Self-Assembled Monolayers of Monofunctionalized Cyclodextrins onto

Sep 15, 1996 - A novel β-cyclodextrin (β-CD) functionalized by a mercaptopropionic acid that was attached to a single 6-deoxyaminoglucose unit has b...
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Anal. Chem. 1996, 68, 3158-3165

Self-Assembled Monolayers of Monofunctionalized Cyclodextrins onto Gold: A Mass Spectrometric Characterization and Impedance Analysis of Host-Guest Interaction Christian Henke, Claudia Steinem, Andreas Janshoff, Gerhard Steffan,† Heinrich Luftmann,‡ Manfred Sieber, and Hans-Joachim Galla*

Institut fu¨ r Biochemie, Westfa¨ lische Wilhelms-Universita¨ t Mu¨ nster, Wilhelm-Klemm-Strasse 2, 48149 Mu¨ nster, Germany

A novel β-cyclodextrin (β-CD) functionalized by a mercaptopropionic acid that was attached to a single 6-deoxyaminoglucose unit has been synthesized in the disulfide form. The flexible single-thiol spacer allows the formation of a monomolecular film by self-assembly onto gold, yielding a high packing density with a surface coverage of 99.6% and a capacitance of 9 µF/cm2, determined by impedance spectroscopy. MALDI-MS and XPS analysis clearly showed that the modified cyclodextrin is chemisorbed on the gold surface by Au-S bonds. Addition of 3-mercaptopropionic acid to the preformed β-CD monolayer considerably improved the intensity of the MALDI mass spectra signals. The incorporation of anilinonaphthalenesulfonates into the β-CD cavity was observable by impedance spectroscopy using the electroactive markers [Fe(CN)6]3-/[Fe(CN)6]4-. Bioanalytical methods based on functionalized monolayers or lipid bilayers with incorporated receptor molecules on solid substrates are gaining widespread importance in the development of biosensor devices, mimicking the mechanisms of molecular recognition and channel conductance of biological membranes.1-5 One strategy involves the immobilization of such a system on an electrical conductive surface in order to apply electrochemical methods. It is possible to functionalize gold surfaces with selfassembled monolayers (SAMs) of chemisorbed thiol molecules carrying different types of headgroups in order to obtain various properties, such as hydrophobicity6-8 or hydrophilicity,9 redox † Present address: University of the Pacific, Department of Microbiology 2155 Webster Street, San Francisco, CA 94115. ‡ Institut fu ¨ r Organische Chemie, Westfa¨lische Wilhelms-Universita¨t Mu ¨ nster, Corrensstr. 40, 48149 Mu ¨ nster, Germany. (1) Sugawara, M.; Kojima, K.; Sazawa, H.; Umezawa, Y. Anal. Chem. 1987, 59, 2842-2846. (2) Terrettaz, S.; Vogel, H.; Gra¨tzel, M. J. Electroanal. Chem. 1992, 326, 161176. (3) Miller, C.; Cuendet, P.; Gra¨tzel, M. J. Electroanal. Chem. 1990, 278, 175192. (4) Reha´k, M.; Snejda´rkova´, M.; and Otto, M. Electroanalysis 1993, 5, 691694. (5) Nikolelis, D.; Krull, U. J. Electroanalysis 1993, 5, 539-545. (6) Sabatani, E.; Rubinstein, I.; Maoz, R.; Sagiv, J. J. Electroanal. Chem. 1987, 219, 365-371. (7) Sabatani, E.; Cohen-Boulakia, J.; Bruening, M.; Rubinstein, I. Langmuir 1993, 9, 2974-2981. (8) Bain, C. D.; Throughton, E. B.; Tao, Y. T.; Evall, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 321-335.

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centers on thiols,10,11 or acidity and basicity.12 SAMs may also be used to cover gold surfaces with hydrophobic or charged molecules that allow adsorption of further lipid mono- or bilayers.13 Moreover, the formation of monomolecular protein layers by molecular recognition processes may be easily achieved, such as the binding of streptavidin to a thiol-functionalized biotin conjugate.14 Concerning biosensor devices, cyclodextrins as a different type of receptor molecules have also gained attention in recent years.15 These toroid-shaped natural cyclic glucopyranose oligomers are used in analytical and industrial applications.16,17 They form hydrophobic cavities lined by the glycoside oxygen bonds, which are responsible for the ability of the CD to include hydrophobic molecules. Binding specificity depends mainly on substrate size and geometry, where the substrate has to fit into the tubular cavity with a diameter of 4.7-8.3 Å, depending on the number of monomeric units in the ring.17 The noncovalent forces such as van der Waals forces, hydrogen bondings, and hydrophobic interactions are responsible for the binding specifity of these inclusion complexes. Attempts have been made by other groups to establish oriented monolayers of CD on solid supports either by use of LangmuirBlodgett techniques or via self-assembly.9,15 In a recent study, Rojas et al. replaced all the primary hydroxyl groups of β-CD with thiol groups.18 This CD derivative chemisorbs onto gold surfaces; however, the obtained monolayers were imperfect, with a considerable fraction of uncovered gold surface. Rojas et al. were able to solve this problem by adding of pentanethiol to increase the apparent surface coverage.18 We report the synthesis of a monofunctionalized thiol cyclodextrin that chemisorbs onto gold substrates with very high (9) Kawabata, Y.; Matsumoto, M.; Nakamura, T.; Tanaka, M.; Manda, E. Thin Solid Films 1988, 159, 353-358. (10) Zhang, L.; Godı´nez, L. A.; Lu, T.; Gokel, G. W.; Kaifer, A. E. Angew. Chem. 1995, 107, 236-237. (11) Chidsey, C. E. D.; Bertozzi, C. R.; Putvinski, T. M.; Mujsce, A. M. J. Am. Chem. Soc. 1990, 112, 4301-4306. (12) Bryant, M. A.; Crooks, R. M. Langmuir 1993, 9, 385-387. (13) Steinem, C.; Janshoff, A.; Ulrich, W.-P.; Sieber, M.; Galla, H.-J.; Biochim. Biophys. Acta 1996, 1279, 169-180. (14) Morgan, H.; Taylor, D. M.; D’Silva, C. Thin Solid Films 1992, 209, 122126. (15) Odashima, K.; Kotato, M.; Sugawara, M.; Umezawa, Y. Anal. Chem. 1993, 65, 927-936. (16) Saenger, W. Angew. Chem. 1980, 92, 343-361. (17) Li, S.; Purdy, W. C. Chem. Rev. 1992, 92, 1457-1470. (18) Rojas, T. M.; Ko ¨niger, R.; Stoddart, J. F.; Kaifer, A. E. J. Am. Chem. Soc. 1995, 117, 336-343. S0003-2700(95)01226-1 CCC: $12.00

