326
Langmuir 1998, 14, 326-334
Vapor Deposition-Polymerization of r-Amino Acid N-Carboxy Anhydride on the Silicon(100) Native Oxide Surface Ying-Chih Chang and Curtis W. Frank* Center on Polymer Interfaces and Macromolecular Assemblies (CPIMA) and Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025 Received August 7, 1997. In Final Form: October 16, 1997X In this study, we introduce a novel dry process for surface deposition-polymerization of vapor phase R-amino acid N-carboxy anhydride (NCA) from a surface-immobilized initiator layer. In particular, the NCA of γ-benzyl-L-glutamate is initiated from (1-aminopropyl)triethoxysilane-modified silicon(100) native oxide substrates to form poly(γ-benzyl-L-glutamate) (PBLG). The progress of the reaction is monitored by the thickness change measured with ellipsometry, the surface composition is analyzed by X-ray photoelectron spectroscopy, the secondary conformation is characterized by attenuated total reflection Fourier transform infrared spectroscopy, and the surface roughness is examined by atomic force microscopy. Under an optimal reaction condition with temperature from 95 to 125 °C in vacuo, PBLG films of 40 nm thickness have been prepared in 4 h with surface roughness rms within 3.5 nm. All films above 4 nm thickness adopt the R-helical conformation.
Introduction Recently, polypeptide thin films covalently grafted on solid substrates have attracted considerable attention in the interfacial polymer science community.1-7 In particular, many attempts have been made to obtain polyglutamates (PGs), such as poly(γ-benzyl-L-glutamate) (PBLG) and poly(γ-methyl-L-glutamate) (PMLG), by grafting on solid substrates. The advantage of utilizing PGs is not only because of their liquid crystalline, amphiphilic properties but also because their ester side chain may be easily modified, which provides the flexibility to further engineer the film structure and behavior at the molecular level. To obtain such films, both “grafting to” and “grafting from” approaches have been examined.1 In the grafting to method, a preformed PG directly reacts with a surfaceimmobilized functional group; in the grafting from procedure, glutamate monomers are reacted with the surface functional group and the subsequent growing chain, thus leading to the PG being polymerized and tethered to the surface. Many surface chemistry routes have been suggested for grafting the preformed PG onto the solid substrates.1-3 However, as a result of the strong physical interaction between PG molecules and the surface, the reacted PG molecules tend to lie parallel to the surface, thus blocking other surface binding sites and hindering subsequent adsorption. Therefore, the resulting film thickness changes are small regardless of the variation of the molecular length of the adsorbed PG. On the other hand, in the * To whom correspondence should be addressed. X Abstract published in Advance ACS Abstracts, December 15, 1997. (1) Chang, Y.-C.; Frank, C. W. Langmuir 1996, 12, 5824. (2) 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. (3) Machida, S.; Urano, T. I.; Sano, K.; Kawata, Y.; Sunohara, K.; Sasaki, H.; Yoshiki, M.; Mori, Y. Langmuir 1995, 11, 4838. (4) Whitesell, J. K.; Chang, H. K. Science 1993, 261, 73. (5) Wieringa, R. H.; Schouten, A. J. Macromolecules 1996, 29, 3032. (6) Worley, C. G.; Enriquez, E. P.; Samulski, E. T.; Linton, R. W. Surf. Interface Anal. 1996, 24, 59. (7) Heise, A.; Menzel, H.; Yim, H.; Foster, M. D.; Wieringa, R. H.; Schouten, A. J.; Erb, V.; Stamm, M. Langmuir 1997, 13, 723.
grafting from method, instead of preformed polymer, the monomers of the polymer are used to react with the surface binding site. Since the molecular size of the monomer is substantially smaller than that of its polymer, the accessibility problem can be eliminated. It has been shown that a ring-opening polymerization of N-carboxy anhydride (NCA) monomers is a much more effective route for obtaining high-molecular-weight homopolymers or random copolymers of the polypeptides than either a stepwise peptide synthesis or an addition polymerization of the corresponding amino acid monomers.8,9 To conduct a surface-initiated ring-opening polymerization, it is essential that there be an immobilized initiator that can be incorporated as part of the resulting polypeptide chains. To meet this requirement, Whitesell et al. first proposed to use a primary-amine-functionalized Au substrate to initiate the surface polymerization of alanine and phenylalanine NCAs.4 However, in past work on the primary-amine-initiated NCA polymerization in bulk solution,8 the maximum value of the degree of polymerization (DP) has been found to be about 100, which corresponds to an overall molecular length of a helical structure of about 15 nm (assuming that each repeating unit contributes 0.15 nm along its molecular axis). This implies that there will be a limitation of the grafted film thickness of the tethered polypeptide chains on solid substrates; i.e., a maximum film thickness of 15 nm would be achievable and then only if the helical axis is perpendicular to the plane of the substrate. More recently, Dorman et al. introduced a rephosgenation procedure for the NCA purification, which yielded higher molecular weight polypeptides.10 By using the ultrapure synthesized NCA of γ-benzyl-L-glutamate (BNCA) for the surface polymerization instead of the traditionally prepared material, Menzel et al. found that the maximum film thickness was increased from ca. 6.5 (8) Kricheldorf, H. R. R-Aminoacid-N-Carboxy-Anhydrides and Related Heterocycles, 1st ed.; Springer-Verlag: Berlin, 1987. (9) Katchalski, E.; Sela, M. Adv. Protein Chem. 1958, 13, 243. (10) Dorman, L. C.; Shiang, W. R.; Meyers, P. A. Synth. Commun. 1992, 22, 3257.
