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Discrimination between N- and C-Termini of Polypeptides by a Two-Dimensional Array of Helical Poly(L-glutamic acid) Rods on Gold Surfaces Masazo Niwa,* Masaaki Morikawa, and Nobuyuki Higashi* Department of Molecular Science & Technology, Faculty of Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan Received December 15, 1998. In Final Form: April 26, 1999 A novel poly(L-glutamic acid) (PLGA) amphiphile, PLGA-SS, which has a disulfide bond at the N-terminus, formed spontaneously an adsorbed monolayer on gold substrates from aqueous solutions with various pHs. The adsorption rates of PLGA-SS were characterized by electrochemistry using a model of a microelectrode array. When the monolayer was prepared at a lower pH, at which the PLGA segment takes the R-helix structure, a densely packed monolayer was formed, and in contrast, the preparation at higher pH was found to provide a less packed monolayer. Subsequently, the interaction of this monolayer with guest PLGAs, PLGA-Fc-N and -C, which have a ferrocenyl group at the N- and C-termini, respectively, was examined by means of the quartz crystal microbalance (QCM) technique. The frequency shift of QCM revealed that a stoichiometric interaction could be caused between the PLGA-SS monolayer and both guest PLGAs. The electron-transfer experiment and angle-dependent X-ray photoelectron spectroscopy were also applied to elucidate such a specific interaction. As a result, the host helix monolayer was found to capture the guest helix PLGAs through an antiparallel, side-by-side helix-macrodipole interaction.
Introduction We report herein the formation of helical poly(L-glutamic acid) (PLGA) mononlayers on gold substrates and their specific interaction with guest PLGAs. Organized monolayers provide peculiar environments for molecular interactions, and as a result, for molecular recognition. In this context, we have devised a strategy in which polymeric amphiphiles carrying a purely synthetic polyelectrolyte segment such as poly(methacrylic acid) (PMAA) and PLGA are aligned on two-dimensional media.1 Au-coated plates have been used as two-dimensional solid substrates since well-organized monolayers can be prepared on Au by spontaneous adsorption of organic thiols and disulfides.2,3 We have already established that self-assembled monolayers of PMAA-based amphiphiles are formed spontaneously on gold substrates from aqueous solutions, and then the molecular-packing density within the monolayers is successfully controlled on the basis of the conformational size of the PMAA segment adopted during adsorption.3a Recently, there has been considerable interest in constructing self-assembled polypeptides on solid substrates. Such structures are expected to be tethered to the surface while having a controlled orientation and molecular conformation. Enriquez and co-authors coupled a S-S moiety to a poly(γ-benzyl-L-glutamate) molecule, so that it could directly react with a gold surface.4 Whitesell et al. have shown that an anisotropic, R-helical film of poly(amino acid)s can be formed on gold by graft polymerization of the corresponding peptide monomers initiated with surface-attached species.5 More recently, we discovered (1) For a recent review, Higashi, N.; Niwa, M. Colloids Surf., A 1997, 123/124, 433. (2) Ulman, A. An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self-Assembly; Academic: Boston, 1991, and references therein. (3) (a) Niwa, M.; Mori, T.; Higashi, N. Macromolecules 1995, 28, 7770. (b) Niwa, M.; Matsui, M.; Koide, K.; Higashi, N. J. Mater. Chem. 1997, 7, 2191. (4) Enriquez, E. P.; Gray, K. H.; Guarisco, V. F.; Linton, R. W.; Mar, K. D.; Samulski, E. T. J. Vac. Sci. Technol., A 1992, 10, 2775.
