Langmuir 1995,11, 3805-3810
3805
Electric-Field-EnhancedSelf-Assembly of a-Helical Polypeptides Christopher G. Worley, Richard W. Linton," and Edward T. Samulski" Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290 Received January 26, 1995. I n Final Form: June 22, 1995@ The rigid, rodlike polypeptide poly(y-benzylL-glutamate)(PBLG)labeled at its N-terminus with a disulfide moiety (PBLGSS)was self-assembled (SA) on gold. In an attempt to influence PBLGSS alignment in situ, the orientation of the dipole moment along the PBLGSS helix axis was biased during self-assembly by applying a voltage between two gold electrodes in a solution of PBLGSS and electrolyte to generate a strong field (the electrical double layer) at the electrode interfaces. The resulting SA films were analyzed with X-ray photoelectron spectroscopy ( X P S )and reflection- absorption Fourier transform infrared spectroscopy (RA-FTIRS). Both techniquesindicatedthat greater polypeptide film coverage-more chemisorption-was present on the negative gold electrode compared with either the positive electrode or a SA control without a voltage applied. This is suggestive of helix orientation during poling, whereby the PBLGSS disulfide moiety is exposed preferentially to the negative electrode. However, there is minimal evidence of polypeptide order in the SA film (the poling field and solvent were removed prior to film analysis); the helix axes appeared to be Gaussian distributed about the substrate normal according to the RA-FTIRS analysis. Elemental percentages determined with XPS on the electrically-poled films were essentially identical to the unpoled control, indicating no redox processes had altered the PBLG molecular composition. Chemisorption was also seen on the positive electrode and is believed to be the result of solvent-mediated lateral aggregation of PBLGSS into (apolar) species with disulfide moieties on both ends.
Introduction Self-assembled (SA) monolayers provide materials scientists with a means of tailoring surfaces to display the desired properties via the spontaneous attachment of an adsorbate species in solution onto a substrate.'I2 Alkanethiol adsorbates on gold substrates are a widely used SA system for modifying surface properties. Strong sulfur-gold interactions and well-ordered packing among the aliphatic component of these adsorbates lead to robust mono1ayers.l Recently, the feasibility of self-assembling the a-helical, rodlike polypeptide poly(y-benzyl L-glutamate) (PBLG)onto a gold substrate was demonstrated by Enriquez et ~ 1 In. that ~ study, the interaction with the substrate was through a disulfide moiety, lipoic acid, covalently coupled to the PBLG amino terminus (PBLGSS; I). Enriquez and Samulski4further demonstrated that the PBLGSS
~ ~ ( c H ~ ) ~ - c o - ( N s ~ HOH -co),S-S
CHzCHzCOOCHzC,H.j
Lipoic acid
PBLG 1
arrangement of the helix axis distribution was essentially isotropic with respect to the substrate normal. The helix
* Corresponding authors. Abstract published in Advance ACS Abstracts, September 1, 1995. (1)Ulman, A. An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self-Assembly; Academic: Boston, 1991 and references cited therein. ( 2 ) Swalen, J. D.; Allara, D. L.; Andrade, J.D.; Chandross, E. A,; Garoff, S.; Israelachvili, J.; McCarthy, T. J.; Murray, R.; Pease, R. F.; Rabolt, J. F.;Wynne, K. J.;Yu, H. Langmuir 1987,3,932andreferences cited therein. (3)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. (4)Enriquez, E. P.; Samulski, E. T. Mat. Res. SOC.Symp.Proc. 1992, 255,423. @
axis disorder derives from the inherent distribution of helix lengths-the MW distribution resulting from condensation chemistry in the synthesis of PBLG-and the absence of specific lateral packing interactions; i.e., a dynamic and chaotic side-chain secondary structure exists on the exterior of the a-helical PBLG backbone. Both of these attributes of PBLG conspire to yield inefficient assembly-incomplete covering of the substrate-and disordered molecular packing in SA films of PBLGSS. Here we examine electrostatic effects which may ultimately influence efforts to fabricate an oriented polypeptide SA monolayer on gold analogous to that exhibited by alkanethiols, i.e., SA films in which all of the polypeptide helices are directionally aligned with the same sense relative to the surface (with the disulfide moiety serving as the substrate binding site). One motivation for fabricating such a directionally aligned SA film is the potential for exploiting peptide sequence control (via the Merrifield protocol5 or genetic expression6) to place chromophores or other chemical species at predetermined positions along a polypeptide helix. Such an aligned SA monolayer of sequentially designed polypeptides would result in a series of strata coplanar with the substrate wherein chemical functionality would be spatially delineated in the intrinsic electric potential of the directionally aligned helices (Figure 1). The latter potential derives from having a uniform dipolar sense of the aligned helices and their associated macrodipole moment^.^^^ Thus, a surface could be engineered to display specifiedproperties such as electron-hole dissociation among donor-acceptor pairs in separate strata. The objective of the present self-assembly study was t o enhance PBLGSS adsorbate coverage and helix alignment with respect to the substrate in situ. PBLG contains a macrodipole moment along its helix axis as a result of (5) Merrifield, B. Science 1986,232,341.