© 1996 American Chemical Society

packing density. SAMs were characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and by X-ray photoelectron spectroscopy (XPS). Two different positional isomers of anilinonaphthalenesulfonates that differ in their steric properties and thus in their ability to form inclusion complexes were used to probe the suitability of these novel β-cyclodextrin-SAMs as interfacial hosts for the analytical determination of guest molecules. Incorporation into the CD cavity was observed by impedance analysis using the electroactive marker molecules [Fe(CN)6]3-/[Fe(CN)6]4-. EXPERIMENTAL SECTION Materials. β-Cyclodextrin (β-CD) was purchased from Roth (Karlsruhe, Germany) and used without further purification. p-Toluenesulfonyl chloride and triphenylphosphine were purchased from Riedel de Hae¨n (Seelze, Germany); IWT-TMD-8 ion exchange resin and 3,3′-dithiomercaptopropionic acid were from Aldrich (Milwaukee, WI); 3,3′-dithiobis(propionic acid N-hydroxysuccinimide ester) and sodium azide were purchased from Fluka (Buchs, Switzerland); 3-mercaptopropionic acid (MPA), 1-anilinonaphthalene-2-sulfonic acid (1,2-ANS), 2-(p-toluidinyl)naphthalene-6-sulfonic acid (2,6-TNS), K3[Fe(CN)6], and K 4[Fe(CN)6] were from Sigma (St. Louis, MI); Sephadex-G10 was from Pharmacia (Uppsala, Sweden); and Hellmanex was from Hellma (Mu¨llheim, Germany). All other chemicals were of the highest available grade of purity. TLC plates were aluminum sheets precoated with silica gel 60 F254 with 0.2 mm layer thickness from Merck (Darmstadt, Germany). TLC eluant was EtOAc/2-propanol/H2O/concentrated NH4OH (7:7:10:0.5). The spots were visualized by spraying the TLC plates with sulfuric acid and subsequent charging. Synthesis of the Thiol-Modified Cyclodextrins. (i) Mono6-deoxy-6-(p-tolylsulfonyl)-β-cyclodextrin (Ts-CD). β-Cyclodextrin (60 g, 52 mmol) was suspended in 500 mL of water. NaOH (6.57 g, 164.0 mmol) dissolved in 20 mL of water was added dropwise over 6 min. The suspension became homogeneous and slightly yellow before the addition was finished. p-Toluenesulfonyl chloride (10.08 g, 52.9 mmol) dissolved in 30 mL of acetonitrile was added dropwise over 8 min, causing immediate formation of a white precipitate. After 2 h of stirring at room temperature, the precipitate was removed by suction filtration, and the filtrate was stirred for 12 h at 4 °C. The resulting second precipitate was recovered by suction filtration, recrystallized from water and lyophilized to give 3 g (4.4%) of a white solid: TLC, one spot, Rf 0.51; positive ion MALDI-MS m/z 1312 for [M + Na]+, calcd (C49H76O37S) 1289. (ii) Mono-6-deoxy-6-azido-β-cyclodextrin (N3-CD ). Ts-CD (1 g, 0.776 mmol) was suspended in 3 mL of dry N,N-dimethylformamide (DMF). After warming to 63 °C, the solution became homogeneous. Crystalline KI (64.8 mg, 0.388 mmol) and NaN3 (504 mg, 7.6 mmol) were added, and the reaction mixture was stirred for 24 h at 65 °C. The mixture was then cooled to room temperature and treated with IWT-TMD-8 ion exchange resin to remove salts. The resin was separated by filtration, and the filtrate was concentrated to 0.5 mL. The addition of acetone resulted in a white precipitate, which was filtered and dried overnight at 40 °C under vacuum, yielding a pure white solid (670 mg, 74%): TLC, one spot, Rf 0.51; positive ion MALDI-MS m/z 1184 for [M + Na]+, calcd (C42H69N3O34) 1160. (iii) Mono-6-deoxy-6-amino-β-cyclodextrin (NH2-CD). N3CD (600 mg, 0.517 mmol) was suspended in 8.8 mL of dry DMF.

