J. Phys. Chem. B 2001, 105, 5223-5230
5223
Self-Assembly of a Rodlike Polypeptide on Solid Surfaces: Role of Solvent, Molecular Weight, and Time of Assembly Alveda J. Williams Department of Chemistry, UniVersity of Illinois, Urbana-Champaign, Illinois
Vinay K. Gupta* Department of Chemical Engineering, UniVersity of Illinois, Urbana-Champaign, Illinois ReceiVed: October 31, 2000; In Final Form: March 26, 2001
The interplay between preparative conditions and molecular characteristics during the self-assembly of a rigid, rodlike, R-helical polypeptide was investigated. Poly(γ-benzyl-L-glutamate) was prepared by a novel synthetic route wherein the polymerization of the N-carboxyanhydride was initiated with an amine terminated disulfide, and the SSPBLG was characterized using light scattering, viscometry, and size-exclusion chromatography. Self-assembly of SSPBLG on gold substrates was found to proceed rapidly and high molecular weight (∼27 kDa) polypeptides formed self-assembled monolayers (SAMs) on a time scale of minutes. Characterization of the SAMs by polarization modulation infrared absorption spectroscopy (PM-IRRAS) was used to explore the effects of the molecular weight of the polypeptide, the time of assembly, and the solvent. An increase in the length of the polypeptide from 66 to 156 Å caused an increase in the average tilt of the helix axis from the normal to the surface. The use of a nonpolar solvent (benzene), where antiparallel aggregation of helices is known to occur, did not prove to be an effective strategy for improving orientation of the longer polypeptides. The time of assembly was found to play a key role, and long times of assembly led to a decrease in surface coverage and increase in tilt angle. This effect was more pronounced for the short polypeptide than the long polypeptide. Self-assembly of a debenzylated SSPBLG showed that physisorption due to hydrophobic interactions was important in the preparation of SAMs formed from long polypeptides. Reduction of physisorption by use of the debenzylated SSPBLG led to an enhancement in surface coverage and decrease in the tilt angle of the higher molecular weight SSPBLG.
Introduction In recent years, self-assembly of simple molecules such as alkanethiols on gold and alkylsilanes on silicon oxide has proven to be a versatile method for surface modification in studies on adhesion, electrochemistry, wetting, and molecular recognition.1 New advances in engineering of interfaces using self-assembled monolayers (SAMs) have increasingly focused on molecules that are complex in structure, design, and function. A major challenge for research here has been the establishment of principles that guide the assembly of these new materials on surfaces and the development of relationships between the molecular organization of the monolayers and their interfacial properties. Self-assembly of R-helical polypeptides is one example where a new structural motif for SAMs has been explored.2-9 These R-helical materials possess a rigid, rodlike structure and a macrodipole moment (100-1000D) parallel to their rod axis, and their assembly at interfaces is relevant to applications in piezoelectric and optical devices.10 In past work, various approaches have been used to promote self-assembly of these polypeptides. For preparation of SAMs on gold, past studies have either coupled disulfide groups to the N-terminus (amine terminated end) of a polypeptide to promote chemisorption on gold2,3,5,8 or used * To whom correspondence should be addressed. Tel: 217-244-2247. Fax: 217-333-5052. Email:
[email protected].
amino groups chemisorbed on gold to initiate graft polymerization at the solid surface.7 Similar strategies for grafting polypeptides on silicon dioxide surfaces have relied on either modifying a preformed polypeptide4 with a silane end group or using surface initiated polymerization.4,6 Studies on assembly of polypeptides such as poly(γ-benzylL-glutamate) (PBLG) on gold frequently found that the molecular orientation and packing of the helices was disordered with large tilt toward the surface. Because the R-helical polypeptides possess a large dipole moment, a general consensus emerged that electrostatic repulsion between neighboring helices caused barriers to self-assembly at surfaces and produced poor orientation.2-4 Recently Fujita et al.5 and Miura et al.8 investigated short (∼8-24 residues) polypeptides of alanine (Ala) and aminoisobutyric acid (AIB) and demonstrated that significant improvement in orientation within the monolayers of poly(Ala-AIB) was possible by inducing association between helices. A similar strategy of helix-helix interactions in antiparallel pairs has been used by Higashi and co-workers11 to assemble poly(L-glutamic acid) (PLGA) on gold and investigate vectorial electron transfer in the monolayer assemblies. To understand the principles governing self-assembly in rodlike polymer systems, we have adopted a different route for self-assembly of PBLG onto gold substrates. We have used an amine-terminated disulfide (Figure 1) to initiate polymerization and prepare SSPBLG that can spontaneously assemble onto
10.1021/jp004043d CCC: $20.00 © 2001 American Chemical Society Published on Web 05/10/2001
5224 J. Phys. Chem. B, Vol. 105, No. 22, 2001
Figure 1. Chemical structures of N-lipoyl-1,3,-diaminopropane, SSPBLG, and SSPLGA.