S0743-7463(97)00891-3 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/20/1998
R-Amino Acid N-Carboxy Anhydride Scheme 1. Surface Polymerization of B-NCA on an APS-modified Silicon Substrate
Langmuir, Vol. 14, No. 2, 1998 327 Scheme 2. Experimental Setup for the Vapor Deposition-Polymerization
secondary conformational structure, X-ray photoelectron spectroscopy (XPS) is used to analyze the surface composition, and atomic force microscopy (AFM) is used to examine the surface roughness. Experimental Section
nm to ca. 12 nm.11 Moreover, polymerization at the melt temperature of the spin-coated ultrapure synthesized NCA of γ-methyl-L-glutamate on a primary amine-modified silicon substrate was first employed by Wieringa and Schouten, who obtained a PMLG film with thickness ca. 20 nm, as estimated by FTIR.5 These experimental results might suggest that the elimination of the impurities and moisture introduced by the solvent or the NCA reactant is critical for achieving a higher degree of surface polymerization. To examine this proposal, and to obtain greater control of the film thickness, which has not been demonstrated in the previous work, we have developed a vapor deposition-polymerization protocol that involves the reaction between the vapor species of the B-NCA and a primaryamine-modified silicon(100) substrate in vacuo (Scheme 1). In contrast to the case for the melt polymerization in which the NCA material is in direct contact with the initiator layer, the vapor reactant in a vapor depositionpolymerization can be mobile in vacuo until it condenses on the substrate; thus, the concentration of the reactant is much more controllable. Moreover, a vacuum process can minimize the oligomerization of the NCA caused by moisture or impurities often introduced by organic solvents or the atmosphere and effectively reduces the reaction temperature that is required to vaporize the NCA monomer. In this study, ellipsometry is used to monitor the film thickness, polarized attenuated total reflection FTIR (polarized ATR-FTIR) is used to characterize the PBLG (11) Menzel, H.; Heise, A.; Yim, H.; Foster, M. D.; Wieringa, R. H.; Schouten, A. J. Organic Thin Films: Structure and Applications; Frank, C. W., Ed.; ACS Symposium Series; in press.
Materials and Substrate Preparation. The monomer B-NCA was synthesized following the method of Daly and Poche´12 and was characterized by H1 NMR. In this experiment, a 50 × 20 mm2 Si(100) single-sided polished wafer (Advanced Technologies) and a silicon ATR crystal (Harrick Co.) were both used as the solid substrates; (1-aminopropyl)triethoxysilane (APS) was used as the initiator. The detailed reaction conditions and the characterization of the deposition of APS on Si(100) have been described elsewhere.1 The average APS thickness measured by ellipsometry is 1.2 ( 0.1 nm (with fixed refractive index Nf ) 1.46). Vapor Deposition-Polymerization. To conduct the surface deposition-polymerization, 10 mg of B-NCA was spread on the bottom of a ca. 260 cm3 glass vessel (Scheme 2), and a freshly prepared APS-modified silicon wafer was placed horizontally on top of the holder with the polished side facing down. The height of the holder is adjustable, so the distance between the substrate and the NCA source can be varied from 5 to 30 mm. The vessel was evacuated and filled with N2 at room temperature for two cycles and then evacuated down to 0.03-0.04 Torr, tightly sealed, and transferred to a temperature-controlled oven or an oil bath for a controlled time period. After the reaction, the substrate was immersed in a dichloroacetic acid (DCA)/chloroform 1/9 (v/ v) mixture or formic acid13 for at least 15 h to remove loosely bound physisorbed materials, rinsed with fresh chloroform, and then dried with N2. Optical Ellipsometry. Ellipsometry measurements were made with a Gaertner variable angle ellipsometer (Model L116A) using a He-Ne laser of wavelength 623.8 nm and an incident angle of 70°. The optical constants of silicon are fixed with Ns ) 3.85 and Ks ) -0.002. The thicknesses of the silicon native oxide, APS, and PG layers were subsequently measured with all layers assumed to have fixed Nf ) 1.46. Polarized Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR). FTIR spectra were recorded on a BIORAD Digilab FTS-60A single-beam spectrometer equipped with a He-Ne laser and a TGS detector. A 25 mm ZnSe infrared grid polarizer (Molectron) was placed in the beam path to adjust the polarized IR beam. A twin parallel mirror reflection ATR attachment (Harrick Co.) was installed in the sample chamber, ATR crystals (50 × 10 mm2) with 60° entry angle were used, and the incidence angle was set normal to the crystal entry surface. For the thin film prepared on silicon wafers, a germanium ATR crystal (refractive index ) 4.0) (Harrick Co.) was used and the silicon wafer was cut into two pieces(50 × 10 (12) Daly, W. H.; Poche´, D. Tetrahedron Lett. 1988, 29, 5859. (13) Block, H. Poly(γ-benzyl-L-glutamate) and Other Glutamic Acid Containing Polymers, 1st ed.; Gordon and Breach Science Publishers, Inc.: New York, 1983.