(6)Creel, H. S.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. Macromolecules 1991,24,1213. (7)Hol, W. G. J.;vanDuijnen, P. T.; Berendsen, H. J.C.Nature 1978, 273,443. (8)Wada, A. Adv. Biophys. 1976,9, 1.
0743-746319512411-3805$09.00/0 0 1995 American Chemical Society
3806 Langmuir, Vol. 11, No. 10, 1995 strata of defined chemical functionality
Worley et al. modified polypeptide groups
trate
Figure 1. Depiction of a n ideal SA monolayer of monodisperse helical polypeptides in which the desired chemical functional groups have been incorporated into specificpeptide side chains.
vector summation of each residue dipole moment (=3.5 D per An attempt was made to orient the PBLGSS macrodipole with E-field poling during selfassembly by utilizing the high electric fields present at the junction of a charged metal and an electrolyte solution (the electrical double layer). Using X-ray photoelectron spectroscopy ( X P S ) and reflection-absorption Fourier transform infrared spectroscopy (RA-FTIRS), a marked increase in film coverage of the poled SA PBLGSS was observed compared with an unpoled PBLGSS SA control, indicative of field-enhanced molecular alignment during self-assemblyin which the SS-capped PBLGSS terminus was biased toward the negatively charged gold electrode.
Electrostatic Considerations When a molecular dipole of magnitude ,uis placed in an electric field ( E )at an angle 8 relative to the field, it will experience a torque (z) which tends to align the dipolar molecule along the field d i r e ~ t i o n : ~
Thermal energy ( k T ) will randomize the molecular orientation distribution. Thus, in a solution of helical polypeptides, the interaction @E) must exceed kTin order for any net alignment to occur. Thus, a reasonable field is necessary to affect helix orientation, and we exploit the double layer for this purpose. The electrode/electrolyte double layer can be modeled by Gouy-Chapman theory:lo The junction between an electrode and an electrolyte solution acts as a parallel plate capacitor with the metal and electrolyte capacitor “plates”separated by some distance. While the charge on a metal electrodeis confined to its surface, thermal motions tend to randomize the positions of the electrolyte ions. The distance between the plates of the double-layer capacitor, then, is an average distance of charge separation from the electrode surface referred to as the d i l s e layer. At this distance, the metal surface charge is completely balanced by oppositely charged ions in solution. By increasing the electrode surface potential or the electrolyte concentration,the diffise layer thickness and the distance over which the double-layer electric field extends are reduced. The electrostatic potential (4) at a distance x from an electrode surface can be related to the electrode surface potential (40) by
tanh(ze@/4k2‘) = exp(-Kx) tanh(ze444k2‘) where
K
is defined as
(9) Serway,R. A. Physics for Scientists & Engineers, 2nd ed.; Saunders College: Philadelphia, PA, 1986; Chapter 26. (10)Bard, A. J.; Faulkner, L. R. Electrochemical Methods, 1st ed.; John Wiley & Sons: New York, 1980; Chapter 12.
(3) Here, no is the bulk electrolyte concentration, z is the charge of an electrolyte ion, e is the electronic charge, E is the solvent dielectric constant, €0 is the permittivity of free space, k is the Boltzmann constant, and T is the absolute temperature. The reciprocal of K is effectively the diffuse layer thickness, and eq 3 reveals that this thickness can be controlled by adjusting the bulk electrolyte ion concentration. An ideal diffuse layer thickness is accurately described by eq 3, which may apply during the initial stages of self-assembly. As increasing numbers of PBLGSS molecules adsorb to the substrate, however, a more complex treatment of the diffuse layer thickness would apply. A diagram of the torque on a PBLGSS helix oriented parallel to a gold electrode surface is illustrated in Figure 2. Calculation of this torque was performed based on a double-layer field magnitude at a distance of half an average helix length, (L)/2, from the electrode. Commercially available polydisperse PBLG with a Mviscosity of 25 kDa was used in these poling experiments, corresponding to (L)= 17 nm. The double-layer field direction relative to the substrate plane is not uniform on a macroscopic scale due to the gold surface roughness. The gold used in this work consisted of grains (approximately atomically flat regions) 50 nm in diameter on average (determined by atomic force microscopy). Hence, on the (L)length scale, the surface would appear flat, and the cartoon presented in Figure 2 is a reasonably valid representation of the field. Increasing 40 applied to an electrode will magnify @(x) experienced by a dipolar molecule and, thus, will amplify the field-induced molecular aligning process. However, at large 4 0 (hundreds of millivolts), 4 decays rapidly with distance from the electrode, and increasing 4 0 no longer increases the field strength for distances over about 1nm. From eqs 1and 2, one can show that voltages larger than 400 mV do not significantly increase the field strength and, therefore, the torque on a PBLGSS molecule (see Table 1).In addition, applying much larger voltages might induce molecular chemical changes as a result of redox processes, which we wanted to avoid in this study. Thus, 400 mV was applied between the electrodes used in this work because this bias provides the maximum orientational energy to overcome kT random motions while simultaneously avoiding the occurrence of electrochemistry.
