observed in the G modes, which was attributed to the competition between the bond expansion and contraction of MWCNTs. In this report, data are presented on the application of compounds 1 and 2 as the recognition layer to the CWA mimics on the MWCNT-ITO surface. The electrostatic interaction between the CWA mimics with phosphonic acid derivatives and the compounds 1 and 2 was utilized for the detection of the mimics. Moreover, it was assumed that a proper balance of hydrophilicity could be obtained within the CWA mimics that allowed for preferential adsorption with the amino acid-modified layer, and this adsorption process is electrochemically detected. We have obtained the concentration-dependent ACV responses of the compound 1-conjugated MWCNT-ITO surface upon titration with MPA. As the concentration of MPA increased in the solution, the capacitace of the surface modified with the ferrocene-amino acid conjugates was affected by the interaction with the negatively charged mimic. The incubation time was 15 min without stirring the electrolyte after each addition of MPA into the electrolyte solution. The determination of the sensor characteristics toward various analytes (CWA mimics) is an important stage in understanding how the MWCNT-ITO sensor with ferrocene-amino acid conjugates perform. The shape of the ACV peak (Ep) resembles a reversible process, where one electron is involved in the reaction (n ) 1). The peak in the ACV current signal occurred at Ep ) E1/2, which is near Ep ∼ 0.46 V (vs Ag/AgCl). As the scanning potential moves away from the Ep, the impedance rises slowly and causes a decrease in the current. It is concluded that the current is controlled by the limiting reagent (the CWA mimic), that is, the smaller of the two surface concentrations (the CWA mimic and the ferrocene-amino acid conjugate). At potentials above Ep, where only small amounts of the ferrocene-amino acid conjugate or CWA mimic exists at the surface, only small or no current responses were observed. Figure 4B shows the capacitance responses upon the titration of CWA mimics on the compound 3-modified MWCNT-ITO surface. To demonstrate the dependence of capacitance on charge, the ACV current-voltage and the capacitance-voltage characteristics of the sensors were studied. Snow et al.22 reported that the conductance (G) of CNTs was proportional to the number (nh) and mobility (µh) of charge carriers at the surface of the CNT

Figure 4. (A) ACV for the interaction of MPA with 1-modified MWCNT-ITO surface. The signal (a) represents the ACV signal obtained in the absence of MPA in blank electrolyte, 2 M NaClO4. ACV response decreased, as the concentration of MPA increased from 1 pM (b), 1 nM (c), and 1 µM (d) to 1 mM (e) MPA. (B) The capacitance responses obtained with the serial titration of the 1-modified MWCNT-ITO surface with MPA at 1 fM (b), 1 pM (c), 1 nM (d), and 1 µM (e) and the titration with blank electrolyte, 2 M NaClO4 (f) after the addition of 1 µM, 1 mM (g) and the titration with blank electrolyte (h) after the addition of 1 mM MPA. The response shown with (a) represents the signal obtained from the 1-modified MWCNT-ITO surface in blank electrolyte. (C) The plot for the dependence of the ∆ACV responses on the concentration of the titrated MPA into the blank electrolyte. ∆ACV represents the data obtained with the subtraction of the initial ACV response from the response obtained after 15 min past the addition of the MPA into the electrolyte (∆ACV ) ACVblank - ACVfinal). The error bars indicate the standard deviation of the three measurements (n ) 3). The measurements of the blank electrolyte were taken after incubating the electrodes for 15 min in the absence of the CWA mimics.

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Figure 5. (A) ACV for the interaction of DECP with 2-modified MWCNT-ITO surface. The signal (a) represents the ACV signal obtained in the absence of DECP. ACV response decreased, as the concentration of DECP increased from 1 nM (b), 1 µM (c), to 1 mM (d). The response shown with (e, gray line) represents the signal obtained after washing of the electrode surface with blank electrolyte in the presence of 1 µM DECP. (B) The capacitance responses obtained with the serial titration of the 2-modified MWCNT-ITO surface with DECP at 1 nM (b), 1 µM (c), and 1 mM (d), and the titration with blank electrolyte, 2 M NaClO4 (e) after the addition of 1 µM. The response shown with (a) represents the signal obtained from the 2-modified MWCNT-ITO surface in blank electrolyte. (C) The plot for the dependence of the ∆ACV responses on the concentration of the titrated DECP into the blank electrolyte.

