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Monofunctionalized β-cyclodextrins as sensor elements for the detection of small molecules
Sensors and Actuators B 70 Ž2000. 243–253 www.elsevier.nlrlocatersensorb
Monofunctionalized b-cyclodextrins as sensor elements for the detection of small molecules Andreas Janshoff, Claudia Steinem, Axel Michalke, Christian Henke, Hans-Joachim Galla) Institut fur Wilhelms-UniÕersitat, Germany ¨ Biochemie, Westfalische ¨ ¨ Wilhelm-Klemm-Str. 2, D-48149 Munster, ¨ Received 14 October 1999; accepted 3 March 2000
Abstract b-Cyclodextrins functionalized by different moieties that were tethered to a single 6-deoxyaminoglucose unit were investigated with respect to their suitability for sensor applications. Derivatizing the cyclodextrins with hydrophobic moieties like dipalmitoylglycerol and cholesterol allowed us to study packing density and orientation of the cyclodextrin tori at the air–water interface. From the pressure-area isotherms, it was concluded that the cyclodextrins are positioned towards the water subphase, with their molecular axis predominately parallel to the interface. By introducing a disulfide group, we managed to immobilize cyclodextrins on gold surfaces via self-assembly. MALDI mass spectrometry ŽMALDI MS. and XPS confirmed that the molecules are chemisorbed on the gold substrate displaying high surface coverage as determined by means of impedance spectroscopy. The inclusion of various charged guest molecules was monitored by changes in the charge transfer resistance of the redox couple wFeŽCN. 6 x 3yrwFeŽCN. 6 x 4y. The charge transfer resistance is sensitive to the surface potential, which leads to either repulsion or attraction of the redox active species. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Host–guest interaction; Monolayer; Impedance spectroscopy; MALDI MS
1. Introduction The quantification of small molecules in aqueous solution by surface bound receptor molecules is an important goal in the research field of chemo- and biosensor development. Prerequisites to create devices suited to monitor the concentration of small molecules are an appropriate functionalization of the sensor surface and a sensitive transduction mechanism to detect the analytes in solution. Robust supramolecular structures exhibiting a distinct affinity for small molecules by forming inclusion complexes are among the most versatile chemical strategies towards small molecule detection. Aside from calixarenes and other cavity-forming supramolecular structures, naturally occurring cyclodextrins have gained widespread attention as affinity molecules for sensor development in the last decades w1–4x. Cyclodextrins are cyclic oligosaccharides composed of at least six a-D-glucose subunits forming a truncated cone. ) Corresponding author. Tel.: q49-251-83-33200; fax: q49-251-8333206. E-mail address: [email protected] ŽH.-J. Galla..
The primary hydroxyl groups are directed to the narrow side and the secondary hydroxyl groups are on the wide side of the torus, which leads to a hydrophobic interior and a hydrophilic outer surface. The hydrophobic cavity of the cyclodextrins is capable of forming inclusion complexes with small hydrophobic guest molecules. Binding specificity depends predominately on the guest size and geometry and is driven by noncovalent forces such as van der Waals forces, hydrogen bondings and hydrophobic interactions w5,6x. Prerequisites for efficiently utilizing cyclodextrins as sensor elements on surfaces are proper orientation of the molecules on one hand and facilitated accessibility of the hydrophobic cavity on the other hand. Attempts have been made by other groups to establish oriented monolayers of b-cyclodextrins on solid supports either by Langmuir– Blodgett technique w7x or via self-assembly w8–13x. In these studies, cyclodextrin derivatives were used, in which all primary hydroxyl groups are derivatized by an anchor for surface immobilization. Though the interaction with the surface is sufficiently strong due to seven contact points, the bulky torus of the cyclodextrin with its molecular axes perpendicular to the interface prevents the establishment of
0925-4005r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 0 0 5 Ž 0 0 . 0 0 5 7 6 - 1
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high packing density. We anticipated that a monofunctionalized cyclodextrin may solve the problem of packing deficiency by simultaneously immobilizing and orienting the molecules on surface. Restricting the functionalization to solely one anchor group renders the cyclodextrin more flexible, thus allowing the torus to fish for appropriate guest molecules in solution w14,15x. In this paper, we report on the synthesis of different monofunctionalized cyclodextrin derivatives in which the anchor is linked to one of the seven primary hydroxyl groups of the b-cyclodextrin. We investigated the packing behavior of derivatives with a hydrophobic anchor at the air–water interface to garner information about orientation and packing density of the molecules. For surface functionalization of gold surface, we synthesized a cyclodextrin derivative bearing a thiol anchor, enabling us to self-assemble those molecules on the electrical conducting surface. Impedance spectroscopy, MALDI mass spectrometry ŽMALDI MS. and XPS were employed to characterize the cyclodextrin films on surface. Since small molecule detection constitutes inherently a problem for most optical and piezoelectric devices in solution, we sought to find a way to quantify inclusion complexes by means of impedance analysis. We managed to quantify the binding of differently charged guest molecules by monitoring changes in the charge transfer resistance of the redox couple wFeŽCN. 6 x 3yr wFeŽCN. 6 x 4y. As the charge transfer resistance strongly depends on the surface potential, we were able to determine the binding constants of the guest molecules by utilizing a well-established electrostatic interaction model w16x, which allows quantification of the electrostatic repulsion or attraction occurring when guest molecules form inclusion complexes with surface-confined receptor molecules.
2. Experimentals 2.1. Materials b-Cyclodextrin was purchased from Roth ŽKarlsruhe, Germany., 3,3X-dithiobisŽpropionic acid-N-hydroxysuccinimide ester., 1-adamantanamine hydrochloride Ž1-ADHC. and 1-adamantanecarboxylic acid Ž1-ADC. were purchased from Fluka ŽBuchs, Switzerland.. Dicyclohexylcarbodiimide, 2,2X-dimercaptoethanol, mercaptopropionic acid, N-hydroxysuccinimide, succinic acidŽcholesteryl.ester-Nhydroxysuccinimide.ester, 1-anilino-naphthalene-2-sulfonate Ž1,2-ANS., 2-Ž p-toluidinyl.naphthalene-6-sulfonate Ž2,6-TNS., potassium ferricyanide ŽwFeŽCN. 6 x 3y ., potassium ferrocyanide ŽwFeŽCN. 6 x 4y ., sodium acetate ŽNaOAc. and cholesterol were from Sigma ŽDeisenhofen, Germany.. Dipalmitoylsuccinylglycerol and 1,2-dipalmitoyl-snglycero-3-phophocholine ŽDPPC. were from Avanti Polar Lipids ŽAlabaster, USA.. Water was first purified by a Millipore water purification system Milli Q RO 10 Plus
and then by Millipore ultrapure water system Milli Q Plus 185 Žspecific resistance: 18 M V.cm.. Gold used for the working electrodes was a generous gift from DEGUSSA ŽHanau, Germany.. Chromium was obtained from Bal Tec ŽBalzers, Liechtenstein.. TLC plates were aluminum sheets pre-coated with silica gel 60 F254 with 0.2-mm layer thickness from Merck ŽDarmstadt, Germany.. TLC eluent was EtOAcr2-propanolrH 2 0rconcentrated NH 4 OH Ž7:7:10:0.5.. The spots were visualized by spraying the TLC plates with sulfuric acid and subsequent charging. 2.2. Synthesis of cyclodextrin deriÕatiÕes All cyclodextrin derivatives were synthesized via mono6-deoxy-6-amino-b-cyclodextrin ŽScheme 1. as described elsewhere w14,17x. In summary, one of the seven primary hydroxyl groups of b-cyclodextrin was first tosylated using p-toluenesulfonyl chloride. For details about regioselective substitution of cyclodextrins, see Petter et al. w17x and Croft and Bartsch w18x. Substitution of the tosyl group by azide and subsequent reduction with triphenylphosphine yields mono-6-deoxy-6-amino-b-cyclodextrin. 2.2.1. Alkane acid-N-mono-6-deoxy-b-cyclodextrin amide (1a r 1b) A total of 1 mmol N-hydroxysuccinimide Ž115 mg. and 1 mmol alkane acid were suspended in 15 ml of ethylacetate. A solution of 1 mmol dicyclohexylcarbodiimide Ž206 mg. in 5 ml of ethylacetate was added dropwise to the suspension and the resulting solution was stirred for 4 h. Precipitated cyclohexyl urea was removed by filtration and the solvent of the filtrate removed by roto-evaporation. A 0.2 mmol of mono-6-deoxy-6-amino-b-cyclodextrin Ž227 mg. was dissolved in 5 ml dry DMF and 0.2 mmol of the activated alkane acid was added. The resulting mixture was stirred for 4 h. Subsequently, the total volume was reduced to 0.5 ml and 5 ml of bi-distilled water was added. The addition of acetone resulted in a white precipitate which was filtered, recrystallized from water and lyophilized. Myristic acid-mono-6-deoxy-b-cyclodextrin amide Ž1a.: yield: 96%, TLC, one spot, R f 0.53, positive ion MALDI MS mrz 1367 for wM q Naxq, calcd. ŽC 56 H 97 NO 35 . 1344. Arachidic acid-mono-6-deoxy-b-cyclodextrin amide Ž1b.: yield: 95%, TLC, one spot, R f 0.55, positive ion MALDI MS mrz 1451 for wM q Naxq, calcd. ŽC 62 H 109NO 35 . 1428. 2.2.2. Succinic acid (cholesteryl)ester-(N-mono-6-deoxyb-cyclodextrin) amide (2) A 0.176 mmol Ž200 mg. mono-6-deoxy-6-amino-bcyclodextrin was dissolved in 10 ml dry DMF. A solution of 100 mg Ž0.176 mmol. succinic acid-Žcholesteryl.esterN-hydroxy-succinimide.ester in 10 ml DMF was added and the solution was stirred for 24 h. Subsequently, the
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Scheme 1.