328 Langmuir, Vol. 14, No. 2, 1998
Figure 1. Progression of the grafted PBLG film thickness, as monitored by ellipsometry. The distance between the APS-Si substrate and the B-NCA source was either 5, 16, or 30 mm, as indicated. mm2) and sandwiched around the germanium crystal. Pressure plates driven by a clamping screw were used to apply a force sufficient to ensure adequate sampling of the film. Thin films can also be directly deposited on the silicon ATR crystal (Harrick Co.). For subsequent use, the silicon crystal was washed with 1% HF (aq) for 4 min, followed by piranha solution for at least 20 min, and then rinsed with DI water and purged with nitrogen until dry. Spectra were recorded at 4 cm-1 resolution, and 2048 scans were coadded. X-ray Photoelectron Spectroscopy (XPS). XPS measurements were done on a Surface Science Model 150 XPS spectrometer equipped with an Al KR X-ray source, a quartz monochromator, a concentric hemispherical analyzer operating in constant analyzer mode, and a multichannel detector. A takeoff angle of 35° from the surface was employed. Spectra were recorded with a 150 eV pass energy and a 250-1000 µm spot size. A charge neutralizer was employed to optimize the binding energy shift. The peak locations were corrected on the basis of the Si-Si(2p) binding energy of 99.3 eV. Atomic Force Microscopy (AFM). AFM images were obtained using a Nanoscope III (Digital Instruments) with an E scanner in tapping mode. Images were acquired at 0 and 90°. Sharpened silicon nitride tips were used.
Chang and Frank
Figure 2. Grafted PBLG film thickness monitored by ellipsometry versus the reaction temperature. The reaction times are 15 and 30 min, as indicated.
Results
Figure 3. Progression of the grafted PBLG film thickness monitored by ellipsometry. The reaction condition is as indicated.
Ellipsometry Study. The feasibility of the vapor deposition-polymerization on the APS-modified silicon surface was first confirmed by the thickness changes monitored by ellipsometry. We verified the existence of the reactive vapor by adjusting the distance between the NCA source and the APS-modified silicon wafer (APS-Si) to ensure that the NCA source had no direct contact with the APS-Si. Figure 1 shows the progression of the resulting PBLG film thickness at 115 ( 5 °C, 0.04 Torr with three different distances from the NCA source. We found that the thickness changes for the two samples at distances of 5 and 16 mm follow the same trend, both yielding 20 nm thickness in only 30 min. On the other hand, the sample with a 30 mm displacement from the vapor source requires a longer time to reach the same thickness. This observation implies that the vapor concentration is fairly constant within the first 16 mm displacement; the thinner film at 30 mm displacement indicates that the vapor concentration depends not only on the reaction time but also on the vertical distance. To simplify the experimental protocol, we fixed the APS-Si height at 16 mm for subsequent experiments, unless otherwise noted.