Triphenylphosphine (539 mg, 2.064 mmol) was added, and after 1 h of stirring at room temperature, the solution was treated with 1.2 mL of concentrated NH4OH. The reaction mixture was stirred for 24 h, concentrated to 0.5 mL, treated with 50 mL of H2O, and filtered. The filtrate was cleared with activated charcoal and then pressed through a 0.2 µm filter to remove traces of charcoal and finally concentrated. Addition of acetone resulted in a precipitate, which was recovered by suction filtration and dried in vacuum to give a white solid (534 mg, 91%): TLC, one spot, Rf 0.26; positive ion MALDI-MS m/z 1157 for [M + Na]+, calcd (C42H71NO34) 1134. (iv) 3,3′-Dithiobis(propan-(N-mono-6-deoxy-β-cyclodextrin)amide) ((MPA-CD)2). NH2-CD (50 mg, 44.1 µmol) was suspended in 5 mL of dry DMF, and 3,3′-dithiobis(propionic acid N-hydroxysuccinimide ester) (8.9 mg, 22.05 µmol) in 10 mL of dry DMF was added dropwise under vigorous stirring. The reaction was followed by TLC. After the reaction was completed, the solution was concentrated. The addition of acetone produced a slightly yellow solid, which was recovered by suction filtration. The solid was dissolved in H2O and subjected to chromatography on an IWT-TMD-8 ion exchange column. Lyophilization of the eluate yielded a fluffy white solid (53.2 mg, 99%): TLC, one spot, Rf 0.20; positive ion MALDI-MS m/z 2466 for [M + Na]+, calcd (C90H148N2O70S2) 2442; ESCA, C96N2O70S2. (v) 3,3′-Dithiopropionic Acid Mono(N-mono-6-deoxy-βcyclodextrin)amide ((MPA)2-CD). 3,3′-Dithiobis(propionic acid N-hydroxysuccinimide ester) (17.8 mg, 44.1 µmol) was suspended in 10 mL of dry DMF, and NH2-CD (25 mg, 22.05 µmol) dissolved in 10 mL of DMF was added dropwise under vigorous stirring. After completion of the reaction, the mixture was concentrated to about 0.5 mL. Addition of acetone produced a white precipitate, which was recovered by suction filtration. The solid was resuspended in water and purified by gel chromatography using a Sephadex-G10 column. Lyophilization of the main fraction yielded a fluffy white solid (26.0 mg, 89%): TLC, one spot, Rf 0.41; positive ion MALDI-MS m/z 1350 for [M + Na]+, calcd (C48H79N2O37S2) 1326. Impedance Analysis. Impedance spectra were taken using the impedance gain/phase analyzer SI 1260 from Schlumberger instruments (Farnborough Hampshire, England). The electrochemical cell consists of two equally designed working electrodes, which were made by evaporation of gold onto glass slides. All data were obtained at zero applied direct voltage and 10-20 mV ac. amplitude to avoid nonlinear responses. The frequency ranged from 0.1 to 100 000 s-1, and the magnitudes of the impedance |Z|(ν) and the phase angle Φ(ν) between potential and current were recorded with a personal computer. Data analysis was carried out using the program BWD (available from M.S.: e-mail [email protected]), which determines the parameters of the assumed elecrical network by a weighted nonlinear leastsquare fit based on the Levenberg-Marquardt algorithm.19 Electrode Preparation. Glass slides (26 mm × 76 mm) were sonicated for 30 min in a detergent solution containing 2% Hellmanex at 70 °C for purification. After the slides were rinsed extensively with ultrapure water (MilliQ, Millipore, Eschborn, Germany) to remove remaining detergent, the electrodes were produced by evaporating gold through a mask onto the slide. To improve the adhesion of the gold layer of about 100 nm thickness, a thin layer of chromium (20 nm) was evaporated first. The metal (19) Bevington, B. R. Data reduction and error analysis for the physical sciences, 1st ed.; McGraw-Hill Co.: New York, 1969; Chapter 11.

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Figure 1. Preparation of (MPA-CD)2 and (MPA)2-CD.

