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Correspondence Anal. Chem. 1996, 68, 402-407
Consecutive Selective Adsorption of Pentamidine and Phosphate Biomolecules on a Self-Assembled Layer: Reversible Formation of a Chemically Selective Coating Bo 1 rje Sellergren,* Aleksander Swietlow,† Thomas Arnebrant,‡ and Klaus Unger
Department of Inorganic and Analytical Chemistry, Johannes Gutenberg University, J. J. Becherweg 24, D-55099 Mainz, Germany
In situ ellipsometric film thickness measurements, FTIR external reflectance spectroscopy, and potentiometric measurements indicated that the amphiphile pentamidine (PAM), a bisbenzamidine, associated by self-assembly with a preformed self-assembled monolayer of a mercaptoalkanoic acid on gold. The structural properties of PAM and the nature of the substrate were ideal for the formation of a densely packed monolayer. This process was fully reversible, as demonstrated by changing the pH of the surrounding medium. Thus, disassembly-reassembly occurred when the pH was cycled between 8.7 and 3. The bilayer structure, featuring a positively charged amidinium surface, was subsequently used for selective adsorption of phosphate biomolecules. Thus, selective binding of adenosine and inositol phosphates (Ka ) 5 × 104 M-1 for ATP) to the surface, as well as strong binding of DNA-oligonucleotides, was monitored by in situ ellipsometry. This system suggests a new approach to chemical sensing, based on the reversible formation of a chemically selective coating. We report an example of a chemically selective coating, reversibly formed by pH-controlled self-assembly of a bolaamphiphile.1 Based on functional group complementarity and layer order, analytes are selectively adsorbed onto the amphiphilemodified surface. This approach to chemical sensing allows repeated use of the substrate in a number of adsorption experiments. Molecular recognition and functional group complementarity are essential in the design and preparation of chemical or biological sensors2,3and analyte-selective chromatographic sup† Permanent address: Pharmacia Consumer Pharma, Box 941, 251 09 Helsingborg, Sweden. ‡ Permanent address: Department of Food Technology, University of Lund, P.O. Box 124, Lund, Sweden. (1) A preliminary account of this research was presented at the ESF meeting: Molecular Recognition from Biology to Materials. 100 years of the Lock and Key concept; Mainz, Germany, August 1994.
ports.4 In this context, organic thin films,5 formed by molecular self-assembly (SA), have been extensively studied.3 The structural order attainable in such films and their simple fabrication allows surface properties to be fine tuned and interfaces to be designed for selective analyte binding as well as signal transduction.3,6-8 The selective coatings are often irreversibly anchored to the sensor interface, preventing regeneration of the coating. In the case of strongly bound analytes, this may limit the reusability of the surface. Chemically selective coatings that can be reversibly attached to a sensor interface would, in this context, be of interest. This paper describes the reversible and selective formation of self-assembled monolayers (SAMs) of pentamidine (PAM), a DNA minor groove binding bisbenzamidine,9 and subsequent on-layer selective adsorption of phosphate biomolecules. The choice of substrate as well as the target analytes is made considering simple functional group complementarity and bulk-phase interactions between the analyte and the amphiphile. Thus, based on our previous experience with molecular imprinting of PAM,10 our initial studies focused on the interactions between the drug and a carboxylic acid-functionalized surface. Because of the inherent benefits associated with SAMs of chemisorbed thiols on gold (2) Biosensors and Chemical Sensors; Edelman, P. G., Wang, J., Eds.; ACS Symposium Series 487; American Chemical Society: Washington, DC, 1992. (3) Interfacial Design and Chemical Sensing; Mallouk, T. E., Harrison, D. J., Eds.; ACS Symposium Series 561; American Chemical Society: Washington DC, 1994. (4) Highly Selective Separations in Biotechnology; Street, G., Ed.; Chapman and Hall: London, 1993. (5) Ulman, A. An Introduction to Ultrathin Organic Films from LangmuirBlodgett to Self-assembly; Academic Press, Inc.: New York, 1991. (6) Sasaki, D. Y.; Kurihara, K.; Kunitake, T. J. Am. Chem. Soc. 1991, 113, 9685-9686. (7) (a) Rubinstein, I.; Steinberg, S.; Tor, Y.; Shanzer, A.; Sagiv, J. Nature 1988, 332, 426-429. (b) Kepley, L. J.; Crooks, R. M.; Ricco, A. J. Anal. Chem. 1992, 64, 3191-3193. (c) Schierbaum, K. D.; Weiss, T.; Thoden van Velzen, E. U.; Engbersen, J. F. J.; Reinhoudt, D. N.; Go ¨pel, W. Science 1994, 265, 1413-1415. (8) Mu ¨ ller, W.; Ringsdorf, H.; Rump, E.; Wildburg, G.; Zhang, X.; Angermaier, L.; Knoll, W.; Liley, M.; Spinke, J. Science 1993, 262, 1706-1708. (9) Tidwell, R. R.; Jones, S. K.; Geratz, J. D.; Ohemeng, K. A.; Cory, M.; Hall, J. E. J. Med. Chem. 1990, 33, 1252-1257. (10) Sellergren, B. Anal. Chem. 1994, 66, 1578.