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solution was concentrated to 1 ml and 100 ml of acetone was added, resulting in a precipitate which was filtrated and dried, yielding 272 mg of 2 Ž96.5%.. TLC, one spot, R f 0.57, positive ion MALDI MS mrz 1626 for wM q Naxq, calcd. ŽC 73 H 119 NO 37 . 1603. 2.2.3. Succinic acid-(2,3-dipalmitoylglyceryl)ester-(Nmono-6-deoxy-b-cyclodextrin) amide (3) 0.15 mmol Ž100 mg. of the sodium salt of dipalmitoylsuccinylglycerol was dissolved in 16 ml methylene chloriderchloroform Ž1:1, vrv.. The solution was acidified by shaking with 20 ml of chloroform and 16 ml of 20 mM phosphatercitrate buffer at pH 5.5. The organic phase was separated and dried with sodium sulfate. After filtration, the solvent was evaporated yielding the free acid of dipalmitoylsuccinylglycerol Žatom absorption spectrometry: 97% exchange rate.. A total of 0.13 mmol Ž84 mg. of the free acid of dipalmitoylsuccinylglycerol were dissolved in a solution of 0.13 mmol Ž15 mg. of N-hydroxysuccinimide in 3.5 ml dry ethylacetatertoluene Ž1:2.5, vrv.. Subsequently, 0.13 mmol Ž26.8 mg. of dicyclohexyl carbodiimide were added and the solution was stirred for 24 h. After removing the precipitated dicyclohexyl urea by filtration, the solvent was removed by roto-evaporation yielding succinic acid-Ž2,3dipalmitoylglyceryl.ester-Ž N-hydroxysuccinimidyl.ester in quantitative yield. 0.13 mmol Ž147 mg. mono-6-deoxy-6-amino-b-cyclodextrin and 0.13 mmol Ž97 mg. succinic acid-Ž2,3-dipalmitoylglyceryl.ester- Ž N-hydroxysuccinimidyl .ester were dissolved in 3 ml dry DMF and stirred for 24 h. The solution was concentrated to 0.5 ml and 100 ml acetone was added. The precipitate was recovered by filtration, yielding 20 mg Ž88%. of 3. TLC, one spot, R f 0.57, positive MALDI MS mrz 1808 for wM q Naxq, calcd. ŽC 81 H 141 NO41 . 1785. 2.2.4. 3,3X-Dithiobis(propane-(N-mono-6-deoxy-b-cyclodextrin) amide (4) The synthesis of 4 has been described elsewhere w14x. Briefly, mono-6-deoxy-6-amino-b-cyclodextrin was reacted with 3,3X-dithiobisŽpropionic acid N-hydroxysuccinimide ester. to yield 99% of 4. 2.3. Film balance measurements Surface pressure-area isotherms were performed on a Teflon trough ŽRiegler–Kirstein film balance, MPI fur ¨ Kolloid-und Grenzflachenforschung, Golm, Germany. with ¨ an area of 144 cm2 equipped with a Wilhelmy system. The trough temperature was controlled with a water bath to T s 208C. After spreading the amphiphiles from a chloroformrmethanol Ž1:1, ÕrÕ . solution on the water subphase ŽMilliQ. using a 100-ml syringe, the solvent was allowed to evaporate for 15 min before compressing the monolayer with a velocity of 9 cm2rmin.
2.4. MALDI MS A time of flight mass spectrometer ŽTOF-MS. was used to separate the masses. Ionization was performed with a N2 laser with an emission wavelength of 327 nm. Light pulses of 3 ns were used, the acceleration voltage was 16 kV and the free drift path 1 m. Ion extraction was performed in one step using a grid. Data were collected with a storage oscilloscope ŽLeCroy DSO 9450A, Chesnut Ridge, USA.. The expected mass accuracy was 0.1%. 2.5. Impedance analysis AC impedance analysis was performed using an impedance gainrphase analyzer from Solartron Instruments ŽSI 1260, Great Britain.. All data were recorded without offset potentials at an AC amplitude of 30 mV. The magnitude of the impedance < ZŽ f .< and the phase angle F Ž f . between voltage and current were recorded in the frequency range from 10y1 to 10 6 Hz. Data analysis was performed by a nonlinear least square fit based on the Levenberg–Marquardt algorithm. Impedance spectra of the 3-mercaptopropionic acid ŽMPA.-CD monolayers were taken in a solution composed of 1.6 mM K 4 wFeŽCN. 6 x, 1.6 mM K 3wFeŽCN. 6 x, 100 mM NaOAc, pH 5.5 in the case of 1-ADHC and pH 8.0 in the case of 1-ADC, to ensure fully charged guest species. In order to follow the time course of the charge transfer resistance while adding guest molecules, the impedance of the system was recorded at a fixed frequency, which was individually determined before each experiment by taking impedance spectra covering the full frequency range.
3. Results and discussion 3.1. Monofunctionalized cyclodextrins at the air–water interface A prerequisite for the high sensitivity of a sensor element is a complete surface coverage with monolayers of highly oriented receptors. We hypothesized that, in particular, monofunctionalized cyclodextrins are capable of forming highly ordered receptor films in which each cyclodextrin maintains sufficient flexibility to form hydrogen bondings between the glucose subunits of neighboring molecules, giving rise to a stable monomolecular film. In order to gather information about the packing density and orientation of monofunctionalized cyclodextrins on surfaces, we first investigated cyclodextrin derivatives at the air–water interface, qualifying us to draw a model about the packing behavior of those molecules. Thus, we synthesized a variety of monofunctionalized cyclodextrins bearing a hydrophobic moiety linked to the amine function of one of the primary hydroxyl groups rendering the molecule amphiphilic ŽScheme 1..
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The first monofunctionalized cyclodextrins investigated at the air–water interface were fatty acid derivatives Žcompounds 1ar1b.. It turned out that independent of the length of the fatty acid linked to the cyclodextrin, no stable monolayer films were formed presumably due to the fact that the alkane chain of the fatty acid is flexible enough to interact with the hydrophobic cavity of the cyclodextrin leading to an intramolecular inclusion complex w19x. In order to prevent intramolecular inclusion, a cyclodextrin derivative with two fatty acid chains was synthesized Žcompound 3., in the following termed as DPG-CD. Because of the steric hindrance of the glycerolipid, the molecule suppresses the undesired formation of self-inclusion complexes and forms, indeed, very stable monolayer films at the air–water interface ŽFig. 1.. Several compression and expansion cycles of a neat DPG-CD monolayer up to a surface pressure of 50 mNrm could be performed without loss of material. At 60 mNrm, the film collapses. Isotherms of neat DPG-CD are characterized by two distinct regions of different compressibility. The first one ˚ 2rmolecule with high comprises the range of 140–100 A ˚ 2rmolecompressibility, the second one starts below 100 A cule, resulting in a rigid film with low compressibility. By extrapolating the area per molecule to zero pressure, the minimum area per molecule of the cyclodextrin derivative ˚ 2rmolecule. Multifunctionalcan be estimated to be 95 A ized cyclodextrins at the air–water interface exhibit a ˚ 2rmolecule, consistent typical area per molecule of 250 A with an orientation of the molecule axes perpendicular to the interface w20–23x. An area per molecule of 95
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˚ 2rmolecule can be explained in terms of CD-tori aligned A parallel to the air–water interface represented by a theoret˚ 2rmolecule. ical value for the area per molecule of 115 A An alternative interpretation, albeit unlikely, can be envisioned by a stacking of CD-molecules beneath the surface, therefore reducing the area per molecule by forming three-dimensional structures in the subphase. In certain applications, it may be advantageous to employ mixtures of CD derivatives and phospholipids since phospholipids are known to suppress nonspecific binding. As a natural component of the cellular membrane, phospholipids are the ideal matrix for receptor molecules such as DPG-CD. Since the structure and length of the hydrophobic part of DPG-CD is similar to DPPC, we also investigated mixtures of these compounds. Fig. 1 gives an overview of isotherms of different mixtures. Starting with neat DPPC, increasing amounts of DPG-CD lead to a fluidization of the monolayer film while at high a surface pressure, the area per molecule remains constant up to a ratio of 1:1 ŽDPG-CDrDPPC.. DPG-CD prevents DPPC to crystallize. Since the area per molecule does not increase up to a ratio of 1:1 ŽDPG-CDrDPPC. at a high surface pressure, it can be concluded that the cyclodextrins are positioned in the subphase, as depicted in the inset of Fig. 1, while the DPPC molecules interact with the hydrophobic fatty acids. Apparently, phase separation of DPPC and DPG-CD does not occur. At higher DPG-CD concentrations in the mixed films, the fatty acids cannot interact with each other because of the bulky cyclodextrin moieties leading to a fluidization of the monolayer.