Figure 2 shows the ellipsometric thickness of PBLG films versus temperature at the slightly lower pressure of 0.03 Torr. There is an obvious optimum for temperature between 95 and 125 °C, where the film thickness can reach ca. 22 nm in 15 min and ca. 28 nm in 30 min. This suggests that the vapor deposition-polymerization process is very robust, in that the resulting film thickness is insensitive to temperature over a wide temperature range but, on the other hand, is easy to manipulate by varying the reaction time. Figure 3 shows the reaction progress of the vapor deposition-polymerization under 0.03 Torr and 105 °C. Here we show that by decreasing the temperature and pressure compared to those of the previous experiments shown in Figure 1 (solid line), we are able to obtain a dramatic increase in PBLG film thickness, reaching a plateau with the film thickness 42 nm in 4 h. XPS Study. From the ellipsometry experiments, we showed that, through manipulating the reaction conditions for the vapor deposition-polymerization, the film thickness can be easily adjusted. Now XPS will be used to
R-Amino Acid N-Carboxy Anhydride
Langmuir, Vol. 14, No. 2, 1998 329
Figure 4. XPS spectrum of the PBLG film prepared at 95 °C in 15 min. Table 1. Composition and Thickness of Vapor-Polymerized Films sample
sample preparation
1 2 3 4 5 APS on Si (theor) PBLG (theor)
105 °C, 4 h 105 °C, 1 h 95 °C, 15 min 85 °C, 30 min 85 °C, 15 min
XPS experimentala nC(1s):nN(1s):nO(1s):nSi(2s)
dPBLGb(ellipsometry) (nm)
calcd dPBLGc(XPS) (nm)
11.7:1.0:3.0:0 11.3:1.0:2.8:0 12.7:1.0:3.0:0 10.0:1.0:3.1:1.6 5.6:1.0:7.3:8.1 C/N ) 2.5 (C/N ) 3) (12:1:3:0)
42 31 22 4.2 0.9 0
d d d 3.2 0.9
a The experimental data obtained by XPS measurement. b The thickness measured by ellipsometry, with fixed N ) 1.46. c The value f calculated by eq 5 with t2 ) 0.8 nm and λC(1S) ) λN(1S) ) 3 nm. d The film thickness exceeds the escape length of Si(2s).
confirm the surface composition of the resulting films and the validity of the film thickness determined by ellipsometry. Figure 4 is the XPS spectrum for the range from 0 to 1100 eV binding energy for the sample prepared at 95 °C for 15 min. The spectrum shows no signals from Si(2s) (150.3 eV) and Si(2p) (99.3 eV), indicating that there is at least a 6 nm overlayer on the top of the silicon substrate. This is because 95% of the observed photoelectron signal is derived from the topmost layer having the thickness 3λ sin θ, where λ is the mean free path of the electrons and is assumed to be 3.5 nm for Si(2s) and θ is the take-off angle of the electrons relative to the surface plane, which is 35° in this XPS setup.14,15 The ellipsometry measurement confirms that the thickness of this particular PBLG sample is 22 nm. Therefore, the signals nC(1s), nN(1s), and nO(1s) all come from the topmost organic layer. The XPS experimental nC(1s):nN(1s):nO(1s) ratio of this sample is 12: 1:3, which is exactly the theoretical stoichiometric ratio for the PBLG molecule. The XPS results thus strongly suggest that the layer grafted by vapor depositionpolymerization is composed of PBLG. Table 1 provides more XPS experimental results with the nC(1s):nN(1s):nO(1s):nSi(2s) ratios and the corresponding ellipsometric thickness data. For the 22, 31, and 42 nm thick PBLG films, the XPS experimental data match the theoretical formula of PBLG. For the 4.2 and 0.9 nm thick (14) Kleshchevnikov, A. M. In-depth profiles of elements concentrations in solids on the basis of X-ray photoelectron spectroscopy data. Synopsis of thesis, Institute of General & Inorganic Chemistry, Academy of Science, Moscow, USSR, 1983. (15) Nefedov, V. I. X-Ray Photoelectron Spectroscopy on Solid Surfaces, 1st ed.; VSP: Netherlands, 1988.
Scheme 3. Depth Profile Model for XPS Measurementa
a Two organic layers are assumed: the top PBLG layer has thickness t1 and C/N ) 12, and the APS layer has thickness t2 ) 1 nm and C/N ) 2.5 based on the experimental results.