(i.e., G ) qnhµh). It was postulated that G would be greatly influenced by charge transfer from an adsorbate and variations in carrier mobility in the CNT. Rosenblatt et al.41 reported that the capacitance response to an analyte was derived from the fieldinduced polarization of the surface dipoles and contained contribu(41) Rosenblatt, S.; Yaish, Y.; Park, J.; Gore, J.; Sazonova, V.; McEuen, P. Nano Lett. 2002, 2, 869.

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Figure 6. (A) ACV for the interaction of EMP with 2-modified MWCNT-ITO surface. The black lines represent the ACV signals obtained with increasing concentrations of EMP from 1 pM (b), 1 nM (c), and 1 µM (d). The response shown in (a) represents the signal obtained after the addition of 1 mM DEES, the mustard agent mimic, which did not have a significant binding affinity to the ferrocene-amino acid conjugates. The gray lines represent the responses obtained after the addition of 1 mM EMP. The measurements were taken at increasing time intervals in every 15 min after the addition of EMP into the electrolyte. (B) The capacitance response shown in (a) was obtained in the absence of EMP, and the response (b) was obtained after the addition of 1 mM DEES onto the 2-modified MWCNT-ITO surface. The serial titration of the 2-modified MWCNT-ITO surface with 1 nM EMP (c), and after the addition of blank electrolyte in the presence of 1 nM EMP (d), after the addition of 1 µM EMP (e), and the gray lines show the signal obtained after the addition of 1 mM EMP and the measurements were taken in every 15-min interval for 1 h (f-i). (C) The plot for the dependence of the ∆ACV responses on the concentration of the titrated EMP into the blank electrolyte.

Figure 7. Plot for the dependence of the ∆ACV responses on the concentration of the titrated PMP and DEES into the blank electrolyte. Table 2. Analytical Calibration Functions for Compounds 1 and 2a CWA mimics MPA decp EMP PMP

1

2

C ) 243.4i + 20.8 R2 ) 0.9985, sr ) 4.7%, DL ) 3.2 × 10-11 M C) 224i + 30 R2 ) 0.9995, sr ) 3.1%, DL ) 4.7 × 10-11 M C) 560i - 745.3 R2 ) 0.9995, sr ) 5.2%, DL ) 5.4 × 10-8 M C) 275.1i - 203 R2 ) 0.9632, sr ) 3.9%, DL ) 5.4 × 10-8 M

C ) 274.6i + 37 R2 ) 0.9987, sr ) 4.2%, D. ) 5.3 × 10-11 M C) 235i + 64 R2 ) 0.9991, sr ) 4.9%, D. ) 5.2 × 10-11 M C ) 647i - 483.2 R2 ) 0.9994, sr ) 5.2%, DL ) 6.8 × 10-8 M C ) 732.6i - 586 R2 ) 0.9645, sr ) 4.5%, DL ) 4.7 × 10-8 M

DEES a The limit of detection, DL, is expressed at the 99% probability level. The units i and C represent the ACV current and concentration.

tions from both the dielectric () and the charge (Q) effects of the analyte (CWA mimic). The electrostatic affinity of the negatively charged CWA mimic on the ferrocene-amino acid conjugate changed the dielectric and the charge characteristics of the MWCNTs. As shown in Figure 5C, the ACV responses showed a linear dependence on the concentration of MPA titrated into the electrolyte solution. ∆ACV represents the data obtained with the subtraction of the initial ACV response from the response