Fig. 1. Surface pressure-area isotherms of mixtures of DPPC and DPG-CD. The isotherms were obtained at 208C on ultrapure water. From low to high area per molecule, the amount of DPG-CD was increased while the total number of molecules was kept constant. The area per molecule was related to pure DPPC. From the left to the right, the ratio of DPG-CDrDPPC is as follows: pure DPPC, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, pure DPG-CD. The inset shows a possible scenario of the packing behavior of a DPPCrDPG-CD mixture at different stages of the isotherm.
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A similar behavior at the air–water interface could be observed with a cholesterol derivative of cyclodextrin Žcompound 2., in the following termed as Ch-CD. An isotherm of pure Ch-CD is shown in Fig. 2. It is characterized by only one region with low compressibility. Extrapolation to zero pressure leads to an area per molecule of 105 ˚ 2 as in the case of DPG-CD indicative of cyclodextrin A moieties aligned parallel to the interface. The film collapses at around 45 mNrm, resulting in a loss of material in the subphase. In order to obtain more information about the influence of the hydrophobic moiety and the succinic acid spacer, we also investigated mixtures of neat cholesterol and Ch-CD. Up to a ratio of 7:4 ŽcholesterolrCh-CD., the isotherms exhibit the same area per molecule at high surface pressure, indicating that the cyclodextrin is positioned in the subphase and is not the space limiting element in the system Žsee inset of Fig. 2.. Since the new cyclodextrin derivatives with one flexible anchor form stable and oriented monolayers with new structural and orientational aspects compared to multifunctionalized cyclodextrins, we were interested in using these monolayer films as sensor elements on surfaces, in particular on gold surfaces, to apply electrochemical transduction methods for the detection of guest molecules. A simple and easy to follow strategy to immobilize molecules on gold surfaces is the well-known self-assembly technique of thiol compounds. Starting again with the amine-functionalized cyclodextrin, we synthesized compound 4, in the following termed as ŽCD-MPA. 2 , to immobilize CDs on
surface via gold sulfur bonds. We decided to invoke a shorter linker instead of a long hydrophobic anchor since the redox reaction used for the detection of small charged molecules relies on the distance between the adsorbed molecule and the surface. 3.2. Characterization of chemisorbed MPA-CD monolayers In order to garner information about the formation of an MPA-CD monolayer via self-assembly of ŽMPA-CD. 2 on clean gold surfaces, we first performed impedance analysis in the presence of the redox active couple wFeŽCN. 6 x 3yr wFeŽCN. 6 x 4y ŽFig. 3.. The obtained impedance spectra were fitted using Randle’s equivalent circuit Žshown in Fig. 3., giving access to three characteristic system parameters — the capacitance Cd of the interfacermonolayer, the charge transfer resistance R ct , which is indicative of the electrode coverage when uncharged molecules are adsorbed and the Warburg impedance s accounting for the mass transfer of the redox ions in solution. Self-assembly of an MPA-CD monolayer results in a very high charge transfer resistance Ž R ct s Ž8300 " 3300. V cm2 ., even higher than the resistance of a OH-terminated monolayer, composed of 11-mercaptoundecanol Ž11-MUD, R ct s Ž300 " 70. V cm2 .. We attribute the tremendous blocking efficiency of the redox reaction on surface to the formation of a well-packed MPA-CD-monolayer, which presumably establishes an intermolecular hydrogen bond-
Fig. 2. Surface pressure-area isotherms of mixtures of cholesterol and Ch-CD. The isotherms were obtained at 208C on ultrapure water. From low to high area per molecule, the amount of Ch-CD was increased while the total number of molecules was kept constant. The area per molecule was related to pure cholesterol. From the left to the right, the ratio of Ch-CDrcholesterol is as follows: pure cholesterol, 1:10, 2:9, 3:8, 4:7, 5:6, 6:5, 7:4, 8:3, 9:2, 10:1, pure Ch-CD. The inset shows a possible scenario of the packing behavior of a cholesterolrCh-CD mixture at different stages of the isotherm.
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Moreover, the chemisorption of MPA-CD was supported by MALDI MS. We compared ŽMPA-CD. 2 physisorbed on stainless steel with MPA-CD monolayers chemisorbed on gold targets by MALDI MS using 2,5 dihydroxybenzoic acid Ž2,5 DHB. as matrix. On stainless steel, the peak with highest intensity relates to the Kq ions of the disulfide ŽMPA-CD. 2 Ž mrz 2481., accompanied by small peaks arising from the presence of Naq and Kq ions of MPA-CD ŽFig. 4A.. However, a MALDI spectrum obtained from a chemisorbed MPA-CD-monolayer displays only one very weak signal at mrz 1245, which is consistent with wMPA-CDq Naxq ŽFig. 4B.. The low Fig. 3. Impedance spectra of an 11-MUD- ŽI. and an MPA-CD monolayer Ž^. on gold in 100 mM NaOAc, pH 5.5, 1.6 mM K 3 wFeŽCN. 6 xrK 4 wFeŽCN. 6 x. For comparison, a gold electrode Ž`. after 24 h in the same solution is shown. The continuous lines represent the results of fitting the parameters of the equivalent circuit to the corresponding spectra. In the case of the MPA-CD monolayer, the Warburg impedance element s was neglected.
ing network. The Warburg impedance dominates the impedance at low frequencies in the impedance spectrum of the 11-MUD monolayer while s can be neglected in the case of MPA-CD monolayer due to its prevailing charge transfer resistance. From the monolayer’s R ct and the one of a pure gold electrode, the electrode coverage can be calculated using an expression first described by Sabatani and Rubinstein w24x. We calculated a surface coverage of 99.9% for the MPA-CD- and 98% for the 11-MUD monolayer. Time-dependent impedance analysis during the formation process of an MPA-CD monolayer using 1 mM ŽMPA-CD. 2 in the absence of redox active species revealed that the self-assembly was completed after approximately 10 h, indicated by an invariant impedance spectrum displaying a typical monolayer capacitance of 9 mFrcm2 . In order to ensure that a possible rearrangement of the molecules on surface is finished before starting the experiments, we incubated the gold surfaces in general for at least 20 h. Notably, the dielectric constant of a MPA-CD monolayer is considerably higher than that of an alkanethiol Ž ´ s 2., resulting in a larger capacitance. A coverage of 99.9% and capacitance of 9 mFrcm2 can be translated in a dielectric constant of 25, assuming a monolayer thickness of 2.5 nm, which is reasonable considering that the molecule contains several polar hydroxyl groups. To further corroborate the chemisorption of MPA-CD monolayers on gold, we performed XPS analysis of the gold sulfur bond. The XPS data exhibited two weak peaks in the sulfur 2p region with maxima at 161.8 eV Ž2p 3r2 . and 163.1 eV Ž2p1r2 .. The peaks confirm the presence of Au`S bonds which are considerably different from those of the disulfide at 163.7 eV Ž2p 3r2 . and 164.9 eV Ž2p1r2 . w25,26x.
Fig. 4. ŽA. MALDI MS spectrum of ŽMPA-CD. 2 on stainless steel targets. A total of 500 fmol of the sample was loaded on the surface. The spectrum was accumulated from seven laser shots Ž337 nm., 2,5 DHB as matrix. ŽB. MALDI MS spectrum of a self-assembled monolayer of MPA-CD on gold covered targets. After the self-assembly process was finished, 2,5 DHB was added. The spectrum was accumulated from 61 single laser shots Ž337 nm.. ŽC. MALDI MS spectrum of self-assembled MPA-CD with a subsequent addition of 2,5 DHB and MPA, accumulated from seven single laser shots Ž337 nm..