gold. Several advantages exist with this route. First, in contrast to past studies on PBLG, no second step of coupling the polypeptide with sulfur-containing molecules is required and the ambiguities due to incomplete coupling can be avoided. Second, the C-terminus of the polypeptide is adjacent to the sulfur moiety (Figure 1). Thus, this method provides a route to preparing a polypeptide where the orientation of the macrodipole is opposite to the case when coupling is used and the N-terminus is adjacent to the sulfur moiety. Third, this method allows us to prepare relatively monodisperse SSPBLG and study the effects of molecular weight on self-assembly at interfaces. In this paper we report the synthesis and characterization of the SSPBLG, and discuss its self-assembly onto gold substrates. Using materials with low polydispersity and two different molecular weights, we discuss the interplay between solvent polarity, time of assembly, and length of the polypeptide rod. Finally, we show that in addition to the dipolar repulsion, hydrophobic interaction with the gold surface poses a second barrier to self-assembly of PBLG on surfaces. Experimental Section Chemicals. All reagents (1,1′-carbonyldiimidazole, lipoic acid, diaminopropane, γ-benzyl-L-glutamate, triphosgene) were purchased from Aldrich Chemicals (Milwaukee, WI) and used without further purification. The solvents used in this study, benzene, dichloromethane (DCM), dimethyl formamide (DMF), dioxane, ether, hexane, methanol, and tetrahydrofuran (THF), were of reagent grade (Fisher Scientific, Pittsburgh, PA). Anhydrous solvents (chloroform, dimethyl sulfoxide) were purchased from Aldrich. Hexadecane (Fisher Scientific) used for contact angle measurements and hexadecanethiol (Aldrich) used for formation of SAMs (to test cleanliness of gold substrates12) were passed through a column of neutral alumina before use. Purified water was from an EasyPure UV system (Barnstead, IA). Preparation of N-Lipoyl-1,3-diaminopropane. The amineterminated disulfide (Figure 1) was prepared according to the procedure reported by Casolaro and Busi.13 To a solution of lipoic acid in dry, alcohol-free chloroform, a small excess of solid carbonyldiimidazole was added over 5 min. This solution was added slowly to a stirred, ice-cooled solution of diaminopropane. Stirring was continued for 30 min over the ice bath followed by mixing for 30 min at room temperature. The yellow solution was washed repeatedly with 10% aqueous NaCl and
Williams and Gupta water and dried over Na2SO4. A rubbery yellow solid was isolated by evaporating the solvent under reduced pressure. Anal. Calcd for C11H22N2OS2: C, 47.1; H, 8.6; N, 9.6. Found: C, 48.3; H, 8.13; N, 10.07. 1H NMR. (DMSO-d6, 400 MHz) δ 7.75 (m, 2H), 3.61-3.58 (m, 1H), 3.30-3.00 (m, 8H), 2.482.38 (m, 2H), 1.87-1.83 (m, 2H), 1.54-1.40 (m, 4H). Preparation of N-Carboxyanhydride (NCA) of γ-BenzylL-glutamate. The NCA was synthesized using the method reported by Dorman and Shiang.14 γ-Benzyl-L-glutamate ester and triphosgene were reacted in a 3:1 equivalent ratio at 50 °C in dry THF for 3 h. After recrystallization from dry hexane, the crude NCA was rephosgenated. Determination of the melting point (86-92°)15 and silver nitrate turbidometric test were used to verify purity. Polymerization of NCA to SSPBLG. Polymerization of the NCA was carried out according to standard literature procedures.14,16 N-Lipoylamine was used to initiate polymerization in a 5% (w/w) solution of the NCA in anhydrous chloroform. The ratio of NCA relative to the N-lipoylamine was varied from 60 to 75 to obtain different molecular weights. The polymer was isolated by precipitation into ether followed by lyophilization from dioxane. The secondary structure of SSPBLG was confirmed by 1H NMR measurements in 92/8 CDCl3/dichloroacetic acid and bulk transmission FTIR spectra in KBr pellets. Molecular weight and intrinsic viscosity ([η]) characterization was performed on a Viscotek Model 300 triple detector array with simultaneous light scattering, viscometry, and size-exclusion chromatography. The column was calibrated with a narrow polystyrene standard and DMF (with 0.05M LiBr) at 60 °C served as the mobile phase. The signals from the three detectors were used in tandem to measure Mw, Mn, [η], and Rg. Briefly, the viscosity (η) is measured as the sample elutes from the column in high dilution with known concentration. Using the Solomon-Ciuta˜ equation,17 the intrinsic viscosity [η] for each slice of the chromatogram was found. Using [η] and the Ptitsyn-Eizner equation18 along with the light-scattering signal allowed estimation of the true molecular weight distribution. Details and evaluation of the triple detector method are available in the literature.19 Preparation of Self-Assembled Surfaces. Gold substrates were prepared by thermal evaporation of gold (A1 Coinbuyers, Champaign, IL, 99.999% purity) onto clean glass microscope slides (Fisher’s Finest, Premium Quality) using a thermal evaporator (Cooke Vacuum Products, South Norwalk, CT). The glass slides were cleaned by immersion in piranha solution (7/3 v/v H2SO4/H2O2). The slides were then rinsed with copious amounts of purified water and dried in a stream of nitrogen. Warning: Piranha solution should be handled with caution; it can detonate unexpectedly.20 Chromium-plated tungsten rods (Kurt J. Lesker, Clariton, PA) were used to deposit an adhesion layer of chromium between the gold and the glass substrate. A 50 Å layer of chromium was first deposited at 0.5 Å/s. Subsequently, 1000 Å of gold was deposited at 1 Å/s under a pressure of 4 × 10-6 Torr. For preparation of SAMs of SSPBLG, substrates were rinsed with ethanol and immersed in 1 mg/mL of benzene or DMF solutions for a period of 30 min to 24 h. SSPLGA (Figure 1) monolayers were formed in a similar manner from DMF only. Upon removal from solution, the substrates were rinsed with fresh solvent, sonicated in DCM to remove any physisorbed material, and dried in a stream of nitrogen. Characterization of Surfaces. Infrared characterization of the SAMs was performed using PM-IRRAS with real time sampling electronics.21 All spectra were collected using a Magna
Self-Assembly of a Rodlike Polypeptide on Solid Surfaces
J. Phys. Chem. B, Vol. 105, No. 22, 2001 5225
TABLE 1: Physical Properties of the Disulfide Inititiated Polymers Mn (g/mol)a Mw (g/mol)a DPnb Rg (Å)c log K (dl/g)d,e ad,e
SSPBLG-1
SSPBLG-2
9700 10 600 44 21 -5.973 1.14
22 700 27 800 104 39 -6.164 1.17
a Uncertainty in molecular weight is (10%.41 b Degree of polymerization estimated from Mn. c Radius of gyration estimated from [η] and Mw using the Ptitsyn-Eisner equation.18 d Mark-Houwink parameter in [η] ) KMa. e PBLG (Mw ) 92600) without disulfide moiety gave log K) -6.376 and a ) 1.24.