the formation of R-helical polypeptide assemblies on water based on helix-macrodipole interactions.6 The dipole moments of amino acid in an R-helical peptide align in the same direction, nearly parallel to the helix axis, and then the resulting macroscopic dipole generates an electrostatic potential, directed from the N-terminus to the C-terminus.7,8 This electrostatic field plays an important role in the structure and functions of proteins. Baldwin et al. have shown that the interactions between the helix macrodipole and charged groups close to the end of the helix are the important determinant of R-helix stability.9-11 Fox et al. have shown that macrodipole affects the efficiency of photoinduced intramolecular fluorescence quenching.12,13 In addition, the orientation of the R-helical peptide in the self-assembled monolayers is also influenced by the intermolecular macrodipole interaction.14,15 Thus, if R-helical peptide assemblies, in which macrodipoles direct in the same sense, are prepared, it could be expected that a specific interaction would be caused between R-helical peptide assemblies and guest peptides. In the present study, a specific interaction of guest PLGAs (PLGA-Fc-N and -C) at the self-assembled monolayer of PLGA-SS will be described. The PLGA-monolayer former (PLGA-SS) consists of the S-S moiety and the (5) (a) Whitesell, J. K.; Chang, H. K. Science 1993, 621, 73. (b) Whitesell, J. K.; Chang, H. K.; Whitesell, C. S. Angew. Chem., Int. Ed. Engl. 1994, 871, 33. (6) Higashi, N.; Sunada, M.; Niwa, M. Langmuir 1995, 11, 1864. (7) Wada, A. Adv. Biophys. 1976, 9, 1. (8) Hol, W. G. J.; van Duijnen, P. T.; Berendsen, H. J. C. Nature 1978, 273, 443. (9) Fairman, R.; Shoemaker, K. R.; York, E. J.; Stewart, J. M.; Baldwin, R. L. Proteins 1989, 5, 1. (10) Chakrabartty, A.; Doig, A. J.; Baldwin, R. L. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 11332. (11) Armstrong, K. M.; Baldwin, R. L. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 11337. (12) Batchelder, T. L.; Fox, R. J., III.; Meier, M. S.; Fox, M. A. J. Org. Chem. 1996, 61, 4206. (13) Fox, M. A.; Galoppini, E. J. Am. Chem. Soc. 1997, 119, 5277. (14) Worley, C. G.; Linton, R. W.; Samulski, E. T. Langmuir 1995, 11, 3805. (15) Chang, Y.-C.; Frank, C. W. Langmuir 1996, 12, 5824.
10.1021/la9817258 CCC: $18.00 © 1999 American Chemical Society Published on Web 06/15/1999
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Chart 1
PLGA segment that are connected with the long methylene chain as a spacer. One terminus (C- or N-terminus) of the guest PLGA is labeled with ferrocene as a redox-active moiety so as to examine such interactions electrochemically. (Chart 1) Results and Discussion Monolayer Formation and Lateral Molecular Mapping of PLGA-SS on a Gold Substrate. The adsorption processes of PLGA-SS on gold are first of all examined by means of the previously established electrochemical method,16 in which Fc-Gly has been employed as a redox probe because it is water-soluble and nonionic and represents an electrochemically reversible, oneelectron redox couple.3 Figure 1A shows changes in the anodic peak current (ipa) of Fc-Gly by immersing clean gold electrodes into aqueous solutions including PLGASS at various pHs. Upon immersion, voltammograms (data not shown here) gave an apparent decrease of redox peak currents and an increase of peak-to-peak separation, indicating that contact of the probe with the electrode surface is restricted because of adsorption of PLGA-SS onto the electrode. In Figure 1A, the adsorption processes are found to depend strongly upon the pH of the solution; the adsorption rate has a tendency to become faster in the lower pH region. This result suggests that the adsorption of PLGA-SS must be governed by the secondary structure such as a helix and random coil and/or the ionization of side-chain COOH groups of the PLGA segment. To reveal the pH-dependent secondary structures of PLGA-SS in water, circular dichroism (CD) spectra were measured and displayed in Figure 1B. At a lower pH of 4.5, the spectrum gives a typical pattern of an R-helical polypeptide with two negative peaks at 208 and 222 nm. With elevating pH, the spectra change to a random coil structure via an isodichroic point of 204 nm. The helix content, which can be evaluated on the basis of the values of molar ellipticity at 222 nm,17 is plotted against the pH of the PLGA-SS solution in Figure 2. The pH of the solution could not be lowered below 4.5 becauase of the appearance of precipitation. At that pH, the helix content shows a considerably high value of about 90%, despite the presence of a long (16) Niwa, M.; Mori, T.; Nishio, E.; Nishimura, H.; Higashi, N. J. Chem. Soc., Chem. Commun. 1992, 547. (17) Ghadiri, M. R.; Fernholz, A. K. J. Am. Chem. Soc. 1990, 112, 9633.
Figure 1. (A) Changes in the anodic peak current (ipa) of FcGly by immersing clean gold electrodes into aqueous solutions containing PLGA-SS at various pHs. The electrolyte solutions are 3 mM Fc-Gly in 1 M KCl. (B) Circular dichroism spectra of PLGA-SS in water at different pHs: 4.5 (a), 5.0 (b), 5.8 (c), 6.2 (d), 6.5 (e), 7.0 and (f), 8.2 (g).
