Anal. Chem. 2003, 75, 5687-5691
β-Cyclodextrin-Based Ferrocene-Imprinted Gold Electrodes Kazimierz Chmurski,* Andrzej Temeriusz, and Renata Bilewicz
Department of Chemistry, University of Warsaw, Pasteura 1, 01-224 Warsaw, Poland
A new stepwise self-assembly procedure is described for the preparation of functional cyclodextrin-modified electrodes. The approach is based on the formation of alkanethiol/lipoylamide-β-cyclodextrin monolayers with the thiol component responsible for blocking of the electrode surface and lipoylamide-β-cyclodextrin moleculessfor controlled opening of the access of the electroactive probe to the electrode. Functionalization of the electrode is achieved by means of a new cyclodextrin derivatives mono(6-deoxy-6-lipoylamide)-per-2,3,6-O-acetyl-β-cyclodextrin-prepared in the peracetyl form and deacetylated directly on the electrode surface following the cyclodextrin self-assembly. The progress of deacetylation was monitored by the MALDI MS technique. Deacetylation caused opening of the active sites toward solution probes. The response toward ferrocene was found to be highly improved when ferrocene was added to the solution following self-assembly of cyclodextrin but prior to the thiol selfassembly step (imprinting method). The proposed synthesis and sequential monolayer formation scheme lead to well-organized and stable modified electrode surfaces with improved sensitivity toward solution species compared to other procedures of electrode modification with the cyclodextrin derivatives. Thiolated cyclodextrins (CDs) have been proved useful as modifiers of electrode surfaces.1,2 They were most commonly used in electrochemical sensors for organic compounds.3,4 There are two ways of modifying the electrode surfaces with monolayers of amphiphilic cyclodextrins. The first is based on the application of the Langmuir-Blodgett technique,5 and the other is a selfassembly procedure.6 The thiol units required to anchor the CD on the solid substrate were introduced either directly into the CD molecules in the C-6 position or into the side chains.7 The thiolated CDs * Corresponding author. Fax:+48228225996. E-mail: chmurkaz@alfa. chem.uw.edu.pl. (1) Kaifer, A. E.; Go´mez-Kaifer, M. Supramolecular Electrochemistry: SelfAssembled Monolayers; Wiley-VCH: Weinheim, 1999; Chapter 15. (2) Choi, S.-J.; Park, S.-M. Bull. Korean Chem. Soc. 2002, 23, 699-704. (3) He, P.; Ye, J.; Fang, Y.; Suzuki, I.; Osa, T. Anal. Chim. Acta 1997, 337, 217-223. (4) Suzuki, I.; Murakami, K.; Anzai, J.; Osa, T.; He, P.; Fang, Y. Mater. Sci. Eng. C 1998, 6, 19-25. (5) Blodgett, K. J. Am. Chem. Soc. 1935, 57, 1007-1022. (6) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 4481-4484. (7) Nells, G.; Weisser, M.; Beck, R.; Wohlfart, P.; Wenz, G.; Mittler-Neher, S. J. Am. Chem. Soc. 1996, 118, 5039-5046. 10.1021/ac0340096 CCC: $25.00 Published on Web 09/23/2003
© 2003 American Chemical Society
differ in the number of thiol groups per molecule, from monosubstituted to persubstituted with all glucopyranoside units possessing the anchoring thiol functionality.8,9 The anchoring groups are thiols, disulfides,10 or other S-S moieties breaking easily during chemisorption on gold, e.g., lipoic acid.11 The cyclic disulfide functionality of lipoic acid affords two binding sites on the gold surface per chemisorbed molecule, which was suggested to lead to more stable SAMs.12 Lipoyl derivatives of β-cyclodextrin with different length of the side chains were described first by He et al.13 Positional isomers of γ-CD derivatives with two lipoic ester groups per molecule were synthesized by Suzuki and co-workers.14 Kitano and Taira presented the synthesis of 6-deoxy-6-lipoylamido-R-CD with the average degree of substitution, 2.5 lipoic units in the CD molecules.15 This synthesis was performed by incubation of respective amines with lipoic acid in THF/water mixtures in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). Similar to the recently published procedure for the per-O-methyl derivative,11 our synthetic approach allows to get multigram amounts of mono(6-deoxy-6-lipoylamide)-per-2,3,6-O-acetyl-β-cyclodextrin (1) without the necessity of using any special separation or cleaning procedures other than routine silica gel column chromatography. (Scheme 1) However, in our case, the introduction of the protective groups on other hydroxyl units in the molecule makes both the synthetic work and the self-assembly of monolayers on electrodes easier, due to good solubility of the derivative 1 in common organic solvents. Moreover, the protective O-acetyl functionalities can be easily removed by treatment with an alkaline solution, even when the compound is already anchored on a solid substrates. The purpose of our investigation was to elaborate an improved sequential self-assembly procedure for the preparation of cyclodextrin-modified gold electrodes. This approach leads to better stability of the monolayers and to enhanced sensitivity of the modified surfaces compared to other procedures of electrode modification with CD derivatives. (8) Maeda, Y.; Fukuda, T.; Yamamoto, H.; Kitano, H. Langmuir 1997, 13, 41874189. (9) Fukuda, T.; Maeda, Y.; Kitano, H. Langmuir 1999, 15, 1887-1890. (10) Henke, C.; Steinem, C.; Janshoff, A.; Steffan, G.; Luftman, H.; Sieber, M.; Galla, H. J. Anal. Chem. 1996, 68, 3158-3165. (11) Carofiglio, T.; Fornasier, R.; Jicsinszky, L.; Tonellato, U.; Turco, C. Tetrahedron Lett. 2001, 42, 5241-5244. (12) Wang, Y.; Kaifer, A. E. J. Phys. Chem B 1998, 102, 9922-9927. (13) He, P.; Ye, J.; Fang, Y.; Suzuki, I.; Osa, T. Electroanalysis 1997, 9, 68-73. (14) Suzuki, I.; Murakami, K.; Anzai, J. Mater. Sci. Eng. C 2001, 17, 143-148. (15) Kitano, H.; Taira, Y. Langmuir 2002, 18, 5835-5840.
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Scheme 1. Synthesis and Structure of Mono(6-deoxy-6-lipoylamide)-per-2,3,6-O-acetyl-β-cyclodextrin (1)
EXPERIMENTAL SECTION Materials. All reagents used were of the highest available purity. Ferrocenecarboxylic acid, ferrocene, and octane- and dodecanethiol were purchased from Aldrich and used as received. Water was distilled and passed through a Milli-Q purification system. The final resistivity of water was 18.2 MΩ cm-1. Synthesis. The starting mono(6-deoxy-6-O-tosyl)-β-cyclodextrin was prepared according to Bittman et al.16 and transformed into mono(6-azido-6-deoxy)-β-cyclodextrin by nucleophilic displacement of the O-tosyl group by azide ion in DMF at 60 °C. The free hydroxyl functionalities were then protected as per-2,3,6O-acetyl esters, using an acetic anhydride/pyridine mixture in the presence of catalytic amounts of 4-(dimethylamino)pyridine. The resulting monoazide derivative was catalytically hydrogenated in the presence Pd/C, yielding the corresponding monoamine derivative. The coupling reaction of this amine and D,L-lipoic acid was performed according to the procedure given by Ponpipom:17 1.8 g (0.91 mmol) of mono(6-amino-6-deoxy)-2,3,6-per-O-acetyl-βcyclodextrin was dissolved in the mixture 1.9 mL of DMF and 11.4 mL of acetonitrile. To this solution, 0.391 g of D,L-lipoic acid (1.9 mmol) and 0.510 g of 1,3-dicyclohexylcarbodiimide were added. The reaction mixture was stirred overnight at ambient temperature, the resulting dicyclohexylurea was filtered off, and the solvents were evaporated. Column chromatography (silica gel, acetone/hexane 1:1, Rf 0.13) afforded the desired 1 in 71% yield (1.4 g, 0.65 mmol). The 13C-1H NMR correlation spectra, performed on Bruker 500-MHz apparatus, showed loss of symmetry of the CD ring and the appearance of separated signals due to a covalently bonded side chain in the aliphatic region indicating 1:1 stoichiometry of the compound. The structure was confirmed by mass spectra: C90H123O55NS2 calcd 2161.12, found MS ESI+, m/z 2226.7 [M + NaN3 + H+]+, 2242.8 [M + KN3 + H]+ Apparatus. Cyclic voltammetry experiments were accomplished using the Autolab potentiostat (ECO Chemie) in a threeelectrode arrangement with the saturated Ag/AgCl as the reference separated from the bulk solution by a double junction filled (16) Zhong, N.; Byun, H.-S.; Bittman, R. Tetrahedron Lett. 1998, 39, 2919-2920. (17) Ponpipom, M. M.; Bugianesi, R. L.; Shen, T. Y. Carbohydr. Res. 1980, 82, 141-148.