860 IR spectrophotometer (Nicolet, Madison, WI) with dual channel capabilities. The beam was polarized with a wire grid polarizer (Optometrics, Ayer, MA) and photoelastically modulated by a ZnSe PEM-90 modulator (Hinds Instruments, Hillsboro, OR). After reflection from the gold surface, the IR beam was focused by a BaF2 lens on a liquid nitrogen cooled MCT detector. The signal was processed by a synchronous sampling demodulator (GWC Instruments, Madison, WI) and then processed with the spectrophotometer. Typically, 1024 scans were collected and averaged at a resolution of 4 cm-1. All the spectra were analyzed using Grams/32 version 5.0 software (Galactic Industries, Salem, NH). Integrated absorbance was obtained by fitting the absorption bands using the nonlinear curve-fitting algorithm provided with the software. Contact angles were measured using a Rame´-Hart goniometer (Model 100-00). Three pairs of advancing and receding angles were measured for each drop. At least two separate drops were used for each surface; the reported angles are an average of all (12 or more) measurements for that sample. Contact angles for monolayers formed from SSPBLG and SSPLGA were measured using water as the probe liquid. Hexadecanethiol monolayers were characterized with hexadecane. A home-built surface plasmon instrument in the Kretschmann configuration was used.22 Briefly, a microstepping motor and a precision rotary stage were used to rotate the prism, substrate and flow-cell assembly. A 3mW HeNe laser diode of wavelength 670 nm was directed onto an equilateral SF10 prism. Index matching fluid was used between the gold coated slide (SF10 glass) and the prism. The intensity of the reflected light was monitored using a wide area silicon photodetector. After a stable value was achieved for the angle corresponding to minimum reflectivity (θp) in pure solvent, SSPBLG solution was pumped through the flow cell and the reflectivity curve was monitored as a function of time. Results and Discussion Characterization of the SSPBLG. It is well-known that for PBLG in solution, properties such as conformation, dipole moments, and diffusion constants vary considerably with molecular weight.23 Because these molecular characteristics can also impact the self-assembly on surfaces, we used measurements from a viscometer, refractometer, and right angle light scattering detector to accurately characterize the molecular weight of SSPBLG. Table 1 shows the molecular weights and the intrinsic viscosity data. Because the triple detector system allows calculation of the molecular weight using RALLS and viscosity data for each retention volume slice, the MarkHouwink parameters can also be accurately estimated from the GPC data.24 The use of N-lipoyl-1,3-diaminopropane as an initiator in the polymerization resulted in SSPBLG materials that possessed
Figure 2. FTIR transmission spectrum (top) of SSPBLG-1 in a KBr pellet in the region 1475-1825 cm-1; symbols are experimental measurements and the solid line is the fit. Individual transition bands (bottom) in the fit are based on known literature values for the frequency of absorption (see text for discussion).
low polydispersity (Mw/Mn < 1.25) (Table 1). The length of the polypeptide (L) can be estimated from the degree of polymerization (DPn) by assuming that each residue contributes 1.5 Å. SSPBLG-1 was found to be approximately 66 Å in length and SSPBLG-2 was 156 Å in length. For a rodlike polymer, the radius of gyration is related to the length of the rod by Rg ) L/x12. Using this relation, the estimated Rg was found to be in good agreement with the values measured in Table 1. The value of the Mark-Houwink exponent (a) was found to lie between 1.15 and 1.25. Blout and co-workers23 correlated [η] over a large molecular weight range (∼20-300 kDa) and reported data that gave a ) 1.7-1.8 while a ) 1.23 has been reported elsewhere.25 Because the method of measurement of molecular weight and [η] influences the estimated values of a and because use of a sensitive viscometer in tandem with the SEC instrument offers higher precision than an Ubbelohde viscometer, we believe that the lower value for a for our materials is reasonable. More importantly, because a is greater than 1 the [η] measurement indicates that SSPBLG-1 and SSPBLG-2 possess an extended rod conformation. Both SSPBLG-1 and SSPBLG-2 differ from materials used in past reports on self-assembly of PBLG on gold substrates. Past studies2,3 used commercially available samples that had large polydispersity, and thereby, gave rise to speculations that the distribution of peptide lengths could play a role in disordered monolayers with low coverage. To overcome this problem, solid-phase peptide synthesis was used by Worley and coworkers26 to prepare low polydispersity materials. However, only short (10-mer and 15-mer) PBLG could be prepared that possessed partial R-helical character and significant β-sheet fraction. For the SSPBLG polypeptides in Table 1, the secondary structure was also verified by FTIR spectra of the material in KBr pellets. Figure 2 (top) shows the typical transmission IR spectrum for SSPBLG-1 in the spectral region 1475-1825 cm-1. Strong and characteristic bands can be observed at 1734 ( 1 cm-1 due to the ester carbonyl (CdO stretching in the side chains), at 1653 ( 1 cm-1 due to the amide I (CdO stretching mode in the amide group), and at 1548 ( 1 cm-1 cm-1 due to the amide II (C-N stretching coupled with N-H bending). The IR bands due to amide I and amide II stretching in R-helical polypeptides are composed, in general, of two overlapping bands
5226 J. Phys. Chem. B, Vol. 105, No. 22, 2001
Figure 3. Shift in SPR angle during the adsorption of SSPBLG-2 on a bare gold surface from a solution in DMF. The curve is offset such that pure DMF is flowing through the cell at t < 0 and the solution of SSPBLG-2 at t > 0.