coatings were made using an evaporation unit Sussex, at a reduced pressure of about 10-5 Torr. The geometrical area of each electrode was 0.13 cm2. To remove remaining adsorbates, all electrodes were purified directly before use by exposing them to an argon plasma of high energy (plasma cleaner, Harrick, Ossining, NY). Formation of Thiol Monolayers via Self-Assembly. Monolayers were formed by placing the gold electrodes into a 1 mM solution of the thiol compound in ultrapure water. Impedance spectra were taken in order to control the process and to check the quality of the monolayer formation. Instrumentation of the MALDI-MS. A self-constructed timeof-flight mass spectrometer (TOF-MS) was used. Ionization was performed with a N2 laser with an emission wavelength of 337 nm. Light pulses of 3 ns were used, and the acceleration voltage was kept at 16 kV. Ion extraction was performed by a grid in one step, and the field free drift path was 1 m. Data were collected with a storage oscilloscope, LeCroy DSO 9450A (Chesnut Ridge, NY) capable of recording 2.5 ns/sample. The expected mass accuracy was (0.1%. Sample Preparation for MALDI-MS. Samples on stainless steel targets were prepared by mixing 0.5 µL of the β-CD solution with a typical concentration of 1 µM with 5 µL of matrix solution (5 g/L 2,5-dihydroxybenzoic acid in acetonitrile) on the target. To investigate the SAMs by MALDI-MS, the stainless steel targets were polished and cleaned as described above and then coated with 20 nm of chromium and 200 nm of gold. The self-assembly was carried out on the prepared targets by exposing them to the (MPA)2-CD or (MPA-CD)2 solution as described above. After rinsing the targets with ultrapure water, 1 µL of the matrix solution was added directly to the surface. For displacement experiments, 1 µL of a 10 mM MPA solution was added to 5 µL of the matrix solution. RESULTS Synthesis of the thiolated β-CD Derivatives. The aim of the synthesis was to obtain a β-CD-derivative with one short spacer 3160 Analytical Chemistry, Vol. 68, No. 18, September 15, 1996

terminated by a thiol group that allows self-assembly onto gold. Moreover, the spacer should give the molecule enough conformational freedom to reach a high packing density on the surface, including a possible hydrogen network between the β-cyclodextrin rings. The corresponding thiolated β-CD derivatives were synthesized as shown in Figure 1. One of the seven primary hydroxyl groups was replaced by an amine group in three steps.20 Yields were considerably high, except for the first tosylation step. Dithiopropionic acid was coupled to the β-CD amine via an amide linkage to form (MPA)2-CD and (MPA-CD)2. These thiol derivatives of β-CD are anticipated to chemisorb on gold surfaces by forming gold-thiol bonds. Kinetic Study of the Formation of the CD Monolayers. The time dependence of the chemisorption of (MPA-CD)2 and (MPA)2-CD onto the gold substrate was followed by impedance spectroscopy without addition of redox-active couples. The values of the capacitances and the standard devitations (σ) of the fit, using the simplest equivalent circuit of a capacitor and a resistor in series, are given in comparison to dithiopropionic acid as a thiol component without β-CD headgroup. Figure 2a shows a representative plot of the time course of the capacitance and of σ for 50 min during the self-assembly of (MPA)2-CD. The decrease in capacitance for the synthesized (MPA)2-CD is much slower compared to that for dithiopropionic acid. For the latter, the final capacitance was reached after 6 min, but it took 80 min for the self-assembly of (MPA)2-CD to reach a final capacitance value for the monolayer of 8-9 µF/cm2. Figure 2b shows the decrease in capacitance arising from the formation of a (MPA-CD)2 monolayer. The time constant for the (MPA-CD)2 adsorption is approximately 30 times smaller than the one obtained for the (MPA)2-CD. The minimal value of 9 µF/ cm2 was reached after 20 h and was constant for at least 3 days. However, the following fact has to be noted: although the capacity (20) Petter, R. C.; Salek, J. S.; Sikorski, C. T.; Kumaravel, G.; Lin, F.-T. J. Am. Chem. Soc. 1990, 112, 3860-3868.

Figure 2. (a) Time-dependent decrease of capacitances during the self-assembly processes of dithiopropionic acid (O) and (MPA)2-CD (b). The values for the capacitances were evaluated from the corresponding impedance spectra using a simple serial RC equivalent circuit. σ is the standard deviation of the fit. (b) Time-dependent decrease of capacitances during the monolayer formation of (MPACD)2 (9). Although the kinetics of the self-assembly is much slower than those for (MPA)2 and (MPA)2-CD, the normal contamination of a clean gold surface takes more time (0). The final capacitances are summarized in Table 1 are all of the same range (7-9 µF/cm2). Deviations of the recorded data from the assumed model of each spectrum are shown in the upper part of the figure (σ vs time). The acquisition time for each spectrum is about 50 s.

of a contaminated Au electrode is of the same value as that of the (MPA-CD)2 monolayer, its kinetics is much slower (Figure 2b). MALDI-MS of the Thiolated Cyclodextrins. We analyzed the thiolated β-CD molecules on conventional stainless steel and as monolayers on gold targets by MALDI-MS using 2,5-dihydroxybenzoic acid (DHB) as matrix molecules. Figure 3 shows the results for thiolated β-CD purely physisorbed onto stainless steel (Figure 3a), chemisorbed monolayers on gold (Figure 3b), and chemisorbed monolayers on gold after addition of 3-mercaptopropionic acid (MPA) to the preformed MPA-CD monolayers (Figure 3c). Each spectrum (1) is obtained from (MPA-CD)2 and each spectrum (2) from (MPA)2-CD. On stainless steel, peaks of high intensities were detected mainly for the respective disulfide [(MPA-CD)2 + K]+ m/z 2481 and [(MPA)2-CD + Na]+ m/z 1350 or [(MPA)2-CD+K]+ m/z 1366, with only small amounts of the monomeric MPA-CD. The spectrum obtained from a (MPA)2CD solution showed an additional small peak of [(MPA-CD)2 + Na]+ at m/z 2466. The corresponding spectra obtained from chemisorbed monolayers on gold surfaces (Figure 3b) differ considerably. For chemisorbed (MPA-CD)2, the accumulation of 61 spectra led to weak peaks at m/z 1245 from [MPA-CD + Na]+. No signal was observable for [(MPA-CD)2 + Na]+ at m/z 2465, which is clearly present on stainless steel (Figure 3a(1)). For chemisorbed molecules obtained from a (MPA)2-CD-solution (Figure 3b(2)), a