films (samples 4 and 5), however, the appearance of the signals from Si(2s) and Si(2p) suggests that the film thickness is less than 6 nm; therefore, the ratio of nC(1s): nN(1s):nO(1s) in these two spectra will not represent the correct composition of the top layer. Instead, the signals from the elements C(1s) and N(1s) are due to both PBLG and the initiator APS layers, and the signal from O(1s) is due to the PBLG, APS, and silicon native oxide layers. Since the nC(1s) and nN(1s) signals provide information on the mixed top two layers in samples 4 and 5, the PBLG layer thickness may be computed by assuming a homogeneous, two-finite-layer model, as Scheme 3 illustrates. The simplest quantitative analysis of the XPS data is found in eq 1
R)
( )( )
nA IA IB ) / nB R A RB
(1)
330 Langmuir, Vol. 14, No. 2, 1998
Chang and Frank
where R is the ratio of the atomic compositions nA and nB found in the XPS spectrum, IA and IB are the intensities of electrons detected at particular energies A and B, and RA and RB are the cross sections of the photoionization of atoms A and B for a given X-ray energy. The intensity dI at a depth z from the surface may be expressed as
dI ) cNRe-z/λ sin θ dz
(2)
where c ) Fk, in which F is the X-ray flux and k is a spectrometer factor, and N is the number of atoms in the volume element. As Scheme 3 illustrates, we assume a two-finite-layer model with homogeneous film thicknesses t1 and t2 corresponding to the PBLG and APS layers, respectively. For an element A that exists in both layers, the intensities I1A and I2A, detected from layers 1 and 2, then can be obtained by the integral of eq 2,
I1A ) cN1ARAλA sin θ (1 - e-t1/λA sin θ) I2A ) cN2ARAλA sin θ (e-t1/λA sin θ - e-(t1+t2)/λA sin θ)
(3) (4)
Therefore, substituting eqs 3 and 4 into eq 1, we then have the ratio R of the elements A and B from both layers as
R)
nA ) nB
N1A(1 - e-t1/λA sin θ) + N2A(e-t1/λA sin θ - e-(t1+t2)/λA sin θ) λA N (1 - e-t1/λB sin θ) + N (e-t1/λB sin θ - e-(t1+t2)/λB sin θ) λB 1B
2B
(5) As Table 1 shows, the ratios R of nC(1s) (composition A refers to C(1s)) and nN(1s) (composition B refers to N(1s)) for samples 4 and 5 are 10 and 5.6, respectively. It is reasonable to assume that the escape depth λc(1s) ≈ λN(1s) ≈ 3 nm and that there is the same atomic packing density in both layers. According to eq 5, we can estimate the PBLG thickness of samples 4 and 5. Table 1 shows the comparison of the ellipsometric experimental results and the XPS calculated thickness. The results are in reasonable agreement with an error not exceeding 20-30%, which is the expected error of eq 1.15 The result also suggests that the assumption of surface homogeneity is valid in the XPS detectable range. Further examination of the surface roughness is conducted by AFM studies as follows. AFM Images. While the XPS and ellipsometry studies describe an area-averaged macroscopic view, AFM provides a more localized view of the microscopic structure of the resulting PBLG films. Parts a and b of Figure 5 are the AFM images of the samples with average ellipsometric film thicknesses 42 and 22 nm. The detectable height ranges span 20 nm, which is smaller than the PBLG layer thickness; this suggests that the images only reflect the morphology of the PBLG molecules. The surface roughness over the whole image size can be estimated by the image rms (root mean square value of the height distribution) (Rq) values. The rms values for these two samples are 1.9 and 2.2 nm, respectively. Compared to the original APS-Si surface with an average rms value 0.5 nm over a 5 nm image size, the rms value is about 3 to 4 times higher. Coupled with other images we obtained, the average rms values for these two samples are in the same magnitude, that is, from 1.5 nm (the lowest) to 3.4 nm (the highest). This result suggests that the surface roughness does not increase as the film thickness increases from 22 to 42 nm.