each.27-29 One band is at a frequency ν|| (0) polarized parallel to the helix axis and two degenerate bands are at frequency ν⊥ (2π/n) polarized perpendicular to the helix with n turns. In the case of PBLG, Miyazawa and Blout28 have shown that the transmission IR spectrum can be further resolved into two amide I bands at 1650 cm-1 and 1652 cm-1 and three amide II bands at 1546, 1516, and 1498 cm-1. Tsuboi29 showed that amide I bands occur at 1652 and 1655 cm-1. The experimentally measured spectrum in Figure 2 was best fit by the CdO stretch at 1735.7 cm-1, two overlapping bands for amide I at 1654 and 1652 cm-1, and amide II bands at 1548, 1522, 1497 cm-1. The IR spectrum of the longer SSPBLG-2 (not shown) revealed the same absorption peaks though the width of the spectral peaks was larger than that for the short SSPBLG-1. The peak positions for both SSPBLG-1 and SSPBLG-2 are consistent with a dominant R-helical conformation. Self-Assembled Monolayers of SSPBLG on Gold Substrates. In past studies on SAMs of PBLG, immersion times of 2-14 days were used because it was assumed that the long time of assembly could improve mass transport and thereby, chemisorption of a rodlike polymer on the substrate.2 Because the usefulness of SAMs frequently relies on their short preparation times, we measured the rate of chemisorption on a bare gold surface to test whether these long times are necessary to achieve self-assembly of a high molecular weight PBLG. We used surface plasmon resonance spectroscopy to monitor the evolution in optical thickness of the SSPBLG-2 monolayer. Figure 3 shows the shift in the surface plasmon resonance angle (∆θp) when a solution of SSPBLG-2 in DMF is placed in contact with a bare gold film. A rapid rise in ∆θp indicates that even for a rodlike polymer that is 156 Å in length self-assembly proceeds at a fast rate. The shift ∆θp reaches a plateau in approximately 1200s (20min) and further changes in the monolayer thickness are slow.30 Fitting the reflectivity data31 using a 4-layer model (glass, gold, monolayer, and DMF) shows that ∆θp∼0.35° corresponds to a thickness of approximately 50 Å for the SSPBLG-2. This optical model for fitting the reflectivity data does not account for the anisotropic structure of the monolayer. In the calculations, a refractive index of 1.50 for SSPBLG was assumed to allow comparison with past studies. Because studies on self-assembly of simple n-alkanethiols on gold have shown that organizational changes such as ordering
Williams and Gupta can occur on long time scales, we focused on the time of immersion and its interplay with molecular weight in determining the orientation and coverage of the polypeptide within the monolayer. For both SSPBLG-1 and SSPBLG-2, we formed SAMs for different immersion times of 30 min, 3 h, and 24 h. The SAMs were then characterized using PM-IRRAS. Because preparative conditions such as the solvent used for assembly can also impact the orientational order within monolayers,32,33 we selected DMF and benzene to investigate the interplay between time of assembly, molecular weight, and solvent polarity. These solvents represent two extremes with DMF being a highly polar solvent ( ) 38.25) and benzene being a relatively nonpolar solvent ( ) 2.28). Figure 4a shows the PM-IRRAS spectra for monolayers formed from solutions of SSPBLG-1 in DMF. In the spectral region from 1500 to 1800 cm-1, strong absorption is observed from the ester carbonyl, amide I, and amide II moieties of SSPBLG chemisorbed on the gold surface. The positions of the absorption peaks are characteristic of the R-helical conformation on the surface and the narrow bandwidth is characteristic of a low degree of disorder. Comparison with the bulk IR spectra of the material in KBr pellets reveals that PM-IRRAS spectra possess a small shift to higher wavenumbers. Specifically, the band due to the ester carbonyl moiety is observed at 1738 ( 1 cm-1 and the band due to the amide I stretching is observed at 1658 ( 1 cm-1. A negligibly small shift is observed for the band due to the amide II stretching at 1549 ( 1 cm-1. These differences are typical of the shift to higher wavenumbers when reflection IR spectra are compared with transmission IR spectra.34 In addition to the conformational analysis, the PM-IRRAS measurements allow characterization of the amount of SSPBLG on the surface and the orientation of the R-helices on the surface. Due to the “surface-selection” rule, reflection absorption spectra of p-polarized light is sensitive to transition moments perpendicular to the surface. Because the transition moment of the ester carbonyl is oriented at approximately 53° to the R-helix axis,29 the absorption intensity (Iν,CdO) of the ester CdO peak at 1738 cm-1 does not change when the average orientation of helix varies relative to the substrate. However, orientation of the transition moments of amide I and amide II absorption is approximately 39° and 75°, respectively.29 Consequently, the absorption intensity Iν,AmI is maximum and Iν,AmII is minimum when the R-helix is normal to the substrate and vice versa when the R-helix is parallel to the substrate. Thus, Iν,CdO is sensitive to the amount of SSPBLG on the surface and the dichroic ratio D ) Iν,AmI/Iν,AmII is sensitive to the tilt angle of the R-helix from the normal to the substrate. Several qualitative features are evident in Figure 4. Figure 4a shows that in DMF solutions the absorption due to the ester carbonyl decreases with time indicating that there is sparser coverage of the SSPBLG-1 at long times compared to short times. Furthermore, because the absorption peak due to amide I is larger than the absorption peak due to the amide II, the spectra indicate that the R-helix is oriented away from the substrate. Comparison with Figure 4b, where the PM-IRRAS spectra are shown for SAMs formed from solutions of SSPBLG-1 in benzene, reveals that here too the R-helices are oriented away from the plane of the substrate. However, no decrease in coverage with time is evident in this case. Figure 4c,d show PM-IRRAS spectra for monolayers formed from the longer peptide, SSPBLG-2. In this case no decrease in the absorption due to the ester carbonyl is evident for DMF solutions
Self-Assembly of a Rodlike Polypeptide on Solid Surfaces
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Figure 4. PM-IRRAS spectra of monolayers formed from (a) solution of SSPBLG-1 in DMF; (b) solution of SSPLBG-1 in benzene; (c) solution of SSPBLG-2 in DMF; (d) SSPBLG-2 in benzene. The resolution is 4 cm-1, 1024 scans have been averaged and the spectra have been acquired using an angle of incidence of 74°.