Figure 3. (a) MALDI-MS spectra of (1) (MPA-CD)2 and (2) (MPA)2CD on stainless steel targets, 500 fmol total sample load, accumulated from 7 (1) or 10 (2) single laser shots, 2,5-DHB matrix (337 nm). (b) MALDI-MS spectra of self-assembled monolayers of MPA-CD on gold obtained from a (MPA-CD)2 (1) and (MPA)2-CD solution (2), with a subsequent addition of 2,5-DHB, accumulated from 61 (1) and 89 (2) single laser shots (337 nm). (c) MALDI-MS spectra of selfassembled monolayers of MPA-CD on gold, with a subsequent addition of 2,5-DHB and MPA, accumulated from 7 (1) (obtained from a (MPA-CD)2 solution) and 41 (2) (obtained from a (MPA)2-CD solution) single laser shots (337 nm).

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peak at m/z 1350 was observable, which is characteristic for [(MPA)2-CD + Na]+. A drastic increase in intensity occurred when mercaptopropionic acid was added to the DHB solution. This procedure led to the low-noise spectra shown in Figure 3c after accumulation of only seven spectra. In the case of (MPA-CD)2 (Figure 3c (1)), we observed the [(MPA-CD)2 + Na]+ peak at m/z 2466 and the [MPA-CD+Na]+ peak at m/z 1245. An unexpected peak at m/z 1350 is due to [(MPA)2-CD+Na]+, which was not present in the solution used for the self-assembly process. The addition of MPA to a self-assembly monolayer obtained from a (MPA)2-CD solution gave similar results. The MALDI spectrum after the addition of MPA is shown in Figure 3c (2). The intensity of the monomer peak of [MPA-CD + Na]+ as well as the one of (MPA)2-CD drastically increased. XPS Analysis of the Chemisorbed MPA-CD Monolayer. To distinguish between chemisorbed and physisorbed cyclodextrins on the gold surface, we determined the binding energies of sulfur in the immobilized CD molecules by XPS. The XPS spectra in the sulfur 2p region exhibit two weak peaks, with maxima at 161.8 (2p3/2) and 163.1 eV (2p1/2), which indicate the presence of Au-S bonds (data not shown). Again, these results show that the modified cyclodextrins are attached to the gold surface via Au-S bonds and that disulfide bridges are not present after binding to the surface. Impedance Analysis in the Presence of [Fe(CN)6]3-/[Fe(CN)6]4-. To obtain information about the electrode coverage with the chemisorbed β-CD monolayers, the suppression of the surface redox reaction of [Fe(CN)6]3-/[Fe(CN)6]4- on gold was investigated. One day of incubation with (MPA-CD)2 was necessary to obtain a close barrier toward the one-electron reaction of the [Fe(CN)6]3-/[Fe(CN)6]4- ions. In general, two frequency regions have to be distinguished in the presence of electroactive species. In the low-frequency region, mass transfer via diffusion has to be taken into account, where the microarray behavior of the pinholes within the passivating monolayers disturbs the 1/ω1/2 dependence of the Warburg impedance.7,21 We focused our attention on the more interesting part of the spectrum at higher frequencies, which reveals the kinetic-controlled charge transfer resistance Rct and indicates the coverage of the gold electrode, assuming that the current is due to defects within the monolayer. The surface coverage can be derived from eq 1,6,7,22 where Rctgold denotes the

Θ ) 1 - Rctgold/RctCD

(1)

charge transfer resistance of the pure gold electrode and RctCD the corresponding resistance of the passivated electrode covered by β-CD. Θ is the apparent surface coverage. Rctgold is calculated to 6 Ω cm2 using

Rctgold ) RT/zFi0

(2)

(21) Finklea, H. O.; Snider, D. A. , Fedyk, J.; Sabatini, E.; Gafni, Y.; Rubinstein, I. Langmuir 1993, 9, 3660-3667. (22) Sabatini, E.; Rubinstein, I. J. Phys. Chem. 1987, 91, 6663-6669.

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Figure 4. Impedance spectra of MPA-CD monolayers formed from a (MPA-CD)2 solution in the absence (0) and in the presence (4) of equal concentrations of [Fe(CN)6]3-/[Fe(CN)6]4- (1.6 mM in 100 mM NaOAc, pH 5.5). For comparison, the spectrum of a gold electrode after the same incubation time (1 day) in pure buffer solution (1.6 mM in 100 mM NaOAc, pH 5.5) is shown (O). The continuous lines represent the NLSQ fit according to Randle’s equivalent circuit. In the case of the CD monolayer, the Warburg part was neglected because of the high charge transfer resistance. For results, see Table 1.