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Figure 1. Ellipsometric thickness (d) measured on a COOH surface (Au-COOH) and a silicon dioxide (SiO2) surface upon addition of incremental amounts of PAM at pH 8.7. Also shown is the associated potentiometric response on the COOH surface. The maximum length of PAM has been indicated. Linear regions of Eadie-Scatchard plots18 of the film thickness and concentration data gave the following binding constants (Ka) and limiting film thicknesses (dmax): Au-COOH, Ka ) (1.9 ( 4) × 105 M-1, dmax ) 21 ( 0.1 Å (results of four experiments using two identically prepared surfaces); SiO2, Ka ) (3.1 ( 0.2) × 104 M-1, dmax ) 6.1 ( 0.1 Å (results of two experiments using the same surface. Note that the error bars are smaller than the size of the symbols). The limiting thickness of PAM on a bare gold surface was dmax ) 4.4 ( 1.4 Å.
surfaces11 (stability, order, ease of preparation), we decided to initially use SAMs of mercaptoalkanoic acids on gold. All processes resulted in thickness changes that were easily monitored by in situ ellipsometry. In this report, we emphasize the aspects of selectivity, affinity, and reversibility in each adsorption step. A more detailed investigation of the layer structure and amphiphile structural criteria for layer formation will be published elsewhere.12 EXPERIMENTAL SECTION Chemicals. The adenosine phosphates (ATP, ADP, AMP; Sigma), octylguanidine hemisulfate (Aldrich), decamethylenebis[guanidine] (Kodak), 5,5′-(1,3-propanediyl)bis[isothiouronium bromide] (Aldrich), pentamidine isethionate (gift from Rhone Poulenc Rorer), the inositol phosphates (IP2, IP3, IP4; gifts from Perstorp AB), absolute ethanol (Merck), and boric acid (Merck) were used as received. The water was purified by double distillation. Ethamidine9 and mercaptoundecanoic acid11d were synthesized as described elsewhere. The pH was adjusted with 0.1 M HCl or 0.1 M NaOH. Substrate Preparation. The gold surfaces were prepared by vapor deposition of gold (2000 Å thickness) onto glass or silicon wafers containing adhesive layers (300 Å) of chromium or titanium. These were then cleaned by brief immersion (15 s) in freshly prepared piranha solution (Caution: Piranha solution, 1:3 H2O2 (30%)/H2SO4 (conc), reacts violently with many organic materials and should not be stored), followed by rinsing in water and ethanol. The carboxylic acid-functionalized surface (COOH (11) (a) Whitesides, G. M.; Ferguson, G. S.; Allara, D.; Scherson, D.; Speaker, L.; Ulman, A. Crit. Rev. Surf. Chem. 1993, 3, 49-65. (b) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559-3568. (c) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 4481-4483. (d) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7164. (12) Sellergren, B.; et al. In preparation.