TABLE 2: Absorbance, Thickness, and Tilt Angle Values for Self-Assembled Monolayers Formed from SSPBLG-1 Iν,CdO,DMF ta (Å) DDMF DMF Iν,CdO,benzene ta (Å) Dbenzene benzene
30 min
3h
24 h
0.221 ( 0.003 45 ( 4 2.43 ( 0.06 43° 0.234 ( 0.014 47 ( 9 2.21 ( 0.25 46°
0.198 ( 0.009 44 ( 4 2.69 ( 0.23 41° 0.235 ( 0.014 49 ( 3 2.56 ( 0.37 42°
0.157 ( 0.001 41 ( 7 2.06 ( 0.10 47° 0.195 ( 0.008 47 ( 3 1.93 ( 0.014 49°
TABLE 3: Absorbance, Thickness, and Tilt Angle Values for Self-Assembled Monolayers Formed from SSPBLG-2 Iν,CdO,DMF ta (Å) DDMF DMF Iν,CdO,benzene ta (Å) Dbenzene benzene
30 min
3h
24 h
0.251 ( 0.011 50 ( 4 1.04 ( 0.09 65° 0.303 ( 0.007 57 ( 1 1.08 ( 0.03 64°
0.206 ( 0.002 44 ( 3 1.02 ( 0.02 65° 0.328 ( 0.026 59 ( 2 1.16 ( 0.09 62°
0.261 ( 0.016 46 ( 1 1.25 ( 0.05 60° 0.334 ( 0.031 67 ( 6 1.34 ( 0.12 57°
a Standard deviations are obtained from a total of at least 12 measurements: four distinct spots on three different samples were measured. The high standard deviations (>4 Å) are possibly influenced by the temperature sensitive LC modulators that we used.37
a Standard deviations are obtained from a total of at least 12 measurements: four distinct spots on three different samples were measured. The high standard deviations (>4 Å) are possibly influenced by the temperature sensitive LC modulators that we used.37
(Figure 4c). The intensity of peak due to amide I is comparable to the peak due to amide II, which indicates that here the R-helix is oriented more toward the substrate. The integrated absorbance of the ester carbonyl peak, Iν,CdO, and the estimated dichroic ratios, D, are summarized in Table 2 for SSPBLG-1 and in Table 3 for SSPBLG-2. The dichroic ratio was used to estimate an average tilt angle of the helix axis from the normal to the substrate.35 The quantities in Tables 2 and 3 reveal that there is a rich interplay between the time of immersion, the length of the polypeptide, and the solvent in determining the self-assembly of the polypeptides. We discuss this interplay below.