obtained after 15 min past the addition of the MPA into the electrolyte (∆ACV)ACVblank - ACVfinal). The concentration-dependent ACV responses of the compound 2-conjugated MWCNT-ITO surface were recorded upon titration with DECP. The incubation time was 15 min without stirring the electrolyte after each addition of DECP. The ACV (Figure 5A) and capacitance (Figure 5B) responses of the sensor was similar to that observed for the interaction of 1 with MPA; however, the linear range was much shorter than the one observed for the detection of MPA (Figure 5C). As shown in Figure 6, ACV responses of the compound 2-conjugated MWCNT-ITO surface were also observed upon titration with EMP. The incubation time was 15 min without stirring the electrolyte after each addition of EMP. The decrease in the ACV responses of compound 2 conjugate also indicates a similar behavior as was observed for compund 1. The peak in the ACV current signal occurred at Ep ) E1/2, which was near Ep ∼0.44 V (vs Ag/AgCl). The impedance rose slowly and caused the decrease in the current as the potential scanned away from the Ep. The effect of DEES, the mustard agent mimic, was also observed using compound 2-modified MWCNT-ITO surface (Figure 6A-a). Since DEES did not have a charge in the electrolyte solution, no significant changes in the electrochemical properties of the compound 2-modified surfaces were detected. As shown in Figure 7, the concentration-dependent ACV responses of the compound 2-conjugated MWCNT-ITO surface were recorded upon titration with PMP and DEES. The incubation time was 15 min without stirring the electrolyte after each addition of PMP or DEES. As expected, DEES did not have a significant affinity toward the compound 4-modified electrodes. On the other hand, PMP showed a concentration dependent increase in ∆ACV responses. The analytical calibration data for the detection of the CWA mimics using 1 and 2 are shown in Table 2. The detection limits reached below subnanomolar levels with wide dynamic linear ranges in most mimics. The linear range for the detection of PMP was not as wide as the other mimics. It can be speculated that nonlinear transport of the analyte to the nanotubes and the threedimensional architecture of the electrode allows for this low detection limit. The linear calibration curves had a wide dynamic range that spanned 3-4 orders of magnitude. After the injection

Table 3. Summary and Comparison of the Properties of Sensors That Are Presently Used for the Detection of Nerve and Mustard Gas Mimicsa detected agent

sensor material SWCNT-modified Si substrate with electrochemical detection19 SWCNT-modified Si substrate with interdigitated electrodes for electrochemical detection22 SWCNT-modified Si substrate with electrochemical detection23 SWCNT-modified PET substrate with electrochemical detection24 SWCNT-modified Si substrate with interdigitated electrodes for electrochemical detection25 MWCNT-modified ITO substrate with electrochemical detectionb a

detection limit

detection time

DMMP

sub-ppb

real time

chemical vapors

sub-ppb

real time

VOCs and low-vapor pressure solids Sarin Soman

0.5 ppb for DMMP

real time

∼25 ppm

real time

chemical vapors

sub-ppb

real time

nerve agent mimics and mustard gas mimics

sub-nM

60-90 min

PET, poly(ethylene terephthalate); VOC, volatile organic compounds; DMMP, dimethyl methylphosphonate. b This report.

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of the mimics into the solution, the changes in the responses were monitored slowly and the signals reached a saturation level in ∼60-90 min. As expected from the electrochemical data, no analytical evaluation could be made for the detection of the mustard agent mimic, DEES. CONCLUSIONS In this paper, we describe the synthesis and characterization of new ferrocene-amino acid conjugates. We demonstrated, for the first time, that the changes in the electrochemical properties of ferrocene-amino acid conjugate-modified MWCNTs can be used for the detection of exposure to CWA mimics in aqueous media. We are currently in the process of synthesizing Gly- and Lys-conjugated ferrocenes that would help better prove the overall mechanism of action for our method. The results using these amino acid conjugates will be reported in a separate submission. While significant challenges remain in optimizing the sensor performance with regard to tunability, stability, detection limit, elimination of false positives, etc., these findings reported here open new opportunities in the design of CWA sensors with onfield applicability.

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ACKNOWLEDGMENT This work was carried out under Public Works Contract W7702-05R104/001/EDM. K.K. is the recipient of a fellowship from the Ontario Ministry of Research and Innovation. The authors also thank J. Maley in Saskatchewan Structural Science Centre for taking Raman spectroscopy and S. Caldwell in the Department of Veterinary Medicine for TEM images and Shatha Qaqish for AFM images. SUPPORTING INFORMATION AVAILABLE 1H and 13C NMR spectra for compounds 1 and 2 are provided. This material is available free of charge via the Internet at http:// pubs.acs.org.

Received for review November 5, 2007. Accepted January 15, 2008. AC7022876