surface) was then prepared by immersing the gold surface in a 1 mM solution of mercaptoundecanoic acid in ethanol for at least 12 h, followed by rinsing with ethanol and drying under a nitrogen stream. Adsorption Experiments. The surfaces were washed consecutively in ethanol, water, 0.1 M HCl, 0.1 M NaOH, and water and immersed in a quartz ellipsometric cuvette containing 5 mL of sodium borate buffer (0.01 M, pH 8.7, prepared from boric acid), thermostated to 25 °C and equipped with a small magnetic stirrer and a pH electrode. Adsorption of compounds was then monitored in situ by ellipsometry13,14 (Rudolph thin-film ellipsometer 43603-200E using an angle of incidence of 68° and a HeNe laser light source, λ ) 633 nm) until a stable value was obtained (usually within 50-200 s). Incremental amounts of compound (2.5 mM stock solutions) were added, and the film thickness (d) (additional to the COOH monolayer) was calculated, assuming a film refractive index of 1.45.11 A change in d of ∼10% was observed when this value was increased or decreased by 0.05. The surfaces were restored by adjusting pH to 2-3 with 0.1 M HCl and then reused after washing with 0.1 M NaOH and water. Reproducibility was regularly checked by PAM-ATP addition to the same surface over a period of several months. The film thicknesses measured in solution agreed with those measured on the corresponding surfaces dried under nitrogen after each addition. Potential Measurements. The cell consisted of the COOH surface (working electrode) partly immersed in the buffer and a Ag/AgCl reference electrode in 3 M KCl solution, connected via an agar-agar gel electrolytic bridge with the buffer solution. Potential changes upon addition of PAM were recorded using a simple pH/mV meter connected to a pen recorder. (13) (a) Arnebrant, T.; Nylander, T. J. Colloid. Interface Sci. 1986, 111, 529. (b) Wahlgren, M.; Arnebrant, T. J. Colloid. Interface Sci. 1991, 142, 503. (14) Azzam, R. M.; Bashara, N. M. Ellipsometry and Polarized Light; NorthHolland: Amsterdam, 1977.
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Figure 2. Simplified scheme depicting the consecutive adsorption of PAM and ATP on a SAM of mercaptoundecanoic acid on gold, together with the layer thicknesses measured by ellipsometry. No claim is made that the structure shown represents the one obtained after adsorption.
FT-IR External Reflection Spectroscopy (FT-IR ERS). Measurements were made in the p-polarized mode using a Nicolet 5DXC instrument equipped with a liquid nitrogen-cooled detector, operating at a resolution of 4 cm-1, and a grazing angle reflectance accessory (Spectratech FT80) with an angle of incidence of 80°. The surfaces for the IR measurements were prepared by drying the modified surface obtained after an adsorption experiment under a stream of nitrogen, followed by vacuum. No washing step was applied, with one exception, the mercaptoundecanoic acid surface. The latter was washed prior to measurement with a 0.01 M borate solution at pH 2. Each spectrum is the sum of 5000 scans on the modified surfaces using an unreacted, cleaned gold substrate as reference.
Chart 1
RESULTS AND DISCUSSION Upon adding PAM to a SAM of mercaptoundecanoic acid on gold (COOH surface) at pH 8.7, we anticipated that attraction between the negatively charged surface15 and the positively charged drug16 would result in the formation of stable hydrogenbonded amidinium-carboxylate ion pairs.17 By investigating this process using in situ ellipsometry,13,14 providing estimates of film thicknesses, information was gained regarding the orientational (15) (a) Wang, J.; Frostman, L. M.; Ward, M. D. J. Phys. Chem. 1992, 96, 52245228. (b) Lee, R. T.; Carey, R. I.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1994, 10, 741-749. (16) pKa (benzamidine) ) 11.6. See: Albert, A.; Goldacre, R.; Philips, J. J. Chem. Soc. 1948, 2240.
preference of the drug relative to the surface. In Figure 1, the adsorption isotherms of PAM on various surfaces are shown, together with the calculated limiting film thicknesses and associa(17) (a) Walker, J. J. Chem. Soc., Chem. Commun. 1949, 1996-2002. (b) Hosseini, M. W.; Schaeffer, P.; De Cian, A.; Kyritsakas, N.; Fischer, J. J. Chem. Soc., Chem. Commun. 1994, 2135.