First, it is clear that the SAMs of the shorter polypeptide SSPBLG-1 have smaller tilt as compared to the longer polypeptide.36 Second, at short times (30 min) the intensity Iν,CdO and, thereby, the coverage is slightly larger in benzene. Measurements of thickness by ellipsometry37 show that the SAMs of SSPBLG-1 have a thickness of approximately 45-47 Å, which is consistent with the value estimated for the average tilt and the length of SSPBLG-1. A similar trend is observed in the ellipsometric thickness of SAMs formed from SSPBLG-2. Third, a significant decrease in coverage is observed with time when SSPBLG-1 is self-assembled from DMF, and values of Iν,CdO show a decrease of nearly 30%. A much smaller decrease
5228 J. Phys. Chem. B, Vol. 105, No. 22, 2001 is evident in SAMs formed from solutions of SSPBLG-1 in benzene. However, no such decreasing trend is observed in Iν,Cd O during self-assembly of the longer polypeptide SSPBLG-2 from either DMF or benzene. Even though a slight increase with time is evident in the dichroic ratio for SSPBLG-2, investigation of SAMs formed for times up to 72 h show that the dichroic ratio remains small (D < 1.3). In all cases, we find that the surfaces are moderately hydrophobic. The advancing contact angles for water are approximately 70-76° and the receding angle is 67-70°. The results in Tables 2 and 3 indicate that molecular weight is an important parameter that determines the nature of selfassembly of polypeptides. The short polypeptide SSPBLG-1 shows a tilt angle of ∼41-49° within self-assembled monolayers irrespective of the solvent used for monolayer preparation. This behavior is similar to recent reports on polar molecules such as 4′-nitro-4-mercaptobiphenyls where it was demonstrated that even O(1) Debye dipole moment of the molecule dominates the stabilization that dipolar interaction with a solvent can provide, and a tilt of 14-17° is observed irrespective of solvent polarity.33 The molecular weight of SSPBLG-1 corresponds to a dipole moment of 155D, which indicates that changes in the solvent polarity will have an insignificant effect on the tilt angle. Because the dipole moment is significantly larger than the small biphenyl materials, electrostatic interactions between helices, plausibly, lead to a higher tilt angle to decrease the net dipole moment of the assembly. For long polypeptides such as SSPBLG-2 with dipole moment ∼363 D an even larger tilt (Table 3) is reasonable. The polarity of solvent appears to play a role and a slightly higher coverage (larger Iν,CdO) is observed upon going from DMF to a nonpolar solvent such as benzene where an increased tendency toward aggregation of the helices exists. However, aggregation of the SSPBLG-2 in benzene does not result in more vertical orientation. In recent studies where high orientation of polypeptides ( ∼28-36°) was reported by promoting antiparallel aggregation of helices at an interface, short polypeptides (∼8-24 residues) with less bulky side chains were used.5,8 The results of our study suggest that this strategy is of limited use with materials such as SSPBLG. The time of assembly emerges as a key parameter that needs to be controlled. For SSPBLG-1 the surface coverage decreases and the tilt angle increases with time when a de-aggregating solvent such as DMF is used. This result is contrary to the commonly held notion that long time of assembly favors improved orientation for SAMs formed from polypeptides. We believe that at short times and low desorption rates, chemisorption on the surface dominates and the empty sites on the gold surface are rapidly filled up with polypeptides possessing lower tilt angles. However, as the surface becomes densely packed, electrostatic interactions drive a reorganization of the monolayer resulting in an increase in the tilt and slight decrease in surface coverage. Because SSPBLG-2 carries a larger dipole moment than SSPBLG-1, the increase in molecular weight should be expected to lead to enhanced desorption. However, Table 3 shows that no decrease with time is observed in the surface coverage when the longer SSPBLG-2 was used. We believe that the lack of desorption of SSPBLG-2 is due to strong physisorption of the hydrophobic backbone onto the gold surface. Enriquez and Samulski2 have speculated that such a physical interaction can diminish chemisorption of PBLG. We believe that the effects we observe due to time of assembly are present in past results8 where large differences in polypeptide orientation have been reported between SAMs
Williams and Gupta
Figure 5. PM-IRRAS spectra of monolayers formed from partially debenzylated SSPBLG-2 (SSPLGA) and SSPBLG-2. DMF was used as a solvent and the time of immersion was 30 min. The resolution of the spectra is 4 cm-1, 1024 scans have been averaged, and the spectra have been acquired using an angle of incidence of 82°.