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peak intensity is probably due to the improved interaction between the molecules and the surface by chemisorption. Thus, a dramatic increase in peak intensity was observed when MPA was added to the 2,5 DHB matrix ŽFig. 4C.. We assume that MPA is capable of displacing chemisorbed cyclodextrin molecules from the surface so that the released MPA-CD becomes readily detectable by MALDI MS. Two additional peaks have been observed arising from ŽMPA-CD. 2 and ŽMPA. 2-CD Ž mrz 1350.. These species might have been formed by recombination processes before, while or after laser desorptionrionization of the molecules due to the higher concentration of MPA-CD and the excess of MPA in the matrix. In order to prove the hypothesis of cyclodextrin displacement by small thiol compounds, we performed an experiment in which we added MPA to an MPA-CD monolayer while taking
impedance spectra in the presence of the redox active couple wFeŽCN. 6 x3yrwFeŽCN. 6 x 4y. Fig. 5A shows the obtained results indicating that the addition of MPA leads to a dramatic decrease of R ct within minutes, which can be explained in terms of cyclodextrin replacement on the surface. Though slower, the same result was obtained by using the neutral disulfide compound 2,2 X -dimercaptoethanol ŽFig. 5B.. 3.3. Quantification of guest molecules by surface-bound cyclodextrins The major goal of this study was the detection of small molecules Ž M - 2000 grmol. in aqueous solution by the surface-bound monofunctionalized cyclodextrins. By determining the charge transfer resistance of the MPA-CD
Fig. 5. ŽA. Time-dependent impedance spectra of an MPA-CD monolayer on gold in 100 mM KCl, 1.6 mM K 3 wFeŽCN. 6 xrK 4 wFeŽCN. 6 x after addition of 1 mM MPA. From top to the bottom: t s 0, 1, 10, 200 min. The inset depicts the change in R ct vs. time. ŽB. Time-dependent impedance spectra of an X MPA-CD monolayer on gold in 100 mM KCl, 1.6 mM K 3 wFeŽCN. 6 xrK 4 wFeŽCN. 6 x after addition of 1 mM 2,2 dimercaptoethanol. From top to the bottom: t s 0, 1, 18, 38, 71, 148, 330 min. The inset depicts the change in R ct vs. time. The continuous lines represent the results of fitting the parameters of the equivalent circuit shown in Fig. 1 to the corresponding spectra.
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Fig. 6. ŽA. Charge transfer resistance of the electron transfer of the redox couple wFeŽCN . 6 x 3yr wFeŽCN . 6 x 4y of an MPA-CD monolayer chemisorbed on gold measured at 0.22 Hz over time. Each step corresponds to the addition of guest molecules Ž1-ADHC.. ŽB. Charge transfer resistance vs. 1-ADHC concentration in solution. Because of the positive charge of 1-ADHC, the negative charges of the redox couple get attracted, increasing the redox reaction on surface. ŽC. Adsorption isotherm of 1-ADHC on an MPA-CD monolayer as the surface charge density vs. concentration of 1-ADHC. The continuous line is the result of fitting an adsorption isotherm taking electrostatic repulsion into account to the data. Only the first part is fitted Žseparated by a dotted line.. The results of the fitting procedures are given in Table 1.
monolayer by impedance spectroscopy in the presence of the redox active species wFeŽCN. 6 x 3yrwFeŽCN. 6 x 4y, we were able to quantify the host–guest interaction of four different guest molecules: two adamantane derivatives, one positively charged Ž1-ADHC. and one negatively charged Ž1-ADC., at the appropriate pH values and two naphthalene derivatives, one which does not fit into the cavity of b-cyclodextrin due to steric hindrance Ž1,2-ANS. and one which binds strongly to the cavity of cyclodextrin Ž2,6TNS. w27x. Adamantane as well as naphthalene derivatives are among the strongest binding guest molecules for bcyclodextrins as revealed by molecular modeling and thermodynamic studies w28–30x. Adamantane derivatives have been used quite frequently as model systems for inclusion complexes. The binding constant for 1-ADC in solution is
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determined to be 33,000 My1 ŽpH 8.3. and 2000 My1 for 1-ADHC ŽpH 6.5. w31x. Since the p K-values are 4.75 for 1-ADC and 9.25 for 1-ADHC, respectively, 99.94% of 1-ADC is negatively charged and 99.98% of 1-ADHC is positively charged. To follow the time course of the charge transfer resistance with considerably higher resolution while adding the guest molecule, the impedance of the system was recorded at a fixed frequency which was individually determined before each experiment by taking complete impedance spectra. In all experiments, the charge transfer resistance of an MPA-CD monolayer was most well-defined in a frequency range of 0.1–1 Hz, indicated by a constant magnitude of the impedance Ž< ZŽ f .< s R ct q R e .. Therefore, it is justified to choose a value from the plateau region and refer to it as the charge transfer resistance unless the spectrum changes dramatically. To prevent a misinterpretation, we checked the spectrum before and after each experiment. Each addition of the positively charged 1-ADHC led to a considerable decrease in R ct ŽFig. 6A., consistent with an attraction of the negatively charged redox couple to the surface. In contrast, the addition of the negatively charged 1-ADC results in a repulsion of the redox couple leading to an increase in R ct w16x. From the obtained charge transfer resistance at any given guest concentration ŽFig. 6B., the surface charge density can be calculated according to Michalke et al. w15x, leading to the plot depicted in Fig. 6C for the adsorption of 1-ADHC. The resulting adsorption isotherms exhibit a two-stage adsorption process, in which the first part is assumed to be specific adsorption while at higher guest concentrations, nonspecific binding occurs. By assuming an adsorption isotherm accounting for the repulsion of charged molecules on surface w32–35x, the first part of the isotherm can be fitted, leading to binding constants which are summarized in Table 1 for the four different guest molecules. 0.068 Crm2 is the maximum charge density of closely packed Žcubic symmetry. CD-molecules assuming a 1:1 host–guest stoichiometry with a net charge of one. Basically, the difference between the calculated and observed value for smax ŽTable 1. can be explained in terms of a reduced influence of the guest molecules on the charge transfer resistance in the case of a defect-driven charge transfer mechanism. Supposing a Debye-length of 1 nm, the interaction radius of the included charges of the
Table 1 Parameters of the adsorption isotherms. The theoretical maximum surface charge density is 68 mCrm2 Guest molecule
K a ŽMy1 . on surface
K a ŽMy1 . in solution w31x
smax ŽmCrm2 .
1-ADHC 1-ADC 2,6-TNS 1,2-ANS
1.3=10 4 0.24=10 4 1.9=10 4 n.b.
0.22=10 4 ŽpH 6.5. 3.30=10 4 ŽpH 8.3. 0.25=10 4 ŽpH 6.0. n.b.
11.0 2.7 2.3 –
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adamantane derivatives with the redox couple is less pronounced in the vicinity of a defect-hole. Therefore, the Frumkin effect is systematically underestimated, yielding in lower apparent charge densities. Another explanation is the limited accessibility of the cavities due to the skewed packing of CD-molecules on surface. Thus, not every binding pocket can be occupied by a guest molecule. It remains to be elucidated which of the explanation applies and how the system can be improved to enhance the signal to noise ratio. In summary, the sensing principle relies on the alteration of the Faradaic current due to changes of the surface charge density. Negatively charged redox active molecules are repelled from the surface if the overall charge is negative and get attracted if the charge is positive. Hence, the surface charge influences the charge transfer resistance. In general, the charge transfer through self-assembled monolayers is based on defects and tunneling of electrons through the barrier formed by the monolayer. Both mechanisms are affected by variations of the charge density. These findings go back to a study of Frumkin who investigated the influence of the surface potential on the charge transfer rates w16x.
4. Conclusions Noncovalent ligand receptor interactions prevail in nature and therefore dominate today’s sensor chemistry. Molecular recognition of low molecular weight ligands plays an important role in pharmacological research, signal transduction and enzymatic transformations. Many toxic substances exhibit a molecular mass well below 2000 grmol. Since most sensitive transducers recognize changes either in mass or refractive index, they are restricted to a molecular weight regime of ) 2000 grmol. Optical methods based on fluorescence spectroscopy are limited to labeled species. We were able to present a strategy based on monofunctionalized cyclodextrins immobilized on metal electrodes. Displaying a highly oriented and densely packed monolayer, we managed to detect small charged guest molecules by means of impedance analysis. Changes in the charge transfer resistance could be attributed to variation in the surface potential due to additional charges.
Acknowledgements This work has been supported by the Deutsche Forschungsgemeinschaft as a contribution from the SFB 424 Žproject B2.. A. Janshoff is supported by a DFGHabilitationsstipendium and C. Steinem by a Lise Meitner fellowship.