Although the surface roughness values for both samples are comparable, we found different morphologies for these two samples. In the 42 nm thick sample, the surface is network-like, while in the 22 nm thick sample there are doughnut-shaped features. Moreover, we found that both structures have similar order of magnitude sizes, that is, ca. 100 nm wide. This observation might imply that the doughnut-shaped features found in the 22 nm thick sample serve as nucleation (or aggregation) sites with the PBLG extending out from those sites, finally forming the network-like structure as found in the 42 nm thick sample. The network-like structure we observed in Figure 5a is seen in many other sets of samples. As the images were taken under dry conditions, we note that it is possible that the network-like structures may be formed due to dewetting after the cleaning treatment with the DCA/ chloroform mixture. ATR-FTIR Study. (1) Secondary Conformation. ATRFTIR is used to examine the surface secondary structure. The secondary structure of PBLG can be easily discerned by the shifts of the bands associated with the changes of the hydrogen-bond formation. In particular, the absorption bands of the amide I (backbone carbonyl stretching) and the amide II (mainly backbone C-N stretching) have high extinction coefficients and are commonly used to identify the secondary conformation of polypeptides. Figure 6a is a typical ATR-FTIR spectrum with p-polarized light of a pure R-helical PBLG material; the sample is prepared by spin-coating a PBLG (Sigma, degree of polymerization ) 100) film on a Si(100) wafer. The average film thickness is 70 nm, and its amide I and amide II bands are at 1655 ( 1 and 1548 ( 1 cm-1, respectively, corresponding to an R-helical conformation.16,17 This spectrum is used as the reference spectrum to compare with the grafted PBLG samples. Parts b and c of Figure 6 show the IR spectra taken before and after the cleaning treatment for a PBLG sample prepared by vapor deposition-polymerization at 117 °C with film thickness ca. 20 nm after cleaning. As Figure 6b shows, the appearance of a small peak at 1785 cm-1 suggests that a side product may be generated during the vapor process. However, this side product can be easily removed and, thus, only chemically-grafted PBLG chains remain after the cleaning treatment (Figure 6c). Other IR spectra taken for the samples prepared by vapor deposition-polymerization under various reaction conditions with final film thicknesses above 6 nm all appear to be in the R-helical conformation on the basis of their peak locations. Interestingly, we found that, for average film thicknesses below 4 ( 1 nm, the grafted PBLG film adopts different conformations. The IR spectra of two samples with film thickness 18 ( 0.7 and 4.1 ( 0.2 nm (prepared at 135 °C for 30 and 15 min, respectively) are compared in Figure 7a-d. For the 18 ( 0.7 nm thick sample, parts a and c of Figure 7 clearly indicate an R-helical dominated conformation under both p- and s-polarization. However, for the 4.1 ( 0.2 nm thick sample, the amide I and the amide II peak regions are broadened as a result of convolution of several peaks. The slight shift of the amide I from 1654 cm-1 (Figure 7a) to 1660 cm-1 (Figure 7b) in p-polarized light suggests the existence of the random coil conformation in the thinner film; moreover, the shoulder at ca. 1632 cm-1 in the s-polarized IR spectrum of the 4.1 ( 0.2 nm film (Figure 7d) indicates the existence of the β-sheet conformation. The observation (16) Tanaka, A.; Ishida, Y. J. Polym. Sci. Polym. Phys. Ed. 1973, 11, 1117. (17) Blout, E. R.; Asadourian, A. J. Am. Chem. Soc. 1956, 78, 955.
R-Amino Acid N-Carboxy Anhydride
Langmuir, Vol. 14, No. 2, 1998 331
Figure 5. AFM height images of the PBLG films with ellipsometry thickness (a, top) 42 nm and (b, bottom) 22 nm.
332 Langmuir, Vol. 14, No. 2, 1998
Chang and Frank Scheme 4. p-ATR-FTIR Setupa
a The incident angle θ ) 60°, with p- or s-polarized light. The ATR crystal can either be a silicon prism or a germanium prism.
Table 2. IR Peak Ratios of the Absorbance Bands from the PBLG-Grafted Silicon ATR Sample p-polarization s-polarization absorbance band (cm-1) Rp (A/ACdO)a (cm-1) Rs (A/ACdO)a Rp/Rs νNH νamide I νamide II
3296.1 1654.3 1546.4
1.41 1.36 0.66
3295.7 1653.7 1545.9
1.46 1.47 0.65
0.97 0.93 1.02
a R and R : ratio of the integrated area of the band ν p s amide I to that of the band νCdO in p- and s-polarized light modes, respectively.
Figure 6. IR spectra of the films of (a) spin-coated PBLG (DP ) 100) and chemically grafted PBLG (b) before and (c) after the cleaning treatment.
Figure 7. IR spectra of the PBLG films: (a) p-polarized, film thickness 18 nm; (b) p-polarized, film thickness 4 nm; (c) s-polarized, film thickness 18 nm; (d) s-polarized, film thickness 4 nm.