formed from a good solvent such as DMF and a poor solvent such as ethanol. Our results indicate that long times of assembly lead to desorption of material from the surface and a decrease in the dichroic ratio, and that these effects are exacerbated with lower molecular weight materials. Because Miura et al.8 formed SAMs from poly(AIB-Ala) that were short (∼12-24 residues) and because they used immersion times of 24 h, it is possible that significant desorption of material occurred during assembly from DMF. To test the hypothesis regarding the effects of physisorption, we converted SSPBLG-2 to a partially hydrophilic material by debenzylation of the hydrophobic side chains.38 The resulting material, SSPLGA (Figure 1), was self-assembled onto gold substrates from solutions in DMF.39 Figure 5 shows a comparison between the SSPLGA monolayers and SSPBLG-2 monolayers after assembly for 30 min. For the SAMs formed from SSPLGA, intense and broad peaks are centered at 1726 ( 1, 1659 ( 1, and 1552 ( 1 cm-1. Two effects are clearly evident in the spectra. First, the integrated absorbance due to CdO stretching in the side chains (ca. 1726 cm-1) is enhanced in the SAMs formed from SSPLGA. Second, the dichroic ratio is significantly larger. From fitting the overlapping amide bands, D is found to be approximately 2.55. Thus, in contrast to the SSPBLG-2, the SSPLGA shows higher surface coverage and higher tilt away from the surface. This suggests that hydrophobic interactions with the gold substrate also play an important role in determining the orientation of the R-helices in the self-assembly process of PBLG. Investigation of the effect of time of assembly for the SSPLGA revealed that the dichroic ratio decreases to 1.84 for a 3 h immersion time and to 1.49 after 24 h.40 Thus, even for the higher molecular weight material, the time of assembly is an important parameter and the lowest tilt from the vertical orientation is observed at short times. Conclusions In conclusion, we have demonstrated the preparation of poly(γ-benzyl-L-glutamate) of low polydispersity by a novel synthetic route where an amine terminated disulfide is used to initiate polymerization of the N-carboxyanhydride. Surface plasmon resonance spectroscopy of the self-assembly of SSPBLG on gold substrates revealed that even high molecular weight polypeptides (∼27 kDa) self-assemble rapidly on a time-
Self-Assembly of a Rodlike Polypeptide on Solid Surfaces scale of minutes. We have found that there is an important interplay between the time of assembly, the molecular weight of the polypeptide, and the solvent in determining the coverage and orientation within the self-assembled monolayers (SAMs). Increase in the length of the polypeptide from 66 to 156 Å caused an increase in the average tilt of the helix axis from the normal to the surface. In contrast to past reports where antiparallel aggregation of helices has been suggested as a way to improve the orientation of polypeptides in SAMs, the use of a nonpolar solvent (benzene) did not prove to be an effective strategy for high molecular weight SSPBLG. The time of assembly was found to play a key role and long times of assembly led to a decrease in surface coverage and slight increase in tilt angle. Self-assembly of a debenzylated SSPBLG (SSPBLGA) caused an enhancement in surface coverage and a more vertical orientation for the high molecular weight SSPBLG. Reduction of physisorption by use of SSPLGA caused the effects of time to be manifested for the higher molecular weight material and an increase in tilt angle at long times of assembly was observed. Our results indicate that physisorption due to hydrophobic interactions is an important barrier in the assembly of the long polypeptides and that short times of assembly are more conducive to a vertical orientation of R-helices within the SAMs. Acknowledgment. This research was carried out with the generous support of NSF (CAREER award CTS-9875467 to V.K.G. and Equipment Grant CTS-9732691), ACS-PRF (Grant 32983G7), and the University of Illinois. We also acknowledge the use of resources developed with the support of the U.S. Department of Energy, Division of Materials Sciences under Award No. DEFG02-ER9645439, through the Frederick Seitz Materials Research Laboratory at the University of Illinois. Financial support in the form of a graduate student fellowship from the David and Lucile Packard Foundation to A.J.W. is acknowledged. We thank John Faull for his help with the SPR and NMR measurements. References and Notes (1) Ulman, A. Chem. ReV. 1996, 96, 1533-1554. Ulman, A. Ultrathin Organic Films; Academic Press: Boston, 1991. Dubois, L. H.; Nuzzo, R. G. Annu. ReV. Phys. Chem. 1992, 43, 437-463. Spinke, J.; Liley, M.; Guder, H. J.; Angermaier, L.; Knoll, W. Langmuir 1993, 9, 1821-1825. Sigal, G. B.; Mrksich, M.; Whitesides, G. M. Langmuir 1997, 13, 2749-2755. Choi, G. Y.; Kang, J. F.; Ulman, A.; Zurawsky, W.; Fleischer, C. Langmuir 1999, 15, 8783-8786. (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, 2775-2782. (3) Worley, C. G.; Linton, R. W.; Samulski, E. T. Langmuir 1995, 11, 3805-3810.Enriquez, E. P.; Samulski, E. T. Mater. Res. Soc. Symp. Proc. 1992, 255, 423-434. (4) Chang, Y.-C.; Frank, C. W. Langmuir 1996, 12, 5824-5829. (5) Fujita, K.; Bunjes, N.; Nakajima, K.; Hara, M.; Sasabe, H.; Knoll, W. Langmuir 1998, 14, 6167-6172. (6) Chang, Y.-C.; Frank, C. W. Langmuir 1998, 14, 326-334. Heise, A.; Menzel, H.; Yim, H.; Foster, M. D.; Wieringa, R. H.; Schouten, A. J.; Erb, V.; Stamm, M. Langmuir 1997, 13, 723-728. Menzel, H.; Heise, A.; Yim, H.; Foster, M. D.; Wieringa, R. H.; Schouten, A. J. In Organic Thin Films; ACS Symposium Series No. 695; American Chemical Society: Washington, DC, 1998; pp 131-141. (7) Kratzmuller, T.; Appelhans, D.; Braun, H.-G. AdV. Mater. 1999, 11, 555-558. Whitesell, J. K.; Chang, H. K. Science 1993, 261, 73-76. (8) Miura, Y.; Kimura, S.; Imanishi, Y.; Umemura, J. Langmuir 1998, 14, 6935-6940. (9) Niwa, M.; Morikawa, M.; Higashi, N. Langmuir 1999, 15, 50885092. Boncheva, M.; Vogel, H. Biophys. J. 1997, 73, 1056-1072. (10) Jaworek, T.; Neher, D.; Wegner, G.; Wieringa, R. H.; Schouten, A. J. Science 1998, 279, 57-60.