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Monofunctionalized b-cyclodextrins as sensor elements for the detection of small molecules Andreas Janshoff, Claudia Steinem, Axel Michalke, Christian Henke, Hans-Joachim Galla) Institut fur Wilhelms-UniÕersitat, Germany ¨ Biochemie, Westfalische ¨ ¨ Wilhelm-Klemm-Str. 2, D-48149 Munster, ¨ Received 14 October 1999; accepted 3 March 2000
Abstract b-Cyclodextrins functionalized by different moieties that were tethered to a single 6-deoxyaminoglucose unit were investigated with respect to their suitability for sensor applications. Derivatizing the cyclodextrins with hydrophobic moieties like dipalmitoylglycerol and cholesterol allowed us to study packing density and orientation of the cyclodextrin tori at the air–water interface. From the pressure-area isotherms, it was concluded that the cyclodextrins are positioned towards the water subphase, with their molecular axis predominately parallel to the interface. By introducing a disulfide group, we managed to immobilize cyclodextrins on gold surfaces via self-assembly. MALDI mass spectrometry ŽMALDI MS. and XPS confirmed that the molecules are chemisorbed on the gold substrate displaying high surface coverage as determined by means of impedance spectroscopy. The inclusion of various charged guest molecules was monitored by changes in the charge transfer resistance of the redox couple wFeŽCN. 6 x 3yrwFeŽCN. 6 x 4y. The charge transfer resistance is sensitive to the surface potential, which leads to either repulsion or attraction of the redox active species. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Host–guest interaction; Monolayer; Impedance spectroscopy; MALDI MS
1. Introduction The quantification of small molecules in aqueous solution by surface bound receptor molecules is an important goal in the research field of chemo- and biosensor development. Prerequisites to create devices suited to monitor the concentration of small molecules are an appropriate functionalization of the sensor surface and a sensitive transduction mechanism to detect the analytes in solution. Robust supramolecular structures exhibiting a distinct affinity for small molecules by forming inclusion complexes are among the most versatile chemical strategies towards small molecule detection. Aside from calixarenes and other cavity-forming supramolecular structures, naturally occurring cyclodextrins have gained widespread attention as affinity molecules for sensor development in the last decades w1–4x. Cyclodextrins are cyclic oligosaccharides composed of at least six a-D-glucose subunits forming a truncated cone. ) Corresponding author. Tel.: q49-251-83-33200; fax: q49-251-8333206. E-mail address: [email protected] ŽH.-J. Galla..
The primary hydroxyl groups are directed to the narrow side and the secondary hydroxyl groups are on the wide side of the torus, which leads to a hydrophobic interior and a hydrophilic outer surface. The hydrophobic cavity of the cyclodextrins is capable of forming inclusion complexes with small hydrophobic guest molecules. Binding specificity depends predominately on the guest size and geometry and is driven by noncovalent forces such as van der Waals forces, hydrogen bondings and hydrophobic interactions w5,6x. Prerequisites for efficiently utilizing cyclodextrins as sensor elements on surfaces are proper orientation of the molecules on one hand and facilitated accessibility of the hydrophobic cavity on the other hand. Attempts have been made by other groups to establish oriented monolayers of b-cyclodextrins on solid supports either by Langmuir– Blodgett technique w7x or via self-assembly w8–13x. In these studies, cyclodextrin derivatives were used, in which all primary hydroxyl groups are derivatized by an anchor for surface immobilization. Though the interaction with the surface is sufficiently strong due to seven contact points, the bulky torus of the cyclodextrin with its molecular axes perpendicular to the interface prevents the establishment of
0925-4005r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 0 0 5 Ž 0 0 . 0 0 5 7 6 - 1
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high packing density. We anticipated that a monofunctionalized cyclodextrin may solve the problem of packing deficiency by simultaneously immobilizing and orienting the molecules on surface. Restricting the functionalization to solely one anchor group renders the cyclodextrin more flexible, thus allowing the torus to fish for appropriate guest molecules in solution w14,15x. In this paper, we report on the synthesis of different monofunctionalized cyclodextrin derivatives in which the anchor is linked to one of the seven primary hydroxyl groups of the b-cyclodextrin. We investigated the packing behavior of derivatives with a hydrophobic anchor at the air–water interface to garner information about orientation and packing density of the molecules. For surface functionalization of gold surface, we synthesized a cyclodextrin derivative bearing a thiol anchor, enabling us to self-assemble those molecules on the electrical conducting surface. Impedance spectroscopy, MALDI mass spectrometry ŽMALDI MS. and XPS were employed to characterize the cyclodextrin films on surface. Since small molecule detection constitutes inherently a problem for most optical and piezoelectric devices in solution, we sought to find a way to quantify inclusion complexes by means of impedance analysis. We managed to quantify the binding of differently charged guest molecules by monitoring changes in the charge transfer resistance of the redox couple wFeŽCN. 6 x 3yr wFeŽCN. 6 x 4y. As the charge transfer resistance strongly depends on the surface potential, we were able to determine the binding constants of the guest molecules by utilizing a well-established electrostatic interaction model w16x, which allows quantification of the electrostatic repulsion or attraction occurring when guest molecules form inclusion complexes with surface-confined receptor molecules.
2. Experimentals 2.1. Materials b-Cyclodextrin was purchased from Roth ŽKarlsruhe, Germany., 3,3X-dithiobisŽpropionic acid-N-hydroxysuccinimide ester., 1-adamantanamine hydrochloride Ž1-ADHC. and 1-adamantanecarboxylic acid Ž1-ADC. were purchased from Fluka ŽBuchs, Switzerland.. Dicyclohexylcarbodiimide, 2,2X-dimercaptoethanol, mercaptopropionic acid, N-hydroxysuccinimide, succinic acidŽcholesteryl.ester-Nhydroxysuccinimide.ester, 1-anilino-naphthalene-2-sulfonate Ž1,2-ANS., 2-Ž p-toluidinyl.naphthalene-6-sulfonate Ž2,6-TNS., potassium ferricyanide ŽwFeŽCN. 6 x 3y ., potassium ferrocyanide ŽwFeŽCN. 6 x 4y ., sodium acetate ŽNaOAc. and cholesterol were from Sigma ŽDeisenhofen, Germany.. Dipalmitoylsuccinylglycerol and 1,2-dipalmitoyl-snglycero-3-phophocholine ŽDPPC. were from Avanti Polar Lipids ŽAlabaster, USA.. Water was first purified by a Millipore water purification system Milli Q RO 10 Plus
and then by Millipore ultrapure water system Milli Q Plus 185 Žspecific resistance: 18 M V.cm.. Gold used for the working electrodes was a generous gift from DEGUSSA ŽHanau, Germany.. Chromium was obtained from Bal Tec ŽBalzers, Liechtenstein.. TLC plates were aluminum sheets pre-coated with silica gel 60 F254 with 0.2-mm layer thickness from Merck ŽDarmstadt, Germany.. TLC eluent was EtOAcr2-propanolrH 2 0rconcentrated NH 4 OH Ž7:7:10:0.5.. The spots were visualized by spraying the TLC plates with sulfuric acid and subsequent charging. 2.2. Synthesis of cyclodextrin deriÕatiÕes All cyclodextrin derivatives were synthesized via mono6-deoxy-6-amino-b-cyclodextrin ŽScheme 1. as described elsewhere w14,17x. In summary, one of the seven primary hydroxyl groups of b-cyclodextrin was first tosylated using p-toluenesulfonyl chloride. For details about regioselective substitution of cyclodextrins, see Petter et al. w17x and Croft and Bartsch w18x. Substitution of the tosyl group by azide and subsequent reduction with triphenylphosphine yields mono-6-deoxy-6-amino-b-cyclodextrin. 2.2.1. Alkane acid-N-mono-6-deoxy-b-cyclodextrin amide (1a r 1b) A total of 1 mmol N-hydroxysuccinimide Ž115 mg. and 1 mmol alkane acid were suspended in 15 ml of ethylacetate. A solution of 1 mmol dicyclohexylcarbodiimide Ž206 mg. in 5 ml of ethylacetate was added dropwise to the suspension and the resulting solution was stirred for 4 h. Precipitated cyclohexyl urea was removed by filtration and the solvent of the filtrate removed by roto-evaporation. A 0.2 mmol of mono-6-deoxy-6-amino-b-cyclodextrin Ž227 mg. was dissolved in 5 ml dry DMF and 0.2 mmol of the activated alkane acid was added. The resulting mixture was stirred for 4 h. Subsequently, the total volume was reduced to 0.5 ml and 5 ml of bi-distilled water was added. The addition of acetone resulted in a white precipitate which was filtered, recrystallized from water and lyophilized. Myristic acid-mono-6-deoxy-b-cyclodextrin amide Ž1a.: yield: 96%, TLC, one spot, R f 0.53, positive ion MALDI MS mrz 1367 for wM q Naxq, calcd. ŽC 56 H 97 NO 35 . 1344. Arachidic acid-mono-6-deoxy-b-cyclodextrin amide Ž1b.: yield: 95%, TLC, one spot, R f 0.55, positive ion MALDI MS mrz 1451 for wM q Naxq, calcd. ŽC 62 H 109NO 35 . 1428. 2.2.2. Succinic acid (cholesteryl)ester-(N-mono-6-deoxyb-cyclodextrin) amide (2) A 0.176 mmol Ž200 mg. mono-6-deoxy-6-amino-bcyclodextrin was dissolved in 10 ml dry DMF. A solution of 100 mg Ž0.176 mmol. succinic acid-Žcholesteryl.esterN-hydroxy-succinimide.ester in 10 ml DMF was added and the solution was stirred for 24 h. Subsequently, the
A. Janshoff et al.r Sensors and Actuators B 70 (2000) 243–253
Scheme 1.