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with 0.2 M Na2SO4. Platinum wire was used as the counter electrode and gold disk (2.85-mm2 surface area) as the working electrode. Argon was used to deaerate the solution, and an argon blanket was maintained over the solution during the experiments. MALDI MS. Mass spectrometry experiments were performed with the Kratos Kompact MALDI 4 ToF V5.2.1 at acceleration voltage of 20 kV and with two-point linear calibration. The timedelayed extraction was set on 3000 Da. An average of 200 scans was analyzed. 2,5-Dihyrobenzoic acid dissolved in acetonitrile was used as a matrix. RESULTS AND DISCUSSION Electrode Modification with CD and the Surface Deacetylation Procedure. The β-CD derivative 1 was dissolved in acetone to form a 2 mM solution and diluted twice with ethanol. After anchoring of the molecules to the gold surfaces, the protective groups were removed by dipping the electrode into an alkaline solution of sodium metoxide in methanol. Efficiency of removal of the acetyl groups was checked directly on the surface of the gold substrate using the MALDI MS technique, and the time required for the removal of all esters was established using this approach. The stainless steel target was coated with 20 nm of chromium and 100 nm of gold and covered next by immersing it into a 1 mM acetone solution of 1 for 24 h, to obtain a monolayer-covered substrate. Next, this substrate was immersed in a freshly prepared 0.5 M methanolic solution of sodium metoxide. Stepwise withdrawing allowed us to modify the length of immersion from 1 to 4 h and to control the time dependence of the deacetylation procedure by MALDI MS. Parts of the target without contact with the alkaline solution gave m/z signals above calculated 2160 M+ (2162, 2175, 2189, 2204, 2235). Parts of the target dipped for 1 h or longer gave already exclusively signals corresponding to free OH groups: m/z 1344, 1360, and 1375 with no signals of the peracetylated CD derivative. This meant that 1 h was sufficiently long to remove all protective groups (20 O-acetyls), and a monolayer of the respective hydroxyl derivative remains on the metal substrate. Following deacetylation, the electrodes were washed with methanol in order to remove the sodium metoxide.
Figure 1. Cyclic voltammetry curves recorded in 0.1 M NaClO4 containing 10-4 M FcCOOH using an Au electrode covered with SAM assembled from the 1 mM solution (99.9% C12H25SH and 0.1% lipoylamide-β-CD 1) as 20 O-acetyls form (solid line) and as 20 OHs form (dashed line). Dotted line, Au bare electrode. Scan rate 0.1 V/s.
Figure 2. Comparison of the voltammetric curves for 5 × 10-6 M Fc in 0.2 M Na2SO4, recorded using a bare gold (dotted line) and CD SAM-modified electrode by the molecular imprinting method (solid line). Scan rate, 0.1 V/s.
Self-assembly of 1 on the gold surface usually took 16 h. Anchoring of lippoic acid derivatives to the gold surface requires a longer time as compared to thiol compounds, and well-organized and stable layers of 1 were obtained after 16 h. Before further characterization, the electrodes were rinsed with acetone, ethanol, and water. The layers were characterized by cyclic voltammetry. After the self-assembly of 1, the background current drops to about half of the initial value. Removal of the protecting groups does not change this current, indicating that the layer is still anchored to the gold surface. If a mixture of 1 and octanethiol in ethanol is used instead of single-component 1, the barrier properties of the layer improve and the calculated double-layer capacitance decreases from 6.86 to 1.59 µF/cm2. Deacetylation of the surface-immobilized CDs opens in a controlled way the surface for solution electrochemical probes.
Upon removal of the protecting groups, the receptor molecules become much more hydrophilic, which explains the channel-like behavior of the CDs. Comparison of voltammetric oxidation curves for ferrocenecarboxylic acid (FcCOOH) in the solution shows clearly the channel opening phenomena (Figure 1). For electrodes modified with receptor in the 20 O-acetyl form, the oxidation peak is irreversible (solid line), while following deacetylation, a much more reversible (dashed line) system of peaks is obtained. After removal of all O-acetyl groups, the FcCOOH molecules have free access to the gold surface and the voltammograms differ only slightly from those recorded using the bare gold electrode (dotted line Figure 1). If a mixed monolayer containing 1 and alkanethiol was used, the blocking efficiency was higher than for the singlecomponent monolayer since the compound approached the electrode only through the open channels of deacetylated 1, while Analytical Chemistry, Vol. 75, No. 21, November 1, 2003
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Figure 3. Cyclic voltammograms recorded for increasing concentrations of ferrocene (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.5, and 4.0 × 10-5 M) in 0.2 M Na2SO4 solution using an electrode modified by the molecular imprinting method with CD 1 and C8H17SH. Scan rate, 0.1 V/s.