with the exchange current

i0 ) zFAk0co(1-R)crR

(3)

where the electron transfer rate k0 at a bare gold electrode7 is 0.026 cm/s, co is the concentration of [Fe(CN)6]3-, and cr is the concentration of [Fe(CN)6]4-. A, R, T, and F denote as usual for the electrode area, the gas constant, the temperature, and the Faraday constant, respectively. The transfer coefficient R amounts to 0.5. Figure 4 shows impedance spectra of a chemisorbed monolayer of the β-cyclodextrin derivative after incubation of a fresh gold electrode with the (MPA-CD)2 solution for 1 day, taken in the absence and in the presence of [Fe(CN)6]3-/[Fe(CN)6]4marker ions. The charge transfer resistance arises below 10 s-1 and shows pure ohmic behavior. To show the effect of typical contamination of a gold surface in the absence of thiol molecules, an electrode was incubated in buffer for the same time and then measured in the presence of [Fe(CN)6]3-/[Fe(CN)6]4-. Although the capacitance decreases significantly, Rct does not change significantly to higher values compared to that for a clean gold surface. From the analysis of the spectra shown in Figure 4, we obtained charge transfer resistances of about 1500 Ω cm2 for the monolayer built from (MPA-CD)2 in the presence of 1.6 mM [Fe(CN)6]3-/[Fe(CN)6]4- in 100 mM NaOAc, pH 5.5. The corresponding electrode coverage Θ is 99.6%. We compared the charge transfer resistances and the coverages obtained with our β-CD thiol with chemisorbed alkanethiols exhibiting different packing densities arising from their differences in chain lengths (Table 1). The value for the transfer resistance of the modified β-cylodextrin monolayer ranges between the values of Θ for butanethiol and hexanethiol. The monolayer from the (MPA)2-CD solution exhibits a low value of Rct ) 90 Ω cm2, which seems to be reasonable if an extended hydrogen network between the CD molecules, neces-

Table 1. Charge Transfer Resistance Rct Obtained by Fitting |Z| (ν) with Randle’s Equivalent Circuit, Neglecting the Warburg Impedance for Cases of Very High Charge Transfer Resistances Which Occur in the Case of MPA-CD (Obtained from a (MPA-CD)2 Solution), Hexanethiol, Octanethiol, and Octadecanthiola Rct/Ω cm2 clean bare gold contaminated gold (MPA)2 (MPA-CD)2 (MPA)2-CD butanethiol hexanethiol octanethiol octadecanethiol

6 2 220 1500 90 660 2800 17000 86000

Θ (%) 0 b 99.60 c 99.10 99.79 99.96 99.99

C (µF/cm2) 30 6.7 8.5 9.0 7.2 6.7 3.7 2.4 1.0

a The concentration of [Fe(CN) ]3-/[Fe(CN) ]4- was 1.6 mM each. 6 6 The electrode coverage is denoted by Θ according to eq 1. b No coverage for the MPA monolayer is calculated because its charged state depends on the pH value. The value for Rct refers to pH 5.5. c The missing value of Θ arises from the fact that the CD monolayer consists of MPA and CD.

sary to get a defect-free monolayer, cannot be established. The Rct is even lower than the value obtained for the monolayer built up from a (MPA)2 solution, which can be explained in terms of a reduced repelling charge density on the surface in the case of the mixed monolayer of MPA-CD/MPA obtained from the (MPA)2-CD solution. Host-Guest Interactions Determined by Impedance Spectroscopy. It is known that the surface charge of a covered electrode influences its charge transfer resistance.23 From impedance spectroscopy using [Fe(CN)6]3-/[Fe(CN)6]4- marker ions, we thus expected an increase in Rct if negatively charged ligands in our system are incorporated into the chemisorbed β-CD monolayer due to repulsive interactions. To test the ability of our newly synthesized β-cyclodextrin derivative to form inclusion complexes, we have chosen two different anilinonaphthalenesulfonates, 2,6-TNS and 1,2-ANS, as guest molecules because of their well-known binding constants to cyclodextrins in solution. Although 2,6-TNS and 1,2-ANS have very similar chemical structures, Catena and Bright24 report a binding constant for TNS of 2500 L/mol, whereas 1,2-ANS does not bind to β-cyclodextrins in solution. The specific binding of the 2,6-TNS molecules to the β-CD-covered surface was determined by impedance spectroscopy using [Fe(CN)6]3-/[Fe(CN)6]4as marker ions again. The charge transfer resistance of the MPACD monolayer is increased by the binding of negatively charged guest molecules. Figure 5 shows the impedance spectra of a MPA-CD monolayer obtained from (MPA-CD)2 in the presence of 1.6 mM [Fe(CN)6]3-/[Fe(CN)6]4- taken before and after addition of 1,2-ANS, with a final concentration of 4.2 × 10-5 M in 100 mM NaOAc buffer at pH 5.5. The charge transfer resistance increases slightly from 1680 to 1890 Ω cm2. In contrast, the addition of 2,6-TNS to the MPA-CD monolayer at the same concentration results in a drastic increase of about 1560 up to 3240 Ω cm2 (Figure 5). It should also be noticed that the capacitance of the monolayer before and after addition of the guest molecules did not change (23) Nagase, S.; Kataoka, M.; Naganawa, R.; Komatsu, R.; Odashima, K.; Umezawa, Y. Anal. Chem. 1990, 62, 1252-1259. (24) Catena, G. C.; Bright, F. V. Anal. Chem. 1989, 61, 905-909.