of different proportions of the secondary conformations revealed under different polarization may be due to their anisotropic orientations; this will be discussed further in the following section. (2) Orientation Study. For an R-helical PBLG, the transition dipole moments in the backbone are anisotropic;
for example, the N-H stretching at ca. 3296 cm-1 and the amide I stretching at ca 1655 cm-1 are more parallel to the R-helix axis, while the amide II stretching at ca. 1549 cm-1 is more perpendicular to the axis. In contrast, the carbonyl stretching from the side chain at ca. 1734 cm-1 is isotropic with respect to the helical axis. To study the anisotropic effect of the transition dipole moments, we applied p- and s-polarized illuminations, which are parallel and perpendicular to the plane of incidence, respectively (see Scheme 4 setup). Consequently, the s-polarized light can only interact with vibrations whose change in dipole moment has a component parallel to the sample surface, and the resulting spectra should resemble the transmission IR spectra if the samples have no in-plane orientation. On the other hand, p-polarized light forms an incident angle relative to the interface, thus interacting with vibrations whose changes in dipole moment are in any direction from the surface. By sequentially applying different polarization on the sample and monitoring its absorbance changes in response to the polarized light, we can determine the preferential orientation of an individual band. The peak deconvolution is accomplished with the software PeakSolve (Galactic), with a mixed GaussianLorentzian (50%-50%) distribution yielding the best peak fitting. Table 2 shows the IR deconvoluted data for the grafted sample with PBLG deposited directly on a silicon ATR crystal (Scheme 4, case 1) after cleaning. Because the side chain carbonyl stretching is randomly distributed, we use it as the reference point for each spectrum; the area ratios ANH/ACdO, Aamide I/ACdO, and Aamide II/ACdO for both p- and s-polarized IR spectra are shown in Table 2. Both amide I and NH stretching bands have somewhat higher absorbance in s-polarization than in p-polarization, while the amide II band is slightly stronger in ppolarization than in s-polarization. This result suggests that, if there is any preferential orientation of the surface PBLG, then it is probably more parallel to the surface. To further understand the surface orientation, additional deconvoluted Aamide I/ACdO ratios in s-polarization for the samples with different film thicknesses prepared on silicon wafers (Scheme 4, case 2) are investigated by the comparison with the results from both spin cast PBLG
R-Amino Acid N-Carboxy Anhydride
Langmuir, Vol. 14, No. 2, 1998 333
Discussion
Figure 8. Area ratios of the IR amide I absorbance to the ester stretching for different PBLG film thicknesses.
(DP ) 100) films and the grafting to sample we took previously in transmission FTIR mode.1 The overall orientation of PBLG molecules prepared by spin-casting is considered to be randomly distributed, as the film thickness is over ca. 50 nm; on the other hand, the overall orientation of the grafting to PBLG sample we took previously in transmission FTIR for the preformed PBLG (DP ) 100, Sigma) grafted on a (trichlorosilyl)propyl chloroformate-modified silicon wafer was considered to be parallel to the substrate, on the basis of both observations of the film thickness by ellipsometry and the orientation study by transmission FTIR. As Figure 8 illustrates, with the PBLG film thickness from 18 to 34 nm, the mean value of the absorbance ratio Aνamide I/AνCdO of ca. 1.4 is close to the values of the spin cast films but smaller than that of the grafting to sample of 1.9. This observation indicates that the overall orientation of the PBLG films prepared by vapor deposition-polymerization is less parallel to the surface than that of the grafting to sample, which implies a higher grafting density of PBLG molecules by the vapor process than by the grafting to technique. However, the tilt angles of the surface PBLG molecules are probably randomly oriented, as the absorbance ratios are similar to the values of the spin cast films. Interestingly, in contrast to the result from the washed sample, the unwashed sample with film thickness ca. 80 nm has a band area ratio of 0.8, which suggests a perpendicular orientation of the surface PBLG. Due to the thermodynamic tendency for the helical chain to be antiparallel to cancel the dipole moments,13 physisorbed PBLG may be anticipated to interdigitate into the chemisorbed layer, thus forming a more perpendicular orientation. However, as the physisorbed material is removed by the washing process, the chemisorbed PBLG will deviate from the unidirectional orientation in order to minimize the surface energy. Overall, combining information from the spectra for both p- and s-polarizations as well as the information from both grafting to and spin cast samples we measured previously, it is reasonable to believe that the overall orientation is rather disordered while having a higher grafting density than the film prepared by the grafting to technique. Moreover, the average tilt angle is probably less than 45° from the surface as compared with the absorbance ratios taken by both p- and s-illuminations.