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Figure 3. Infrared external reflectance spectra of gold surfaces modified consecutively with (a) mercaptoundecanoic acid (acid form), (b) mercaptoundecanoic acid-PAM, and (c) mercaptoundecanoic acid-PAM-ATP as described in the Experimental Section. In spectrum a, the band at 1700 cm-1 is attributed to the CdO stretch and the bands at 1400-1500 cm-1 to a combination of CsH bending modes and the CsO stretch. In spectrum b, the band at 1685 cm-1 is assigned to the CsN stretch and the sharp bands at 1611 and 1490 cm-1 to the benzamidine ring breathing. At 1611 cm-1, this is superimposed on a broad band probably arising from the COOasymmetric stretch. The band at 1266 cm-1 is assigned to the CO ether vibration. In spectrum c, the 1685 cm-1 band has increased in strength, presumably due to addition of NH bending modes of ATP. The multiple broad bands at 1000-1250 cm-1 is assigned to POand POC- vibrations of the phosphate groups.21 The switch in the relative band heights at 1685 and 1611 cm-1 upon ATP adsorption remains to be investigated.
tion constants.18 The agreement between the observed thickness increase upon addition of the drug and the maximum length of the molecule (21 Å, Chart 1)19 suggests that the drug forms a closely packed monolayer on the COOH monolayer surface. As depicted in Figure 2, this process would transform the originally negatively charged COOH surface to a positively charged amidinium surface. FT-IR external reflectance spectra of the PAMmodified COOH surface showed CH stretching vibrations at 2851 and 2926 cm-1, strong broad bands at 3000-3600 cm-1 arising from the OH and NH stretch, and strong bands characteristic of PAM in the low-frequency region (Figure 3).20 The position and intensity of these bands indicate that PAM binds to the surface by hydrogen bonding6 with a disordered structure21,11b of the detectable alkyl chains. There is other supporting evidence for a self-assembly process. First, the thickness measured for the COOH monolayer (13 ( 2 Å) agreed with the expected thickness,11 indicating a certain reliability of the ellipsometric data. Second, upon repeating the PAM experiment using a silicon dioxide containing ionizable silanol groups or a bare gold surface, only small layer thicknesses and weak binding were observed (Figure 1). Moreover, among several studied basic amphiphiles or bolaamphiphiles (Chart 1), only bisbenzamidines gave large (18) Connors, K. A. Binding Constants; Wiley: New York, 1987. (19) HN-HN′ distance based on molecular modeling of the bis-charged drug using the AMBER force field and known structural information. See: Lowe, P. R.; Sansom, C. E.; Schwalbe, C. H.; Stevens, M. F. G. J. Chem. Soc., Chem. Commun. 1989, 1164-1165. (20) Bellamy, L. J. The Infrared Spectra of Complex Molecules; Wiley: New York, 1975.
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Figure 4. Ellipsometric thickness (d) measured on a COOH surface (Au-COOH) upon addition of incremental amounts of PAM, ethamidine (EAM), decamethylenebis[guanidine] (DMBG), octamidine (OG), or 5,5′-(1,3-propanediyl)bis[isothiouronium bromide] (PBIB) at pH 8.7 (for structures, see Chart 1). The maximum length of PAM has been indicated.
thickness changes, approaching the length of the molecules (Figure 4). The adsorption of PAM could furthermore be detected by potentiometric measurements. As seen in Figure 1, the resulting curve exhibited a shape similar to that of the adsorption isotherm obtained from the thickness measurements. Surface potentials of monolayers depend on layer thickness and density of surface charges and dipoles.5 Related conclusions however, cannot be drawn at this stage. In Figure 5, the influence of the pH on the layer thickness is shown. Interestingly, the resulting curve resembles pH titration curves determined previously for COOH SAMs on gold,15 with the largest increase in layer thickness occurring between pH 7 and 10. This shows that charged carboxylic acid groups are predominantly responsible for the anchoring of the amphiphile head groups. As expected from the monolayer model, the layer thickness levels off just above 20 Å between pH 9 and 10. Related systems aimed at the buildup of self-assembled multilayered films have recently been described.22 These involve consecutive adsorption of bipolar cationic and anionic amphiphiles or polyelectrolytes onto a charged solid surface. It was pointed out that short or flexible amphiphiles prefer to interact by a twopoint electrostatic interaction with the oppositely charged surface and that only amphiphiles with a rigid core connected to the ionic end groups by long alkyl chains were able to form self-assembled structures.22a,d The structural requirements concerning the amphiphile seem to be different in our case. First, a relatively rapid layer formation is observed in spite of the amphiphiles being (21) Bertilsson, L.; Liedberg, B. Langmuir 1993, 9, 141-149.