Figure 5. Impedance spectra of a self-assembled MPA-CD monolayer obtained from a (MPA-CD)2 solution in the presence of 1.6 mM [Fe(CN)6]3-/[Fe(CN)6]4- in 100 mM NaOAc, pH 5.5 (0). An increasing charge transfer resistance reveals the repelling effect of the immobilized charge due to the attachment of 1,2-ANS (O) and 2,6-TNS (4). The continuous lines represent the NLSQ fits according to a parallel RC subcircuit and a resistor in series.

significantly. Only a small decrease of capacitance could be observed after addition of TNS. DISCUSSION Different functionalized β-cyclodextrins have been described in the literature that allow the formation of monolayers on an electrode surface.9,15,18,23 A prerequisite for a reliable use as a bioanalytical tool based on impedance spectroscopy is the formation of a monolayer. But it also has to be guaranteed that only a minor amount of defects leading to otherwise uncovered electrode surface areas is present. In the present paper, we introduced a novel β-CD-derivative that fullfills these demands. Kinetics of the MPA-CD Monolayer Formation Studied with Impedance Analysis. The results of our experiments concerning the time dependence of the chemisorption allow some qualitative conclusions about the adsorption process. First of all, it is interesting to note that the time necessary for monolayer formation increases in the order (MPA)2 < (MPA)2-CD < (MPACD)2, due to the increasing geometrical size of the molecules. The CD molecules require more space than the MPA, which may be responsible for the fact that the largest disulfide (MPA-CD)2 is adsorbed much more slowly on the gold surface than (MPA)2CD or dithiopropionic acid. Especially if monolayers are formed almost free of defects and form an interaction network that covers the complete surface, it remains to discuss whether the limiting factor of the kinetics of the self-assembly process relating to the recorded capacitances is the attachment of the disulfide to the gold surface itself or, in a second step, the arrangement of the adsorbed species on the electrode. Due to the large standard deviations of the fit in the time range of interest (e.g., Figure 2a and b), detailed kinetic information on the adsorption process is not available from our data. Characterization of the CD Monolayers by MALDI-MS and XPS. Our MALDI-MS analysis of the different CD monolayers yields completely new aspects of mass spectrometry performed on these organic films. On a normal stainless steel target, where the synthesized (MPA-CD)2 and the asymmetric (MPA)2-CD are only physisorbed, it is possible to detect the molecular peaks of Analytical Chemistry, Vol. 68, No. 18, September 15, 1996

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the disulfides with high intensities and small peaks of the monomeric thiol components. The additional peak of [(MPA-CD)2 + Na]+ obtained from the (MPA)2-CD solution (Figure 3a(2)) gives evidence of an impurity which was not detectable by TLC. The situation changes completely if gold surfaces are used (Figure 3b) with the CD derivatives chemisorbed on the target surface. The XPS analysis clearly showed that the disulfides are reduced at the surface and form a monolayer consisting of pure MPA-CD in the case of (MPA-CD)2 and a mixed monolayer of MPA-CD and MPA in the case of (MPA)2-CD. Due to the improved interaction with the surface by chemisorption, the intensity of the mass spectrum decreases drastically. However, the peak corresponding to MPA-CD is clearly visible. Surprisingly, we also observed a (MPA)2-CD peak (Figure 3b(2)), which is not easy to explain since only thiols and no disulfides are observable by XPS. The most important result of our mass spectrometric analysis is the drastic gain in intensity by displacement of film-forming molecules via addition of another thiol component to the solution of the matrix molecules. Mercaptopropionic acid added in excess to the DHB solution fulfilled the expectation. MPA-CD molecules chemisorbed onto gold are obviously replaced on the surface by MPA, and thus they are embedded into the matrix, allowing a better laser-induced desorption, which explains the recovered intensity. However, the situation at the surface seems to be more complicated. In both cases, (MPA-CD)2 and (MPA)2-CD, we observed not only the monomeric MPA-CD peak but also the disulfides (MPA)2-CD and (MPA-CD)2. We thus have to consider recombination processes at the surface after laser desorption/ ionization in the gas phase or directly at the surface. Although the mechanism of desorption is not yet clear, this new strategy of displacing the molecules under consideration by simply adding the displacer to the matrix solution may stimulate further applications in mass spectrometry of thin organic films. Characterization of the CD Monolayer with Impedance Spectroscopy. Capacitances of thin organic films strongly depend on the monolayer defectiveness. Equation 4 shows a simple relation between surface coverage and the measured capacitance,22 where Θ denotes the surface coverage, Cgold the

C ) ΘCCD + (1 - Θ)Cgold

(4)

capacitance of the pure gold electrode, and CCD the capacitance of a complete CD monolayer assuming 100% coverage. In general, capacitances are determined by the dielectric permittivity, the thickness of the adsorbed layer, and the packing density of the molecules. All those parameters have to be determined in more than one experiment. In addition to the capacitance C, independent measurements of the thickness and the electrode coverage are necessary to evaluate the dielectric permittivity of the adsorbed monolayers of β-CD derivatives. In our case, it seems to be appropriate to estimate the average thickness d of the adsorbed monolayer in a range of 20-25 Å from the geometrical size of the CD molecules and the length of the chemical bonds within the spacer. Starting with those values, only the coverage remains to be determined in a second experiment. We determined the surface coverage by impedance analysis, making use of the electroactive marker ions [Fe(CN)6]3-/[Fe(CN)6]4-. Those ions tend to penetrate through the defects of the monolayer; therefore, impedance measurements yield the information about the uncov3164