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solution was concentrated to 1 ml and 100 ml of acetone was added, resulting in a precipitate which was filtrated and dried, yielding 272 mg of 2 Ž96.5%.. TLC, one spot, R f 0.57, positive ion MALDI MS mrz 1626 for wM q Naxq, calcd. ŽC 73 H 119 NO 37 . 1603. 2.2.3. Succinic acid-(2,3-dipalmitoylglyceryl)ester-(Nmono-6-deoxy-b-cyclodextrin) amide (3) 0.15 mmol Ž100 mg. of the sodium salt of dipalmitoylsuccinylglycerol was dissolved in 16 ml methylene chloriderchloroform Ž1:1, vrv.. The solution was acidified by shaking with 20 ml of chloroform and 16 ml of 20 mM phosphatercitrate buffer at pH 5.5. The organic phase was separated and dried with sodium sulfate. After filtration, the solvent was evaporated yielding the free acid of dipalmitoylsuccinylglycerol Žatom absorption spectrometry: 97% exchange rate.. A total of 0.13 mmol Ž84 mg. of the free acid of dipalmitoylsuccinylglycerol were dissolved in a solution of 0.13 mmol Ž15 mg. of N-hydroxysuccinimide in 3.5 ml dry ethylacetatertoluene Ž1:2.5, vrv.. Subsequently, 0.13 mmol Ž26.8 mg. of dicyclohexyl carbodiimide were added and the solution was stirred for 24 h. After removing the precipitated dicyclohexyl urea by filtration, the solvent was removed by roto-evaporation yielding succinic acid-Ž2,3dipalmitoylglyceryl.ester-Ž N-hydroxysuccinimidyl.ester in quantitative yield. 0.13 mmol Ž147 mg. mono-6-deoxy-6-amino-b-cyclodextrin and 0.13 mmol Ž97 mg. succinic acid-Ž2,3-dipalmitoylglyceryl.ester- Ž N-hydroxysuccinimidyl .ester were dissolved in 3 ml dry DMF and stirred for 24 h. The solution was concentrated to 0.5 ml and 100 ml acetone was added. The precipitate was recovered by filtration, yielding 20 mg Ž88%. of 3. TLC, one spot, R f 0.57, positive MALDI MS mrz 1808 for wM q Naxq, calcd. ŽC 81 H 141 NO41 . 1785. 2.2.4. 3,3X-Dithiobis(propane-(N-mono-6-deoxy-b-cyclodextrin) amide (4) The synthesis of 4 has been described elsewhere w14x. Briefly, mono-6-deoxy-6-amino-b-cyclodextrin was reacted with 3,3X-dithiobisŽpropionic acid N-hydroxysuccinimide ester. to yield 99% of 4. 2.3. Film balance measurements Surface pressure-area isotherms were performed on a Teflon trough ŽRiegler–Kirstein film balance, MPI fur ¨ Kolloid-und Grenzflachenforschung, Golm, Germany. with ¨ an area of 144 cm2 equipped with a Wilhelmy system. The trough temperature was controlled with a water bath to T s 208C. After spreading the amphiphiles from a chloroformrmethanol Ž1:1, ÕrÕ . solution on the water subphase ŽMilliQ. using a 100-ml syringe, the solvent was allowed to evaporate for 15 min before compressing the monolayer with a velocity of 9 cm2rmin.
2.4. MALDI MS A time of flight mass spectrometer ŽTOF-MS. was used to separate the masses. Ionization was performed with a N2 laser with an emission wavelength of 327 nm. Light pulses of 3 ns were used, the acceleration voltage was 16 kV and the free drift path 1 m. Ion extraction was performed in one step using a grid. Data were collected with a storage oscilloscope ŽLeCroy DSO 9450A, Chesnut Ridge, USA.. The expected mass accuracy was 0.1%. 2.5. Impedance analysis AC impedance analysis was performed using an impedance gainrphase analyzer from Solartron Instruments ŽSI 1260, Great Britain.. All data were recorded without offset potentials at an AC amplitude of 30 mV. The magnitude of the impedance < ZŽ f .< and the phase angle F Ž f . between voltage and current were recorded in the frequency range from 10y1 to 10 6 Hz. Data analysis was performed by a nonlinear least square fit based on the Levenberg–Marquardt algorithm. Impedance spectra of the 3-mercaptopropionic acid ŽMPA.-CD monolayers were taken in a solution composed of 1.6 mM K 4 wFeŽCN. 6 x, 1.6 mM K 3wFeŽCN. 6 x, 100 mM NaOAc, pH 5.5 in the case of 1-ADHC and pH 8.0 in the case of 1-ADC, to ensure fully charged guest species. In order to follow the time course of the charge transfer resistance while adding guest molecules, the impedance of the system was recorded at a fixed frequency, which was individually determined before each experiment by taking impedance spectra covering the full frequency range.
3. Results and discussion 3.1. Monofunctionalized cyclodextrins at the air–water interface A prerequisite for the high sensitivity of a sensor element is a complete surface coverage with monolayers of highly oriented receptors. We hypothesized that, in particular, monofunctionalized cyclodextrins are capable of forming highly ordered receptor films in which each cyclodextrin maintains sufficient flexibility to form hydrogen bondings between the glucose subunits of neighboring molecules, giving rise to a stable monomolecular film. In order to gather information about the packing density and orientation of monofunctionalized cyclodextrins on surfaces, we first investigated cyclodextrin derivatives at the air–water interface, qualifying us to draw a model about the packing behavior of those molecules. Thus, we synthesized a variety of monofunctionalized cyclodextrins bearing a hydrophobic moiety linked to the amine function of one of the primary hydroxyl groups rendering the molecule amphiphilic ŽScheme 1..
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The first monofunctionalized cyclodextrins investigated at the air–water interface were fatty acid derivatives Žcompounds 1ar1b.. It turned out that independent of the length of the fatty acid linked to the cyclodextrin, no stable monolayer films were formed presumably due to the fact that the alkane chain of the fatty acid is flexible enough to interact with the hydrophobic cavity of the cyclodextrin leading to an intramolecular inclusion complex w19x. In order to prevent intramolecular inclusion, a cyclodextrin derivative with two fatty acid chains was synthesized Žcompound 3., in the following termed as DPG-CD. Because of the steric hindrance of the glycerolipid, the molecule suppresses the undesired formation of self-inclusion complexes and forms, indeed, very stable monolayer films at the air–water interface ŽFig. 1.. Several compression and expansion cycles of a neat DPG-CD monolayer up to a surface pressure of 50 mNrm could be performed without loss of material. At 60 mNrm, the film collapses. Isotherms of neat DPG-CD are characterized by two distinct regions of different compressibility. The first one ˚ 2rmolecule with high comprises the range of 140–100 A ˚ 2rmolecompressibility, the second one starts below 100 A cule, resulting in a rigid film with low compressibility. By extrapolating the area per molecule to zero pressure, the minimum area per molecule of the cyclodextrin derivative ˚ 2rmolecule. Multifunctionalcan be estimated to be 95 A ized cyclodextrins at the air–water interface exhibit a ˚ 2rmolecule, consistent typical area per molecule of 250 A with an orientation of the molecule axes perpendicular to the interface w20–23x. An area per molecule of 95
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˚ 2rmolecule can be explained in terms of CD-tori aligned A parallel to the air–water interface represented by a theoret˚ 2rmolecule. ical value for the area per molecule of 115 A An alternative interpretation, albeit unlikely, can be envisioned by a stacking of CD-molecules beneath the surface, therefore reducing the area per molecule by forming three-dimensional structures in the subphase. In certain applications, it may be advantageous to employ mixtures of CD derivatives and phospholipids since phospholipids are known to suppress nonspecific binding. As a natural component of the cellular membrane, phospholipids are the ideal matrix for receptor molecules such as DPG-CD. Since the structure and length of the hydrophobic part of DPG-CD is similar to DPPC, we also investigated mixtures of these compounds. Fig. 1 gives an overview of isotherms of different mixtures. Starting with neat DPPC, increasing amounts of DPG-CD lead to a fluidization of the monolayer film while at high a surface pressure, the area per molecule remains constant up to a ratio of 1:1 ŽDPG-CDrDPPC.. DPG-CD prevents DPPC to crystallize. Since the area per molecule does not increase up to a ratio of 1:1 ŽDPG-CDrDPPC. at a high surface pressure, it can be concluded that the cyclodextrins are positioned in the subphase, as depicted in the inset of Fig. 1, while the DPPC molecules interact with the hydrophobic fatty acids. Apparently, phase separation of DPPC and DPG-CD does not occur. At higher DPG-CD concentrations in the mixed films, the fatty acids cannot interact with each other because of the bulky cyclodextrin moieties leading to a fluidization of the monolayer.