General Comments. We have demonstrated the feasibility of vapor deposition-polymerization involving a reaction between the NCA vapor species and surfacebound amine groups. By applying high temperature and vacuum, it is possible to polymerize the NCA species to a considerable extent. For example, considering the 42 nm thick PBLG film with an average tilt angle less than 45° from the surface, which is a reasonable assumption based on our polarized ATR-FTIR study, then the actual molecular length of PBLG on the surface is larger than ca. 60 nm. This molecular length is comparable to the helical persistence length (ca. 50-70 nm) reported previously in solution.18,19 Furthermore, considering a helical conformation with each repeating unit contributing 0.15 nm along its molecular axis, then the degree of polymerization of the surface PBLG is ca. 400, which is much higher than one would expect for the primary-amineinitiated polymerization in bulk solution.8 It is important to optimize the reaction condition to maximize the productivity. One can anticipate that the NCA monomers can undergo polymerization in two ways: react with the surface initiator layer and the subsequent grafted chain or self-polymerize either in the melt or vapor state. The latter side reactions are the main source of physisorbed materials. They cannot be easily avoided, but they can be minimized by adjusting the reaction temperature, because melt polymerization is favored at low temperature and vapor self-polymerization is favored at high temperature. For example, under a particular reaction condition as shown in Figure 2, 85 °C is too low to evaporate enough B-NCA monomers to reach the APSmodified substrate, while it is high enough to undergo thermal polymerization on the bottom of the reaction, thus consuming most of the monomers. On the other hand, at temperature as high as 135 °C, the evaporation rate of the monomer is rapid compared to the surface polymerization rate. As a result, an excess amount of monomer is accumulated on the APS-modified substrate undergoing the thermal polymerization, thus forming the physisorbed material on the substrate and blocking the active chains propagating from the initiator layer. Grafting Density. The ATR-FTIR study shows that the molecular orientation (estimated by Rp/Rs) has no apparent correlation to its corresponding film thickness, for film thicknesses larger than 18 nm (Figure 8). This result might suggest that at this stage the surface deposition-polymerization only involves chain propagation rather than chain initiation. In other words, the number of surface PBLG chains may be saturated, so that there is no space to allow further chain initiation from the surface; thus, the overall chain orientation is identical for thicknesses above 18 nm. Surface roughness studies by AFM further support this postulate. As mentioned, the roughness values estimated by the rms of the image height for thicknesses of 20 and 42 nm are in the same range; i.e., the surface does not become more or less rough as the film thickness increases. Secondary Conformation Change. The IR study shows that a secondary conformation transition does occur during the surface deposition-polymerization procedure. The transition occurs as the film thickness is at around 4 nm. For a β-sheet material or an extended oligomer chain, each repeating unit contributes ca. 0.683 nm or (18) Block, H.; Hayes, E. F.; North, A. M. Trans. Faraday Soc. 1970, 66, 1095. (19) Nakamura, H.; Husimi, Y.; Jones, G. P.; Wada, A. J. Chem. Soc., Faraday Trans. 2 1977, 73, 1178.
334 Langmuir, Vol. 14, No. 2, 1998
more along its molecular axis.20 On the basis of this number, we may estimate the degree of polymerization (DP) of this 4 nm thick film to have a minimum value of six (if the sheet is perpendicular to the surface plane) or up to 12 (if it is 30° tilted from the surface). This result is consistent with the past observation of the conformation transition in solution polymerization: the β-sheet structure is found only in oligomers with 10 > DP g 4, and the longer PBLG chains adopt exclusively the R-helical structure.21 Therefore, in our case, the film adopts the β-sheet or random coil structures only at the early reaction stage; as the reaction time increases, the film is always dominated by the R-helical structure. We have tested the stability of the helicity of the PBLGgrafted film (>20 nm) by immersing the substrate into helix-breaking solvents such as dichloroacetic acid and formic acid for 1 day. In contrast to the grafted PMLG, whose conformation is reversible after such solvent exposure,22 the PBLG helical structure is retained after such treatments. This shows that the PBLG R-helical conformation is very stable. Moreover, the feasibility of preparation of random or block copolymers grafted on solid substrates by the vapor deposition-polymerization method has also been examined and is addressed elsewhere.23 Conclusion Here we propose a new approach to graft polypeptide chains to solid substrates through vapor deposition(20) Brown, L.; Trotter, I. F. Trans. Faraday Soc. 1956, 52, 537. (21) Rinaudo, M.; Domard, A. Biopolymer 1976, 15, 2185. (22) Chang, Y.-C.; Frank, C. W. In preparation. (23) Chang, Y.-C.; Frank, C. W. Organic Thin Films: Structure and Applications; Frank, C. W., Ed.; ACS Symposium Series; in press.
Chang and Frank
polymerization. Compared to methods used in the past, this vapor process provides a completely solvent-free environment, thus minimizing the amount of water or impurities in the reaction, which often leads to early reaction termination. Second, compared to the melt state in which the local reactant concentration is not easy to vary, it is possible to change the reaction rate or monomer concentration by changing the temperature, substrate-monomer separation distance, pressure, and reaction time, to obtain the desired film properties. Third, the vapor process is easy and reproducible. Here we show that by simply changing the reaction time, the PBLG film thickness can be varied from 4 to 40 nm, reproducibly. Moreover, all the PBLG films with film thicknesses above 4 nm adopt a pure R-helical conformation and have a homogeneous surface structure. In the future, efforts will be made to understand the reaction mechanism of the vapor deposition-polymerization process and to further examine its versatility for possible applications. Acknowledgment. This work is supported by the NSF Materials Research Science and Engineering Center Program through the Center on Polymer Interfaces and Macromolecular Assemblies (CPIMA). We wish to acknowledge the hospitality of the group of W. Knoll at the Max Planck Institute for Polymer Research, at which the final manuscript was prepared. LA970891X