Experimental Section Lipoic Acid-PBLG Condensation. In order to anchor PBLG to a gold substrate, the disulfide moiety lipoic acid (dlthioctic acid, Aldrich Chemical Co.) was chemically attached to the PBLG N-terminus (PBLGSS). The details of this reaction can be found in a previous p~blication.~ Commercially available PBLG from Sigma Chemical Co. was used in this condensation reaction. The MWD of the PBLG used was 11.3, and the molecular masses provided by Sigma were 25 kDa determined by viscosity measurements and 19 kDa found by light scattering. Preparation of Substrates. Gold substrates used for the electrodes and self-assembly experiments were prepared by thermal evaporation on glass microscope slides using a Key High Vacuum Products Model KV-301 vacuum evaporator. Prior to depositing the gold, the slides were washed with soap and distilled, deionized (Nanopure 11) water. They were then immersed in piranha solution (30%H202 and concentrated HzSO*,1:4 by volume) for 20 min. Caution: Piranha solution is extremely corrosive and can react violently with organic compounds. Finally, the slides were thoroughly rinsed with Nanopure water and oven dried. To provide better gold adhesion to
Field-Enhanced Polypeptide Self-Assembly
Langmuir, Vol. 11, No. 10, 1995 3807 4f were acquired a t a pass energy of 35 eV. Each sample was analyzed for a n equivalent period of time. Thus, any possible damage induced by X-ray exposure should have been normalized away between the samples. All spectra were obtained a t a takeoff angle (0)between the sample and spectrometer of 70" in order to maximize the XPS information depth of the thick films analyzed. Relative atomic concentrations were calculated based on empirically derived sensitivity factors.13J4 Film thicknesses were determined by comparing the gold substrate signal attenuated by an organic overlayer to a gold blank sputter cleaned in situ with a n Ar ion beam. Relation 415
L E Figure 2. Model of the alignment of a PBLGSS helix normal to a n electrode surface due to the torque applied to the helix dipole moment by the electrode double-layer field.
Table 1. Field Strength ( E ) and Torque (z) Calculated as a Function of $0 at a Distance of 85 A (Half an Average PBLGSS Helix Length) from an Electrode with 1/K = 85 (Diffuse Layer Thickness)"
a
dn. mV
10 50 100 200 300 400 500 a
E , V/m 4.32 2.03 3.42 4.46 4.63 4.66 4.66
105
x x x x x x
lo6 lo6 lo6 lo6 lo6 lo6
z, kJ/mol
0.4 1.6 2.7 3.6 3.71 3.73 3.73
kT thermal energy = 2.48 kJ/mol a t 25 "C.
the glass substrates, -10 nm of chromium was initially evaporation deposited on the glass. Approximately 150 nm of gold was then deposited over the Cr layer with evaporation pressures of -2.5 x Pa. The gold was used in self-assembly experiments within a few days of deposition and was cleaned immediately prior to an experiment using ozone generated from a UV lampll (Boekel Industries, Inc.). Electric Poling Cell. All solutions were prepared with methylene chloride solvent (Fisher Scientific, optima grade). A 1.48 x M tetrabutylammonium hexafluorophosphate (TBAP) electrolyte solution was used which should produce an 8.5-nm diffuse layer a t room temperature according to eq 3. A bias of 400 mV generated by a dc source was applied between two gold electrodes immersed in the electrolyte solution, and a n ammeter was placed in the circuit to monitor any current during the experiment. After approximately 2 min, 0.5 mL of a 5 mg/mL PBLGSS stock solution was added, which provided a n excess of molecules necessary to form a complete monolayer on the electrodes used in this study (-1 cm2). The PBLGSS aliquot was added after the electrodes had been exposed to the TBAP solution in order for the double layer to form and produce the electric field necessary for molecular alignment. The poling voltage was applied for 48 h to allow ample time for a complete SA monolayer to form.12 The electrodes were then removed from solution, rinsed thoroughly with fresh solvent to remove any physisorbed molecules, and dried with a stream of Ar gas. Control Films. Two PBLGSS SA controls were run without applying a voltage. In one control, the gold was immersed in a solution of TBAP electrolyte, PBLGSS, and methylene chloride solvent. In the second control, the gold substrate was placed in a solution of PBLGSS in methylene chloride. All control concentrations were identical to those used in the poling experiment. Two gold blanks were also prepared: one exposed to a TBAP/methylene chloride solution and one immersed in pure methylene chloride. All PBLGSS controls and gold blanks were immersed in solution for 48 h and subsequently rinsed with copious amounts of fresh solvent. Film Characterization. The XPS data were acquired with a Physical Electronics Industries Model 5400 spectrometer using a Mg Ka X-ray anode a t 15 kV and 400 W. Survey spectra (01000eV) were taken a t a constant analyzer pass energy of 89 eV, and binding energy windows for C Is, N Is, 0 Is, S 2p, and Au (11)Vig, J. R. J . Vac. Sci. Technol. A 1985, 3, 1027. (12) In studies of PBLGSS SA as a function of time, no increase in film coverage for self-assembly over 48 h was observed (unpublished).