Fig. 1. Surface pressure-area isotherms of mixtures of DPPC and DPG-CD. The isotherms were obtained at 208C on ultrapure water. From low to high area per molecule, the amount of DPG-CD was increased while the total number of molecules was kept constant. The area per molecule was related to pure DPPC. From the left to the right, the ratio of DPG-CDrDPPC is as follows: pure DPPC, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, pure DPG-CD. The inset shows a possible scenario of the packing behavior of a DPPCrDPG-CD mixture at different stages of the isotherm.
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A similar behavior at the air–water interface could be observed with a cholesterol derivative of cyclodextrin Žcompound 2., in the following termed as Ch-CD. An isotherm of pure Ch-CD is shown in Fig. 2. It is characterized by only one region with low compressibility. Extrapolation to zero pressure leads to an area per molecule of 105 ˚ 2 as in the case of DPG-CD indicative of cyclodextrin A moieties aligned parallel to the interface. The film collapses at around 45 mNrm, resulting in a loss of material in the subphase. In order to obtain more information about the influence of the hydrophobic moiety and the succinic acid spacer, we also investigated mixtures of neat cholesterol and Ch-CD. Up to a ratio of 7:4 ŽcholesterolrCh-CD., the isotherms exhibit the same area per molecule at high surface pressure, indicating that the cyclodextrin is positioned in the subphase and is not the space limiting element in the system Žsee inset of Fig. 2.. Since the new cyclodextrin derivatives with one flexible anchor form stable and oriented monolayers with new structural and orientational aspects compared to multifunctionalized cyclodextrins, we were interested in using these monolayer films as sensor elements on surfaces, in particular on gold surfaces, to apply electrochemical transduction methods for the detection of guest molecules. A simple and easy to follow strategy to immobilize molecules on gold surfaces is the well-known self-assembly technique of thiol compounds. Starting again with the amine-functionalized cyclodextrin, we synthesized compound 4, in the following termed as ŽCD-MPA. 2 , to immobilize CDs on
surface via gold sulfur bonds. We decided to invoke a shorter linker instead of a long hydrophobic anchor since the redox reaction used for the detection of small charged molecules relies on the distance between the adsorbed molecule and the surface. 3.2. Characterization of chemisorbed MPA-CD monolayers In order to garner information about the formation of an MPA-CD monolayer via self-assembly of ŽMPA-CD. 2 on clean gold surfaces, we first performed impedance analysis in the presence of the redox active couple wFeŽCN. 6 x 3yr wFeŽCN. 6 x 4y ŽFig. 3.. The obtained impedance spectra were fitted using Randle’s equivalent circuit Žshown in Fig. 3., giving access to three characteristic system parameters — the capacitance Cd of the interfacermonolayer, the charge transfer resistance R ct , which is indicative of the electrode coverage when uncharged molecules are adsorbed and the Warburg impedance s accounting for the mass transfer of the redox ions in solution. Self-assembly of an MPA-CD monolayer results in a very high charge transfer resistance Ž R ct s Ž8300 " 3300. V cm2 ., even higher than the resistance of a OH-terminated monolayer, composed of 11-mercaptoundecanol Ž11-MUD, R ct s Ž300 " 70. V cm2 .. We attribute the tremendous blocking efficiency of the redox reaction on surface to the formation of a well-packed MPA-CD-monolayer, which presumably establishes an intermolecular hydrogen bond-
Fig. 2. Surface pressure-area isotherms of mixtures of cholesterol and Ch-CD. The isotherms were obtained at 208C on ultrapure water. From low to high area per molecule, the amount of Ch-CD was increased while the total number of molecules was kept constant. The area per molecule was related to pure cholesterol. From the left to the right, the ratio of Ch-CDrcholesterol is as follows: pure cholesterol, 1:10, 2:9, 3:8, 4:7, 5:6, 6:5, 7:4, 8:3, 9:2, 10:1, pure Ch-CD. The inset shows a possible scenario of the packing behavior of a cholesterolrCh-CD mixture at different stages of the isotherm.
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Moreover, the chemisorption of MPA-CD was supported by MALDI MS. We compared ŽMPA-CD. 2 physisorbed on stainless steel with MPA-CD monolayers chemisorbed on gold targets by MALDI MS using 2,5 dihydroxybenzoic acid Ž2,5 DHB. as matrix. On stainless steel, the peak with highest intensity relates to the Kq ions of the disulfide ŽMPA-CD. 2 Ž mrz 2481., accompanied by small peaks arising from the presence of Naq and Kq ions of MPA-CD ŽFig. 4A.. However, a MALDI spectrum obtained from a chemisorbed MPA-CD-monolayer displays only one very weak signal at mrz 1245, which is consistent with wMPA-CDq Naxq ŽFig. 4B.. The low Fig. 3. Impedance spectra of an 11-MUD- ŽI. and an MPA-CD monolayer Ž^. on gold in 100 mM NaOAc, pH 5.5, 1.6 mM K 3 wFeŽCN. 6 xrK 4 wFeŽCN. 6 x. For comparison, a gold electrode Ž`. after 24 h in the same solution is shown. The continuous lines represent the results of fitting the parameters of the equivalent circuit to the corresponding spectra. In the case of the MPA-CD monolayer, the Warburg impedance element s was neglected.
ing network. The Warburg impedance dominates the impedance at low frequencies in the impedance spectrum of the 11-MUD monolayer while s can be neglected in the case of MPA-CD monolayer due to its prevailing charge transfer resistance. From the monolayer’s R ct and the one of a pure gold electrode, the electrode coverage can be calculated using an expression first described by Sabatani and Rubinstein w24x. We calculated a surface coverage of 99.9% for the MPA-CD- and 98% for the 11-MUD monolayer. Time-dependent impedance analysis during the formation process of an MPA-CD monolayer using 1 mM ŽMPA-CD. 2 in the absence of redox active species revealed that the self-assembly was completed after approximately 10 h, indicated by an invariant impedance spectrum displaying a typical monolayer capacitance of 9 mFrcm2 . In order to ensure that a possible rearrangement of the molecules on surface is finished before starting the experiments, we incubated the gold surfaces in general for at least 20 h. Notably, the dielectric constant of a MPA-CD monolayer is considerably higher than that of an alkanethiol Ž ´ s 2., resulting in a larger capacitance. A coverage of 99.9% and capacitance of 9 mFrcm2 can be translated in a dielectric constant of 25, assuming a monolayer thickness of 2.5 nm, which is reasonable considering that the molecule contains several polar hydroxyl groups. To further corroborate the chemisorption of MPA-CD monolayers on gold, we performed XPS analysis of the gold sulfur bond. The XPS data exhibited two weak peaks in the sulfur 2p region with maxima at 161.8 eV Ž2p 3r2 . and 163.1 eV Ž2p1r2 .. The peaks confirm the presence of Au`S bonds which are considerably different from those of the disulfide at 163.7 eV Ž2p 3r2 . and 164.9 eV Ž2p1r2 . w25,26x.
Fig. 4. ŽA. MALDI MS spectrum of ŽMPA-CD. 2 on stainless steel targets. A total of 500 fmol of the sample was loaded on the surface. The spectrum was accumulated from seven laser shots Ž337 nm., 2,5 DHB as matrix. ŽB. MALDI MS spectrum of a self-assembled monolayer of MPA-CD on gold covered targets. After the self-assembly process was finished, 2,5 DHB was added. The spectrum was accumulated from 61 single laser shots Ž337 nm.. ŽC. MALDI MS spectrum of self-assembled MPA-CD with a subsequent addition of 2,5 DHB and MPA, accumulated from seven single laser shots Ž337 nm..