Figure 2. The fractional coverage of the electroactive site, (1-θ), estimated by assuming a disk-shaped microelectrode model (top) or the helix content evaluated by the CD spectra in Figure 1A as a function of pH during adsorption.
alkyl group at the PLGA chain end. Upon an increment of pH, the helix content gives a steep decrease between pHs 6 and 7, suggesting the occurrence of a helix-coil transition of the PLGA segment at such a pH region. In Figure 2, the fractional coverage of an electroactive site, (1-θ), estimated by employing a disk-shaped microelectrode model18,19 is also displayed as a function of (18) Amatore, C.; Saveant, J. M.; Tessier, D. J. Electroanal. Chem. 1983, 147, 39. (19) Finklea, H. O.; Snider, D. A.; Fedyk, J. Langmuir 1990, 6, 371.
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pH during adsorption. Quantitative information about a lateral molecular distribution in the resultant PLGA-SS monolayers can be extracted by fitting the voltammograms with the theoretical treatment of such a model. Ru(NH3)63+/2+ was used as a reference redox couple for probing the active sites in the monolayers, and the electrochemical measurements were performed at pH 4.5, at which the PLGA segment should take an R-helical conformation. In this model, the electrode is assumed to be covered with disk-shaped active sites with an average radius of Ra and an area fraction of (1-θ), where θ is the fractional coverage of an inactive site. Each active site is surrounded by an inactive area with an average radius of R0. For small area fractions of active sites, Ra and R0 are related to (1-θ) by the following equation: (1-θ) ) Ra2/R02. The detailed procedures have been described elsewhere.20 It can be seen from the figure that with elevating pH the area fraction of active sites (1-θ) increases steeply at around pH 6, while it corresponds well with that of the helix-coil conformational transition of the PLGA segment. These results clearly demonstrate that the lateral molecular distribution in the monolayer is successfully controlled by the secondary structure of the PLGA segment during adsorption. Discrimination between N- and C-Termini of Guest PLGAs by a PLGA-SS Monolayer. To examine the interaction of the PLGA-SS monolayer and the guest PLGAs (PLGA-Fc-N and PLGA-Fc-C), we prepared PLGA-SS monolayers on clean gold electrodes or goldcoated quartz crystal microbalances (QCM) from aqueous solutions containing PLGA-SS at pH 9.0, at which molecular packing in the resulting monolayer is predicted to be relatively loose because of ionized, expanded conformation of the PLGA segment. In fact, the surface coverage (Γ) was evaluated to be 3.7 × 10-11 mol cm-2 on the basis of analysis of the cathodic peak in the cyclic voltammogram due to reductive desorption21 of PLGASS from the electrode surface. On one hand, by using this value, an occupied area of the PLGA-SS molecule can be calculated to be about 400 Å2. On the other hand, the occupied area for the PLGA-SS monolayer prepared at pH 4.5, where the PLGA segment is expected to be in an R-helix structure, can also be estimated in the same way to be 110 Å2, which is consistent with that for the theoretical cross-sectional area of the R-helical PLGA rod. Therefore, the former monolayer would have a suitable cavity to receive guest helical PLGAs. To reveal the secondary structure of PLGA segments at such immobilized monolayer surfaces, FTIR-RAS spectra were measured for the PLGA-SS monolayers prepared at pH 4.5 and prepared at pH 9.0 and then treated with water of pH 4.5. Figure 3 displays the spectra of the CdO stretching band region. Absorbance for the latter monolayer (b) is considerably smaller, comparable with that for the former one (a), probably because of more sparse surface coverage as described above. The peaks that appear in the region of 1700-1750 cm-1 can be assigned to the CdO stretching band of side-chain COOH groups. In both spectra, two peaks corresponding to the amide I and II bands are observed at 1663 and 1550 cm-1, respectively, which are characteristic absorptions based upon an R-helix structure. These results demonstrate that the PLGA segments should practically maintain the helical structure at the monolayer surface prepared at pH 4.5 and even at that prepared at pH 9.0, which was then treated with water of pH 4.5 after adsorption on the gold substrate. (20) Niwa, M.; Mori, T.; Higashi, N. J. Chem. Soc., Chem. Commun. 1993, 1081.
Niwa et al.
Figure 3. FTIR-RAS spectra for the PLGA-SS monolayers prepared at pH 4.5 (a) prepared at pH 9.0, and then treated with water at pH 4.5 for 1 h at room temperature (b).