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Figure 5. Ellipsometric thickness (d) measured on a COOH surface in the presence of either PAM (50 µM) (average of three experiments) or PAM (50 µM) and IP4 (150 µM) as a function of pH. Titration was started from either the acidic side (PAM) or the basic side (PAMIP4, from point 1 to point 2) by pH adjustments using 0.1 M NaOH or HCl. PAM-IP4 was back-titrated from point 2 to point 3.
smaller than those previously used.22,23 Second, as shown by the large binding constant for PAM (Figure 1), the monolayer is formed at a lower concentration than the amphiphile concentration previously used for the buildup of multiple layers. We therefore propose that the amidinium-carboxylate interaction, being not only attractive but also directional in nature,17 is the main driving force for the layer formation in this work. It occurred to us that the reversible nature of the self-assembly process could be used in more general terms for reversible functionalization of a given surface. In this way, a surface may be reversibly provided with functionalities suitable for molecular recognition or selective adsorption. In the present case, the positively charged amidinium surface should be able to serve as receptor sites for phosphate biomolecules.6 Selective adsorption of such compounds would thus provide an additional probe for this structural arrangement. Addition of adenosine phosphates (Figure 6) and inositol phosphates (Figure 7) to the PAM-modified COOH surface produced an additional thickness increase related to the number of phosphate groups of the compound. Thus, ATP and inositol tetraphosphate (IP4) showed the strongest binding, producing also the largest thickness increases. As controls, ATP was added to the PAM-modified silicon dioxide or gold surfaces (Figure 6), to the unmodified surfaces, or to COOH surfaces modified with the other dibasic amphiphiles. Only on COOH surfaces modified with bisbenzamidines was strong binding accompanied by a large thickness increase observed. This (22) (a) Decher, G.; Hong, J.-D. Macromol. Chem., Macromol. Symp. 1991, 46, 321-327. (b) Decher, G.; Hong, J. D. Ber. Bunsenges. Phys. Chem. 1991, 95, 1430-1434. (c) Lvov, Y.; Decher, G.; Sukhorukov, G. Macromolecules 1993, 26, 5396-5399. (d) Mao, G.; Tsao, Y.; Tirrel, M.; Davis, H. T.; Hessel, V.; Ringsdorf, H. Langmuir 1993, 9, 3461. (e) Zhang, X.; Gao, M.; Kong, X.; Sun, Y.; Shen, J. J. Chem. Soc., Chem. Commun. 1994, 1055-1056. (23) Mao, G.; Tsao, Y.; Tirell, M.; Davis, H. T.; Hessel, V.; van Esch, J.; Ringsdorf, H. Langmuir 1994, 10, 4174-4184.
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Figure 6. Ellipsometric thickness (d) increase measured upon addition of incremental amounts of adenosine phosphates to a PAMmodified ([PAM] ) 50 µM) COOH and silicon dioxide surface. Linear regions of Eadie-Scatchard plots of the film thickness and concentration data gave the following binding constants (Ka) and limiting film thicknesses (dmax) in the cases where saturation was observed: on the Au-COOH-PAM surface, ATP, Ka ) (5.3 ( 0.2) × 104 M-1, dmax ) 14 ( 2.5 Å (results of four experiments using two identically prepared surfaces); ADP, Ka ) (0.9 ( 0.6) × 104 M-1, dmax ) 7.1 ( 2.0 Å (results of two experiments using the same surface).
suggests that a densely packed monolayer of PAM can serve as binding sites for phosphate biomolecules, as depicted in Figure 2. It is noted that the selectivity for ATP is higher on the PAMmodified surfaces than on ATP-selective interfaces based on polyamine receptors.24 This may be due to a favorable multiply hydrogen-bonded ion pair interaction between ATP and the surface, similar to that proposed by Sasaki et al. for the association of ATP and LB films of alkylguanidines at the air-water interface.6 Supporting evidence for the presence of such interactions was provided by the FT-IR characterization. The spectra of the ATPmodified COOH-PAM surface (Figure 3) show, in addition to the PAM characteristic bands, also bands characteristic for ATP. In particular, the strong broad bands at 2700-3600 cm-1, arising from the NH and OH stretching vibrations, indicate a considerable contribution from hydrogen bonding to the adsorption of ATP.6,20 Commonly, we observed that addition of ATP to COOH surfaces modified with the dibasic amphiphiles used in the control experiment (Chart 1) produced a thickness decrease. This indicates a loose solvated structure22a and again suggests that PAM forms a densely packed monolayer. Interestingly, the limiting thickness of IP4 was approximately 2 times larger than those of IP3 and IP2. One explanation is that IP4, due to its multiple charges and for steric reasons, may act as a bridge between two layers of PAM. Such multilayer formation may serve as a response enhancement mechanism for particular analytes. We noted further that the PAM-phosphate layers were stable at a lower pH than the single PAM layer. One example of this effect is shown in Figure 5. This finding supports the dense layer (24) See Odashima, et al. In ref 3, Chapter 11.