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peak intensity is probably due to the improved interaction between the molecules and the surface by chemisorption. Thus, a dramatic increase in peak intensity was observed when MPA was added to the 2,5 DHB matrix ŽFig. 4C.. We assume that MPA is capable of displacing chemisorbed cyclodextrin molecules from the surface so that the released MPA-CD becomes readily detectable by MALDI MS. Two additional peaks have been observed arising from ŽMPA-CD. 2 and ŽMPA. 2-CD Ž mrz 1350.. These species might have been formed by recombination processes before, while or after laser desorptionrionization of the molecules due to the higher concentration of MPA-CD and the excess of MPA in the matrix. In order to prove the hypothesis of cyclodextrin displacement by small thiol compounds, we performed an experiment in which we added MPA to an MPA-CD monolayer while taking
impedance spectra in the presence of the redox active couple wFeŽCN. 6 x3yrwFeŽCN. 6 x 4y. Fig. 5A shows the obtained results indicating that the addition of MPA leads to a dramatic decrease of R ct within minutes, which can be explained in terms of cyclodextrin replacement on the surface. Though slower, the same result was obtained by using the neutral disulfide compound 2,2 X -dimercaptoethanol ŽFig. 5B.. 3.3. Quantification of guest molecules by surface-bound cyclodextrins The major goal of this study was the detection of small molecules Ž M - 2000 grmol. in aqueous solution by the surface-bound monofunctionalized cyclodextrins. By determining the charge transfer resistance of the MPA-CD
Fig. 5. ŽA. Time-dependent impedance spectra of an MPA-CD monolayer on gold in 100 mM KCl, 1.6 mM K 3 wFeŽCN. 6 xrK 4 wFeŽCN. 6 x after addition of 1 mM MPA. From top to the bottom: t s 0, 1, 10, 200 min. The inset depicts the change in R ct vs. time. ŽB. Time-dependent impedance spectra of an X MPA-CD monolayer on gold in 100 mM KCl, 1.6 mM K 3 wFeŽCN. 6 xrK 4 wFeŽCN. 6 x after addition of 1 mM 2,2 dimercaptoethanol. From top to the bottom: t s 0, 1, 18, 38, 71, 148, 330 min. The inset depicts the change in R ct vs. time. The continuous lines represent the results of fitting the parameters of the equivalent circuit shown in Fig. 1 to the corresponding spectra.
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Fig. 6. ŽA. Charge transfer resistance of the electron transfer of the redox couple wFeŽCN . 6 x 3yr wFeŽCN . 6 x 4y of an MPA-CD monolayer chemisorbed on gold measured at 0.22 Hz over time. Each step corresponds to the addition of guest molecules Ž1-ADHC.. ŽB. Charge transfer resistance vs. 1-ADHC concentration in solution. Because of the positive charge of 1-ADHC, the negative charges of the redox couple get attracted, increasing the redox reaction on surface. ŽC. Adsorption isotherm of 1-ADHC on an MPA-CD monolayer as the surface charge density vs. concentration of 1-ADHC. The continuous line is the result of fitting an adsorption isotherm taking electrostatic repulsion into account to the data. Only the first part is fitted Žseparated by a dotted line.. The results of the fitting procedures are given in Table 1.
monolayer by impedance spectroscopy in the presence of the redox active species wFeŽCN. 6 x 3yrwFeŽCN. 6 x 4y, we were able to quantify the host–guest interaction of four different guest molecules: two adamantane derivatives, one positively charged Ž1-ADHC. and one negatively charged Ž1-ADC., at the appropriate pH values and two naphthalene derivatives, one which does not fit into the cavity of b-cyclodextrin due to steric hindrance Ž1,2-ANS. and one which binds strongly to the cavity of cyclodextrin Ž2,6TNS. w27x. Adamantane as well as naphthalene derivatives are among the strongest binding guest molecules for bcyclodextrins as revealed by molecular modeling and thermodynamic studies w28–30x. Adamantane derivatives have been used quite frequently as model systems for inclusion complexes. The binding constant for 1-ADC in solution is
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determined to be 33,000 My1 ŽpH 8.3. and 2000 My1 for 1-ADHC ŽpH 6.5. w31x. Since the p K-values are 4.75 for 1-ADC and 9.25 for 1-ADHC, respectively, 99.94% of 1-ADC is negatively charged and 99.98% of 1-ADHC is positively charged. To follow the time course of the charge transfer resistance with considerably higher resolution while adding the guest molecule, the impedance of the system was recorded at a fixed frequency which was individually determined before each experiment by taking complete impedance spectra. In all experiments, the charge transfer resistance of an MPA-CD monolayer was most well-defined in a frequency range of 0.1–1 Hz, indicated by a constant magnitude of the impedance Ž< ZŽ f .< s R ct q R e .. Therefore, it is justified to choose a value from the plateau region and refer to it as the charge transfer resistance unless the spectrum changes dramatically. To prevent a misinterpretation, we checked the spectrum before and after each experiment. Each addition of the positively charged 1-ADHC led to a considerable decrease in R ct ŽFig. 6A., consistent with an attraction of the negatively charged redox couple to the surface. In contrast, the addition of the negatively charged 1-ADC results in a repulsion of the redox couple leading to an increase in R ct w16x. From the obtained charge transfer resistance at any given guest concentration ŽFig. 6B., the surface charge density can be calculated according to Michalke et al. w15x, leading to the plot depicted in Fig. 6C for the adsorption of 1-ADHC. The resulting adsorption isotherms exhibit a two-stage adsorption process, in which the first part is assumed to be specific adsorption while at higher guest concentrations, nonspecific binding occurs. By assuming an adsorption isotherm accounting for the repulsion of charged molecules on surface w32–35x, the first part of the isotherm can be fitted, leading to binding constants which are summarized in Table 1 for the four different guest molecules. 0.068 Crm2 is the maximum charge density of closely packed Žcubic symmetry. CD-molecules assuming a 1:1 host–guest stoichiometry with a net charge of one. Basically, the difference between the calculated and observed value for smax ŽTable 1. can be explained in terms of a reduced influence of the guest molecules on the charge transfer resistance in the case of a defect-driven charge transfer mechanism. Supposing a Debye-length of 1 nm, the interaction radius of the included charges of the
Table 1 Parameters of the adsorption isotherms. The theoretical maximum surface charge density is 68 mCrm2 Guest molecule
K a ŽMy1 . on surface
K a ŽMy1 . in solution w31x
smax ŽmCrm2 .
1-ADHC 1-ADC 2,6-TNS 1,2-ANS
1.3=10 4 0.24=10 4 1.9=10 4 n.b.
0.22=10 4 ŽpH 6.5. 3.30=10 4 ŽpH 8.3. 0.25=10 4 ŽpH 6.0. n.b.
11.0 2.7 2.3 –
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adamantane derivatives with the redox couple is less pronounced in the vicinity of a defect-hole. Therefore, the Frumkin effect is systematically underestimated, yielding in lower apparent charge densities. Another explanation is the limited accessibility of the cavities due to the skewed packing of CD-molecules on surface. Thus, not every binding pocket can be occupied by a guest molecule. It remains to be elucidated which of the explanation applies and how the system can be improved to enhance the signal to noise ratio. In summary, the sensing principle relies on the alteration of the Faradaic current due to changes of the surface charge density. Negatively charged redox active molecules are repelled from the surface if the overall charge is negative and get attracted if the charge is positive. Hence, the surface charge influences the charge transfer resistance. In general, the charge transfer through self-assembled monolayers is based on defects and tunneling of electrons through the barrier formed by the monolayer. Both mechanisms are affected by variations of the charge density. These findings go back to a study of Frumkin who investigated the influence of the surface potential on the charge transfer rates w16x.
4. Conclusions Noncovalent ligand receptor interactions prevail in nature and therefore dominate today’s sensor chemistry. Molecular recognition of low molecular weight ligands plays an important role in pharmacological research, signal transduction and enzymatic transformations. Many toxic substances exhibit a molecular mass well below 2000 grmol. Since most sensitive transducers recognize changes either in mass or refractive index, they are restricted to a molecular weight regime of ) 2000 grmol. Optical methods based on fluorescence spectroscopy are limited to labeled species. We were able to present a strategy based on monofunctionalized cyclodextrins immobilized on metal electrodes. Displaying a highly oriented and densely packed monolayer, we managed to detect small charged guest molecules by means of impedance analysis. Changes in the charge transfer resistance could be attributed to variation in the surface potential due to additional charges.
Acknowledgements This work has been supported by the Deutsche Forschungsgemeinschaft as a contribution from the SFB 424 Žproject B2.. A. Janshoff is supported by a DFGHabilitationsstipendium and C. Steinem by a Lise Meitner fellowship.
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