Figure 4. Resonance frequency changes of the PLGA-SS monolayer-covered QCM by addition of the aqueous solution of PLGA-Fc-C (a) and -N (b) at pH 4.5. The arrow indicates the time at which guest PLGA solutions were injected.
Subsequently, the interaction between this monolayer and PLGA-Fc-N or PLGA-Fc-C, which has a ferrocenyl group at the N- or C-terminus of the PLGA chain, was examined by both QCM and electrochemical methods. Figure 4 shows resonance frequency changes of the PLGA-SS monolayer-modified QCM by addition of the aqueous solution of PLGA-Fc-N or PLGA-Fc-C at pH 4.5. For both guest PLGAs, the resonance frequency is found to decrease steeply upon addition, followed by a gradual decrease, and then becomes unchanged around 3 h after addition. Such a frequency decrease must be due to a mass increase, resulting from adsorption of guest PLGAs onto the PLGA-SS monolayer. The total mass increase (∆m) can be calculated from Sauerbrey’s equation,23 and the ∆m values thus obtained are summarized in Table 1, in which the data for the PLGA-SS monolayer is also included. The molar ratio (r) of the guest PLGA molecule per PLGA-SS molecule of the host monolayer is close to unity for each combination, implying the formation of a 1:1 complex probably through a helixhelix interaction. If such an interaction is based upon a macrodipole moment of the helix rods and works more tightly in head-to-tail antiparallel orientation, the distance of ferrocenyl groups of guest PLGAs located from the gold (21) Walczak, M. M.; Popenoe, D. D.; Deinhammer, R. S.; Lamp, B. D.; Chung, C.; Porter, M. D. Langmuir 1991, 7, 2687. (22) Reda, T.; Hermel, H.; Holtje, H.-D. Langmuir 1996, 12, 6452. (23) Sauerbrey, G. Z. Phys. 1959, 155, 206.
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Table 1. Total Frequency Changes of a PLGA-SS Monolayer-Covered QCM Electrode by Adsorption of Guest PLGAs adsorbate
∆Fa (Hz)
∆mb × 107 (g cm-2)
rc
bare + PLGA-SS PLGA-SS + PLGA-Fc-N PLGA-SS + PLGA-Fc-C
-34 ( 2 -32 ( 3 -32 ( 2
1.8 ( 0.1 1.7 ( 0.2 1.7 ( 0.1
0.9 ( 0.2 0.9 ( 0.1
a The ∆F values are the average of three repeated runs. b Total mass change (∆m) was calculated by the following Sauerbrey’s equation: ∆m (g cm-2) ) -5.45 × 10-9 ∆F (Hz). c Molar ratio, r ) [∆mguest/MWguest]/[∆mPLGA-SS/MWPLCA-SS]; ∆mguest and ∆mPLGA-SS denote total mass increases by adsorption of guest PLGAs (PLGAFc-N and -C) onto the PLGA-SS monolayer and by adsorption of PLGA-SS onto bare gold QCM, respectively, and MW shows the molecular weight of the corresponding molecules.
Figure 6. The content of Fe (Fe2p) normalized with the total carbon (C1s) for PLGA-SS monolayers after complexation with PLGA-Fc-N (a) or PLGA-Fc-C (b) as a function of the takeoff angle of the photoemmitted electron.
Figure 5. (A) Cyclic voltammograms after immersion of PLGA-SS monolayer-covered electrodes into PLGA-Fc-N (dashed line) and PLGA-Fc-C (solid line) aqueous solutions at pH 4.5. (B) Relation between peak current (ip) and sweep rate (v).