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Figure 6. Possible mechanism of electrode passivation caused by (a) the self-assembled CD monolayer itself, where penetration of the [Fe(CN)6]3-/[Fe(CN)6]4- ions occurs only through defects in the monolayer, (b) enhanced electrostatic repulsion caused by the specific host-guest interaction of 2,6-TNS with the hydrophobic cavity of the CD-molecules, and (c) the incomplete repulsion of the marker ions due to unspecific adsorption of 1,2-ANS, which is structurally very similar to 2,6-TNS but not capable of forming a strong complex with the β-cyclodextrin cavity.

ered surface fraction that could be derived from eq 1. With a calculated surface coverage of 99.6% and a measured final capacitance of 9 µF/cm2, the dielectric permittivity r is 20-25 using eq 5, where CCD is obtained from eq 4. This high value for

r ) dCCD/oA

(5)

r is reasonable, considering the hydrophilicity of the cyclodextrin molecule compared with that of the hydrophobic alkanethiols, with

r ) 2.1-2.5.25 One important result to discuss is the high packing density of the MPA-CD monolayers, with a coverage of 99.6%. This is close to the coverage obtained with hexanethiol, a compound that forms very close packed monolayers. Here, bulky polysaccharide rings obviously orient themselves in a way on the surface that prevents the marker ions from penetrating the surface layer. This result deserves attention, since Rojas et al. synthesized a β-cyclodextrin with seven thiol groups at the primary hydroxyl groups that also chemisorbed onto gold but led to imperfect monolayers in which a substantial fraction of gold surface remained uncovered. Rojas et al. assumed that missing favorable side-to-side interactions among the β-CD units and the inability of the molecules to undergo lateral displacement on the surface after chemisorption may be responsible for the formation of defects. They solved the problem by patching the defects with an alkanethiol. Our β-CD derivative, with only one thiol group terminating a medium-sized spacer instead of the one with seven thiol groups without spacers used by Rojas et al., obviously gives the cyclodextrins enough orientational freedom to self-assemble in a proper way, allowing a hydrogen bond network to form between the outer spheres. The single thiol group may also allow lateral displacement, leading to a high packing density after chemisorption. Host-Guest Interactions Determined by Impedance Analysis. The obtained close-packed monolayers of self-assembled β-CD derivatives gave us reason to assume that these molecules may serve as a basis for the measurement of specific inclusions into the CD cavity. Cyclic voltammetry as well as impedance spectroscopy offers the possibility to measure faradaic impedances with high precision. We have chosen the latter one to study the response of this analytical device consisting of chemisorbed CD monolayers to distinguish between a specific and a nonspecific interaction with guest molecules. Two anilinonaphthalenesulfonates were chosen, differing in their structural configuration. One is 2-(p-toluidinyl)naphthalene-6-sulfonic acid (2,6-TNS), which is known to bind to cyclodextrin with a KD of 2500 L/mol, and the other is 1-anilinonaphthalene-2-sulfonic acid, which is known not to bind to β-CD. It should be mentioned that both substances (25) Ullman, A. An introduction to ultrathin organic films, 1st ed.; Academic Press: San Diego, 1991. (26) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; Wiley: New York, 1980.

carry a negative charge and therefore form a charge barrier for anions at the surface if inclusion occurs. Specific adsorption of ions affects the redox reaction at the electrode.21 So, considering a negatively charged electrode surface, electroactive anions will be repelled, which increases the charge transfer resistance. This fact can be expressed using the Frumkin correction for the electron transfer rate,26

k0 ) k0,t e(Rz-n)FΦ2/RT

(6)

which shows the relation between the apparent transfer rate, k0, and the true rate, k0,t. z denotes the number of electrons per molecule oxidized or reduced, n the charge magnitude of each ion, and Φ2 the additional potential to the Galvani potential arising from charged surfaces. Inserting this equation in eq 3 for the exchange current, it could be easily shown that Φ2 controls the repulsion or attraction between the surface charges and the electroactive ions. In fact, a drastic increase in the charge transfer resistance Rct was observed after addition of 2,6-TNS, but not in the presence of 1,2-ANS. The small increase of Rct after addition of 1,2-ANS can be discussed as a nonspecific adsorption of the molecule on the CD monolayer. The possible mechanism of electrode passivation caused by the CD monolayer itself and by the analyzed molecules is summarized in Figure 6. ACKNOWLEDGMENT This work has been financially supported by the Bundesminister fu¨r Bildung und Forschung (BMBF). C.S. is a recepient of an award of the Graduiertenfo¨rderung des Landes Nordrhein Westfalen, A.J. is supported by an award of the Fonds der Chemischen Industrie, and G.S. is supported by a travel grant from the Deutsche Forschungsgemeinschaft (DFG). Support of the Degussa GmbH is gratefully acknowledged. We thank Dipl.Ing. W. Willenbrink for his professional support in electronics. Received for review December 19, 1995. Accepted April 24, 1996.X AC9512261 X

Abstract published in Advance ACS Abstracts, August 1, 1996.

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