Figure 4. Linear part of the plots [Fc]/I vs [Fc] for scan rates of (9) 10, (b) 20, and (2) 50 mV/s with correlation coefficients 0.985, 0.987, and 0.981, respectively.
the remaining part of the electrode was blocked with alkanethiol. Molecular Imprinting Procedure. Based on the voltammograms, the thiol component was preferentially adsorbed from a solution containing both 1 and alkanethiol. Therefore, the stepwise self-assembly procedure was employed. First the lipoylamide-βCD 1 was self-assembled for ∼16 h, then the molecules of the layer were deacetylated by placing the electrode in the alkaline solution for 1 h, and next, the electrode was washed with methanol and transferred to a solution of 0.2 mM ferrocene (Fc) in EtOH/ H2O (50:50%) for 2 h, to prevent inclusion of the alkanethiol molecules into the CD cavities in the following step of the procedure.1,2 In this final step, ethanol solution of alkanethiol was added and the electrode was left for a couple of hours in order to block the surface not occupied by 1. 5690 Analytical Chemistry, Vol. 75, No. 21, November 1, 2003
The procedure was advantageous in terms of monolayer quality compared to the common one-step self-assembly procedures and was used to enhance molecular recognition. Adding the guest as a template following the step of self-assembly of CDs and prior to the thiol self-assembly was found favorable for the construction of surfaces responding to the same guest placed in the solution. This was because under these conditions most of the surface was blocked by the thiol and the background current remained much lower than for the bare gold electrode while the CD sites were open for the guest. This allowed us to detect much lower concentrations of solutes (as low as 5 × 10-7 M) than by using the unmodified surface (Figure 2) and hence increased the sensitivity of the electrode toward the selected analyte in the solution.
Anodic peak current recorded for 5 × 10-6 Fc in the solution was linear versus scan rate rather than versus the square root of the scan rate, which indicated surface binding of ferrocene by the CD monolayer. The plot of peak current versus concentration of Fc is linear until ∼2 × 10-5 M, and for higher concentration, leveling of the current is observed. Since the solubility of ferrocene is 6 × 10-5 M, it means that equilibrium concentration is attained earlier than the solubility limit. The maximum surface concentration of ferrocene based on the charge of the ferrocene oxidation peak was Γ ) 4.3 × 10-11 mol/cm2. The β-CD derivative 1 upon compression at the air-water interface shows a stable monolayer.18 The Langmuir isotherm showed a collapse surface pressure of 25 mN/m and a mean molecular area of 300 Å2. Approximating the latter value as surface of the single receptor and using the area of the electrode, the surface concentration was Γ ) 5.53 × 10-11 mol/cm2. This value is in quite good agreement with the previous one taking into account that polycrystalline gold electrodes were used. Cyclic voltammograms recorded for increasing concentration of ferrocene in 0.2 M Na2SO4 using the modified electrode are shown in Figure 3. The binding constant is determined for ferrocene by fitting the experimental data to the following equation:8,9
[Fc]/I ) 1/K1Imax + [Fc]/Imax
where I is the anodic peak current for a given concentration of Fc in the linear part of the plot (Figure 4), the concentration of (18) Unpublished results.
Fc is [Fc], Imax is the maximum peak current when the current attains constancy, and K1 is the association constant of Fc with a surface-confined compound, calculated from the x-intercept of the linear part of the plots for different scan rates (Figure 4). The association constant was found to be 3.3 × 104 M-1, which is similar to the value 3.9 × 104 M-1 obtained by Rojas et al.1 for Fc and per(6-deoxy-6-thio)-β-CD anchored at the gold electrode surface. CONCLUSIONS The proposed scheme of synthesis and monolayer formation allows us to improve the selectivity and sensitivity of the electrode compared to other procedures of electrode modification with CD derivatives. The monolayer was formed using acetylated CD moleculesswhich enhanced the solubility of this receptor in common organic solvents, thus making the synthesis, separation, and cleaning, as well as self-assembly, much easier. On the other hand, gating of the CD molecules is achieved by deacetylation carried out directly on the electrode and checked by the MALDI MS technique. Stepwise self-assembly with the CD deposition and thiol deposition separated by immersion of the electrode into ferrocene solution allows us to open all the CD sites and to obtain a sensitive analyte-responsive modified electrode. ACKNOWLEDGMENT This work was supported by Warsaw University BW-1562/02/ 2002 and KBN Grant 3T09A 12219. The authors thank Dr. I. Wielgus (Chemistry Department of Technical University of Warsaw) for MALDI MS measurements. Received for review January 6, 2003. Accepted August 20, 2003. AC0340096
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