surface after complexation should be different between PLGA-Fc-N and -C. To elucidate this interpretation, electrochemical measurements for the complexed monolayers were performed. The preparation of the PLGA-SS monolayer and interaction with guest PLGAs were carried out on gold electrodes in the same manner as that employed for the QCM technique. Figure 5A shows the cyclic voltammograms obtained for the PLGA-SS-modified electrode after adsorption of PLGA-Fc-N or PLGAFc-C, in 1 M KCl solution at pH 4.5. The observed peaks are due to the one-electron oxidation and reduction of the ferrocene moiety of adsorbed PLGA-Fc-C. Figure 5B displays a plot of the peak current (ip) of this electrode acquired at different potential scan rates against scan rate (v). On one hand, the peak current is found to vary linearly with the scan rate, indicating that PLGA-Fc-C is surface-bound to the PLGA-SS monolayer and is not
subjected to diffusion to the electrode. On the other hand, in the case of PLGA-Fc-N as a guest, no peak is observed in the cyclic voltammogram drawn as a dotted line in Figure 5A. In general, electron transfer is known to be governed by the distance between electroactive species and the electrode surface. It is, therefore, clear that the ferrocenyl moiety of PLGA-Fc-C exists within the range that electron transfer can take place but that of PLGAFc-N exists too far away from the electrode surface to cause electron transfer. These results indicate that the ferrocenyl group of PLGA-Fc-C is located near the electrode surface and that of PLGA-Fc-N is exposed to the bulk phase, resulting from an antiparallel, helix macrodipole interaction working between the PLGA-SS monolayer and guest PLGAs. To obtain further information about the vertical location of ferrocenyl groups in the complexed monolayers, XPS spectra were measured with various takeoff angles of the photoemmitted electron. The formation of PLGA-SS monolayers and then interaction with guest PLGAs were confirmed by the appearance of S2P, C1S, N1S, O1S, and Fe2P signals from the corresponding XPS spectra (data not show). Figure 6 shows the relation between the takeoff angle and the content of Fe(Fe2P) based upon the ferrocenyl group normalized with the total carbon (C1S), as estimated from the relative peak intensity. The Fe content clearly changes with the takeoff angle. For the complexed monolayer with PLGA-Fc-N, the Fe content decreases with increasing the takeoff angle, and in contrast, for the monolayer with PLGA-Fc-C, the Fe content has a completely reversed trend to increase with increasing the takeoff angle. Because the sampling depth increases with increasing the takeoff angle, these results strongly suggest that the ferrocenyl moiety of PLGAFc-N exists predominately near the monolayer surface and that of PLGA-Fc-C prefers the reverse, to be close to the gold surface. Therefore, the driving force of such a complexation among helical PLGA segments can again be concluded to be helix-helix macrodipole interaction. Conclusions The present study has demonstrated that (i) a novel PLGA amphiphile, PLGA-SS forms a spontaneously adsorbed monolayer on gold substrates from aqueous solutions with various pHs, (ii) the lateral molecular distribution within the monolayers is successfully controlled by the secondary structural transition of the PLGA segment, and (iii) these monolayers can discriminate
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between N- and C-termini of guest, helix PLGAs on the basis of a specific interaction between helix macrodipoles. Further characteristics of the monolayers before and after complexation will follow after investigation of the helix rod orientation. Experimental Section Materials. PLGA-SS was synthesized as follows. γ-Benzyl N-carboxylic anhydride (BLG-NCA) was first synthesized by reacting triphosgene with γ-benzyl L-glutamate in THF.24 BLG-NCA and n-propylamine was added to make a 40:1 molar ratio in CHCl3. The solution was stirred for 3 h at room temperature and poly(γ-benzyl L-glutamate) (C3N-PBLG) thus obtained was purified by washing with diethyl ether. The chemical structure of C3N-PBLG was confirmed by 1H NMR in CDCl3, and the number-average degree of polymerization (n) was determined to be 45. Condensation of the terminal amino group of C3N-PBLG with 11-(ethyldithio)undecanoic acid25 was achieved in the presence of dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine as a coupling agent in CH2Cl2. The resulting mixture was stirred for 5 days at room temperature and disulfide group-modified C3N-PBLG was purified by washing with methanol. Successful capping of the N-terminal of C3NPBLG with 11-(ethyldithio)undecanoic acid was confirmed by 1H NMR in CDCl . Finally, the objective PLGA-SS was obtained 3 by removal of the benzyl groups in 5% HBr acetic acid solution.26 PLGA-Fc-N, which has a ferrocenyl moiety at the N-terminal of the PLGA segment, was synthesized as follows. Condensation of the N-terminal of C3N-PBLG (n ) 45) with 3-ferrocenoylpropionic acid was performed in the same way described above. The benzyl groups were removed by hydrogenation using palladium black in DMF for 12 h. The chemical structure of PLGA-Fc-N thus obtained was confirmed by 1H NMR in d6DMSO. Half-wave potential (E1/2) of the ferrocenyl moiety of PLGA-Fc-N was measured by means of cyclic voltammetry for an aqueous solution (pH 9.0) of PLGA-Fc-N and determined to be 0.45 V (vs a Ag/AgCl reference electrode). PLGA-Fc-C, which has a ferrocenyl moiety at the C-terminal of the PLGA segment, was synthesized as follows. First, a dichloromethane solution of ferrocenoyl chloride was added dropwise with ice cooling to a CH2Cl2 solution containing excess ethylenediamine. The mixture was stirred for 12 h at room temperature. After removal of the solvent and excess ethylenediamine, a THF solution of triethylamine equimolar with ferrocenoyl chloride was added to the residue and stirred for several hours. The resulting triethylamine hydrochloride was filtered off and the solvent was removed. The crude product was purified by a chromatographic technique with a silica gel column, eluting with methanol. N-Ferrocenoylethylenediamine thus obtained was immediately used as an initiator of the polymerization described above. On the basis of the 1H NMR spectrum, the degree of polymerization, n, was determined to be 43. Finally, the benzyl groups were removed by hydrogenation in the same way as described above. E1/2 of the ferrocenyl moiety of PLGAFc-C was determined to be 0.39 V (vs a Ag/AgCl reference electrode). Preparation of Gold Substrates. A gold disk electrode (BAS, surface area 0.02 cm-2) was cleaned by sonication in 0.1 M KOH L-glutamate
(24) Daly, W. H.; Poche, D. Tetrahedron Lett. 1988, 29, 5859.