J. Phys. Chem. B, Vol. 105, No. 22, 2001 5229 (11) Niwa, M.; Morikawa, M.-A.; Higashi, N. Angew. Chem., Int. Ed. 2000, 39, 960-963. (12) Every gold-coated glass slide that was used was tested by preparation of a densely packed monolayer of CH3(CH2)15SH on one piece of the slide. A fully formed CH3(CH2)15SH surface is known to exhibit a contact angle of 46-47° with hexadecane. (13) Casolaro, M.; Busi, E. Polymer 1994, 35, 360-366. (14) Dorman, L. C.; Shiang, W. R.; Meyers, P. A. Synth. Commun. 1992, 22, 3257-3262. (15) Daly, W. H.; Poche, D. Tetrahedron Lett. 1988, 29, 5859-5862. (16) Dessipri, E.; Tirrell, D. A.; Atkins, E. D. T. Macromolecules 1996, 29, 3545-3551. (17) Solomon, O. F.; Ciut, I. Z. J. Appl. Polym. Sci. 1983, 6, 683-686. (18) Ptitsyn, O. B.; Eisner, Y. E. SoV. Phys., Technol. Phys. 1960, 4, 1020-1036. (19) TriSEC GPC Version 3 Software Manual; Viscotek Corp.: Houston, 1999. Detection and Data Analysis in Size Exclusion Chromatography; Provder, T., Ed.; ACS Symposium Series No. 352; American Chemical Society: Washington, DC, 1987. Chromatography of Polymers: Hyphenated and Multidimensional Techniques; Provder, T., Ed.; ACS Symposium Series 731; American Chemical Society: Washington, DC, 1999. Yi, H.; Bu, L.; Bu, L.; Zhang, D.; Su, C.; Xu, Z.; Yip, L. W.; Tang, B.; Mays, J. W. Polym. Bull. (Berlin) 2000, 44, 539-546. (20) Dobbs, D. A.; Bergman, R. G.; Theopold, K. H. Chem. Eng. News 1990, 2. (21) Barner, B. J.; Green, M. J.; Saez, E. I.; Corn, R. M. Anal. Chem. 1991, 63, 55-60. Green, M. J.; Barner, B. J.; Corn, R. M. ReV. Sci. Instrum. 1991, 62, 1426-1430. (22) Kretschmann, E.; Raether, H. Z. Naturforsch. A 1968, 23, 21352136. (23) Blout, E. R.; Karlson, R. H. J. Am. Chem. Soc. 1956, 78, 941946. (24) Dutta, P. K.; Gillespie, D.; Hammons, K.; Haney, M. A. J. Pharm. Biomed. Anal. 1991, 9, 865-870. Kirkland, J. J.; Rementer, S. W.; Yau, W. W. J. Appl. Polym. Sci.: Appl. Polym. Symp. 1991, 48, 39-56. (25) Biochemical and Reagents for Life Sciences Research Catalog, Sigma St. Louis, MO, 1999, p2152. (26) Worley, C. G.; Enriquez, E. P.; Samulski, E. T.; Linton, R. W. Surf. Interface Anal. 1996, 24, 59-67. (27) Cantor, C. R.; Schimmel, P. R. Biophysical Chemistry; W. H. Freeman: San Francisco, 1980; Vol. 2. (28) Miyazawa, T.; Blout, E. R. J. Am. Chem. Soc. 1961, 83, 712-719. (29) Tsuboi, M. J. Polym. Sci. 1962, 59, 139. (30) We did not perform long time SPR experiments as the solvent DMF tends to swell Viton O-rings used in our sample cell. (31) Hansen, W. N. J. Opt. Soc. Am. 1968, 58, 380-390. (32) Kang, J. F.; Ulman, A.; Jordan, R.; Kurth, D. G. Langmuir 1999, 15, 5555-5559. Kang, J. F.; Liao, S.; Jordan, R.; Ulman, A. J. Am. Chem. Soc. 1998, 120, 9662-9667. (33) Kang, J. F.; Ulman, A.; Liao, S.; Jordan, R. Langmuir 1999, 15, 2095-2098. (34) Reflection-absorption spectra of thin films on metal films can differ considerably in frequency, intensity, and line shape from spectra obtained in transmission from KBr pellets or solutions (see: Allara, D. L.; Baca, A.; Pryde, C. A. J. Am. Chem. Soc. 1978, 11, 1215-1220. Yen, Y. S.; Wong, J. S. J. Phys. Chem., 1978, 93, 7208-7216). The physical basis of this distortion is optical effects associated with the reflection of radiation, the optical anisotropy of the film, and variation in the excitation of individual bands by surface modes of the electric field. (35) For estimating the tilt angle, we used the equation D ) K(2SHS1 + 1)/(2SHS2 + 1) where SH is the orientational order parameter of the helix axis and S1, S2 are the orientational order parameters of the amide I and amide II transition moments. This relation has been derived elsewhere by Enriquez et al.3 and Boncheva et al.9 Enriquez and Samulski3 have reported a value of K ) 1.5 ( 0.2 from reflection-absorption spectra of unidirectionally aligned PBLG in a Langmuir-Blodgett (LB) film on a gold surface. Because optical distortion accompanies reflection of IR radiation from a thin film on a metallic substrate the value for K from the LB films is believed to be a better approximation compared to estimation from a spectrum of a bulk solid. Coincidentally, fitting of the spectrum of the KBr pellet (cf. Figure 2) also showed that the ratio of integrated absorbance of amide I to amide II peaks was 1.51. We used K ) 1.5 in our estimates of the tilt angle. (36) Enriquez and co-workers modified a PBLG of Mw ) 20 kDa by coupling a disulfide to its N-terminus. They reported a dichroic ratio of 1.6 and a thickness of 41 Å.
5230 J. Phys. Chem. B, Vol. 105, No. 22, 2001 (37) We used a home-built ellipsometer that relies on a liquid crystal phase modulator. Variations in laboratory temperature caused a large variance in ellipsometric thickness. (38) Debenzylation was performed by treating SSPBLG with 30% hydrogen bromide in acetic acid at 50 °C for 30 min (see Kuroyanagi, Y.; Kim, K. Y.; Seno, M. J. Polym. Sci., Polym. Chem. Ed. 1983, 21, 1289). (39) Benzene was not used as SSPLGA was only partially soluble in benzene. Contact angles for water on the SSPLGA surfaces were measured to be 27°(advancing) and 23°(receding). (40) The estimated value of the dichroic ratio showed large variance ((0.9) in case of assembly for 30 min. Errors in spectral fitting of the
Williams and Gupta broad peaks for SSPLGA also contribute to the large variance. For longer times, smaller variance ((0.01) was observed. (41) We estimated the uncertainty in the molecular weight by characterizing a series of six polystyrene narrow molecular-weight standards (3.7212 kDa) with the triple detector GPC system. Comparison with the manufacturer supplied molecular weight (no error estimates provided) shows that the disagreement between the two values is typically 3-11% for Mw and 2-10% for Mn with the higher discrepancy when the samples have molecular weights smaller than 4000 and larger than 200, 000. An upper bound of 10% was chosen for the uncertainty in the molecular weights reported in Table 1.