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Figure 7. Ellipsometric thickness (d) measured on a PAM-modified ([PAM] ) 50 µM) COOH surface upon addition of incremental amounts of inositol phosphates having the following ionized structures: IP2, R1 ) PO32-, R2 ) R3 ) H; IP3, R1 ) R2 ) PO32-, R3 ) H; IP4, R1 ) R2 ) R3 ) PO32-. The binding constants (Ka) and limiting film thicknesses (dmax) were as follows: IP4, Ka1 ) (2.7 ( 3) × 105 M-1, Ka2 ) (0.9 ( 1.0) × 105 M-1, dmax1 ) 17 ( 2 Å, dmax2 ) 23 ( 1 Å (where 1 and 2 indicate the first and second concentration intervals of a biphasic plot); IP3, Ka ) (8.5 ( 0.4) × 104 M-1, dmax ) 9.0 ( 1.2 Å. Values are averages of two experiments.
structure proposed in Figure 2, where the phosphates associate strongly with at least one amidinium group. The DNA binding properties of bisbenzamidines9 suggested that oligonucleotides would also bind to the surface. Consecutive addition of PAM and a 15-bases-long (15-mer) random oligonucleotide (Figure 8a) produced thus an additional thickness increase for the oligonucleotide of ∼30 Å. This indicates that more than one layer of DNA is adsorbed to the surface. From Figure 8, it appears clearly that the PAM-phosphate layer formation was completely reversible. Thus, when acidifying the solution to pH 3, the thickness decreased to the value measured for the bare COOH surface, and when pH was again raised, the thickness returned to its original value for the PAM-phosphate system. This important observation is confirmed by the PAM-IP4 experiment (Figure 8b), where a similar pH-dependent film thickness is seen. The absence of hysteresis in the corresponding pH-film thickness curve (Figure 5) indicates that the layer structure is reproduced after one pH cycle. In the case of a single PAM layer, this was further confirmed by FT-IR ERS characterization. CONCLUSIONS Consecutive selective adsorption on a self-assembled monolayer has been demonstrated, where adsorption of one compound induces binding sites for a second compound. This leads to the reversible formation of a layered structure, as demonstrated in the consecutive adsorption of pentamidine and phosphate biomolecules. The selective binding of ATP, IP4, and oligonucleotides and the reversible surface functionalization are interesting features in the context of chemical sensors or detector devices.2,3
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Figure 8. Real-time thickness measurements by ellipsometry in an experiment with consecutive addition of PAM (final concentration, 50 µM) and (a) a random DNA 15-mer (5 µg per addition) or (b) IP4 (1 × 10 µL, 4 × 20 µL, and 2 × 100 µL additions of the 2.5 mM stock solution), followed by pH adjustments with acid (0.1 M HCl) and base (0.1 M NaOH).
In another perspective, the system may have potential for the buildup of defined multilayered structures. ACKNOWLEDGMENT This work was supported by a grant from the Bank of Sweden Tercentenary Foundation. The gifts of pentamidine isethionate from Rhone Poulenc Rorer, Sweden; inositol phosphates from Perstorp AB, Sweden; gold surfaces from Dr. Thomas Zettera, IMM, Mainz; and the DNA samples from Dr. Michael Mecklenburg, University of Lund, Sweden are gratefully acknowledged. Finally, we are grateful to Dr. Bo Liedberg, University of Linko¨ping, Sweden, and the group of Prof. Helmut Ringsdorf, University of Mainz, for valuable assistance. SUPPORTING INFORMATION AVAILABLE Eadie-Scatchard plots for the calculation of the individual binding constants and the limiting film thicknesses (6 pages). Ordering information is given on any current masthead page.
Received for review August 17, 1995. Accepted October 27, 1995.X AC9508356 X
Abstract published in Advance ACS Abstracts, December 1, 1995.