Niwa et al. aqueous solution for a few minutes and careful polish with diamond and alumina paste (particle size 1 mm and 0.05 mm, respectively) and then rinsed with water (purified with a Milli-Q purification system, Millipore). A gold-coated electrode for quartz crystal microbalance (QCM) measurement was cleaned by rinsing with CHCl3 and Milli-Q water, respectively, until a frequency change in dry air was not found and then it was immediately used. Gold substrates on clean glass slides used for reflection absorption FT-IR and X-ray photoelectron spectroscopy (XPS) measurement were prepared by thermal evaporation of gold at the pressure of about 10-6 Torr and cleaned by rinsing with CHCl3 and Milli-Q water prior to use. Measurements. Circular dichroism (CD) spectra of the aqueous solutions were recorded at room temperature on a spectropolarimeter (JASCO J-720) with a 1-cm quartz cell. The pHs of solutions were adjusted with the required amounts of HCl and KOH. Cyclic voltammetry (CV) was performed at 25 °C with a CV1B cyclic voltammo graph (BAS) connected with an RW-21 X-Y recorder (Rikadenki, Tokyo). A standard three-electrode configuration was used with the monolayer sample on gold as the working electrode, Ag/AgCl (3 M NaCl) as the reference electrode, and a platinum wire as the counter electrode. K4[Fe(CN)6]‚3H2O and Ru(NH3)6Cl3 were obtained commercially and used as received. Preparation of ferrocenecarboxylic acid 1-glyceryl ester (Fc-Gly) was described elsewhere.3a The solutions containing 1 M KCl as a supporting electrolyte were deoxgenated by purging with nitrogen. Gold-coated AT-cut quartz crystals (USI System, Fukuoka) with a fundamental resonance frequency of 9 MHz, in which only one side of the resonator was in contact with the surface of the solution, were employed for QCM measurement. The frequencies of bare crystals were first measured in dry air and then immersed in PLGA-SS solutions with a prescribed pH for the required period. After adsorption of PLGA-SS, the crystals were withdrawn from the solution and rinsed with water and the frequency was then measured in dry air. The frequency changes (∆F) in air were used to determine the mass of the absorbent.23 The PLGA-SS-modified crystals were immersed in PLGA-Fc-N or -C solutions (pH 4.5) for the required period, and the crystals were withdrawn from solution and rinsed with Milli-Q water of pH 4.5 and the frequency was measured in dry air. FTIR was performed on a Nicolet System 800 spectrometer with a mercury-cadmium-tellurium (MCT) detector. The measurements were carried out with the 1024 scans of interferogram accumulations using a bare gold substrate as a reference for reflection absorption spectroscopy (RAS) measurement. The optical path was purged with dry air before and during measurements. A reflection attachment at an incident angle of 80°, together with a polarizer, was used. XPS was performed on a Shimadzu ESCA-1000 system using a Mg KR source. Angle-dependent XPS studies were carried out by varying the takeoff angle from the surface. The peak locations were corrected based on the C1s line emitted from neutral hydrocarbon.
LA9817258 (25) Samuel, N. K. P.; Singh, M.; Yamaguchi, K.; Regen, S. L. J. Am. Chem. Soc. 1985, 107, 42. (26) Kuroyanagi, Y.; Kim, K.; Seno, M. J. Polym. Sci., Polym. Chem. Ed. 1983, 21, 1289.