::I :[
d = -1 sin(@)In -
(4)
was utilized to determine these film thicknesses (d). Here, A is the gold photoelectron inelastic mean free path derived from a compilation of A values from XPS studies of many organic f i l m ~ . ~0~ is J ~the take-off angle, Auf is the gold substrate intensity through a n organic overlayer, and Aub is the intensity of a gold blank (sputter cleaned in situ). Using eq 4 requires a precise measurement of the blank and sample gold signals. To accomplish this, the X-ray source was maintained a t a constant position while analyzing all the samples, and all sample heights were equivalent relative to the analyzer. To obtain accurate peak areas, high-resolution energy windows were acquired of the Au 4f peak. The RA-FTIR spectra were obtained with an evacuable Bomem DA3 FTIR spectrometer using unpolarized light from a Globar source and a MCT detector. A Harrick reflection attachment was used for reflectiodabsorption information in which incident light was reflected off the sample surface a t an incident angle of 14". To remove ambient water vapor, all spectra were acquired a t a chamber pressure of ~ 2 . 6x lo2 Pa. Each spectrum was comprised of 500 scans coadded a t 4-cm-l resolution, and all spectra were referenced to a gold blank that was exposed only to pure solvent. Cyclic voltammograms (CVs) of gold immersed in TBAP/ methylene chloride and PBLGSSmBAP/methylene chloride solutions were obtained using a three-electrode cell with a Ag wire reference. The scan rate was 100 mV/s for both experiments. A CV of gold in the TBAP/methylene chloride solution was taken first using identical concentrations to the poling experiments. An equivalent amount of PBLGSS used in the poling experiments was then added to the cell, and another CV was done. All voltammograms were acquired with a EG&G Princeton Applied Research potentiostatlgalvanostat Model 273A.
Results and Discussion Film Coverage. XPS was employed to measure the gold substrate signal of both electrodes in the electric poling experiment as well as the PBLGSS SA control sample and gold blanks. As a molecular film will mask the XPS gold substrate signal, the degree of gold attenuation is proportional to film overlayer thickness (eq 4). While the gold signal of the SA control was less intense than a gold blank due to substrate signal attenuation by the SA polypeptide molecules, the substrate intensities of both electrically-poled electrode monolayers were considerably lower than the control sample, indicating more extensive polypeptide overlayers.18 A comparison of XPS survey spectra for the SA PBLGSS control and the two (13)Moulder, J. F.; Stickle, W. F.; Sobol, P. E.; Bomben, K. D. In Handbook ofx-ray Photoelectron Spectroscopy; Chastain,J.,Ed.; PerkinElmer: Eden Prairie, MN, 1992. (14) Wagner, C. D.; Davis, L. E.; Zeller,M. V.; Taylor, J.A.;Raymond, R. H.; Gale, L. H. Surf. Interface Anal. 1981, 3, 211. (15)Fadley, C. S. Prog. Surf. Sci. 1984, 16, 3. (16) Seah, M. P.; Dench, W. A. Surf Interface Anal. 1979, 1, 2. (17) Seah, M. P. Surf. Interface Anal. 1986,9, 85. (18) If, after rinsing with solvent, significant quantities of TBAP electrolyte were adsorbed on the gold substrates used in the poled films, perhaps this might explain some of the increase in film coverage observed. TBAP is completely soluble in methylene chloride solvent, however, and any adsorbed ions should be completely removed after
Worley et al.
3808 Langmuir, Vol. 11,No. 10, 1995
h
I
Au4f
PBLG Helix
N-terminus
1000 900
800 700 600
500 400 300 200 Binding Energy (ev)
100
0
+
