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J. Phys. Chem. C 2007, 111, 3710-3718
Formation and Characterization of Homogeneous and Mixed Self-Assembled Monolayers of Peptides and Alkanethiols on Indium Phosphide Surfaces Heeyeon H. Park† and Albena Ivanisevic*,†,‡ Department of Chemistry and Weldon School of Biomedical Engineering, Purdue UniVersity, West Lafayette, Indiana 47907 ReceiVed: September 18, 2006; In Final Form: January 3, 2007
This report describes the detailed characterization of alkanethiol and cysteine-terminated peptide layers on InP(100). Contact angle measurements, atomic force microscopy, X-ray photoelectron spectroscopy, and ellipsometry were performed. The surface characterization techniques showed that the composition of the mixed monolyers can be controlled through the concentration of each adsorbate solution. The surface coverage of the adsorbates on the InP(100) surface was lower than that of similar self-assembled monolayers (SAMs) on gold. The tilt angles for all adsorbates were higher than those of similar adsorbates on metal surfaces. The data from this report is important for the successful chemical functionalization of III-V semiconductor surfaces that are part of biosensor platforms such as light-emitting diodes.
Introduction The attachment of peptides to inorganic surfaces has been the subject of intense research in the past decade.1-4 A variety of substrates have been explored. In particular, the attachment of these biomolecules to semiconductor surfaces is of interest because of the possibility to construct hybrid inorganic-organic devices. Such devices can be used in either fundamental studies geared toward understanding the self-assembly process or in applied tasks such as the construction of better biosensors.5 The semiconductor surfaces studied so far have included II-VI and III-V materials.6,7 The majority of the systematic investigations of modified semiconductor substrates have centered on understanding the nature of binding of adsorbates to the surface of interest. Factors considered have included the surface chemical composition, crystal orientation, and the peptide size and sequence makeup. All of the published studies have looked at peptide adhesion to the semiconductor materials8 rather than at the use of specific functional groups that can potentially bond to the surface via covalent or ionic bonds. Covalent binding of molecules, and especially biomolecules, to semiconductors offers several advantages for subsequent biosensor applications.9 Among these advantages, stability and control over the conformation of the biomolecule rank very high. These two parameters are linked to device longevity, specificity, and selectivity. Covalent attachment of biomolecules to III-V semiconductors has not been as frequently employed as covalent attachment of adsorbates to metal surfaces. This is due to the presence of native oxide species on many of the technologically important materials such as GaAs, InP, and GaP.10-13 Recent interest in these surfaces has led to several systematic studies involving GaAs.14,15 Covalent attachment of molecules to GaAs via thiol functionalities has offered the opportunity to compare and contrast the quality, stability, and structure of the selfassembled monolayers (SAMs) with respect to the ones frequently formed on metals such as gold. Researchers have * To whom correspondence should be addressed. E-mail: albena@ purdue.edu. † Department of Chemistry. ‡ Weldon School of Biomedical Engineering.
presented strong evidence toward the conclusion that under the appropriate conditions direct substrate-molecule attachment via a covalent bonding is possible on this surface.14 Several similarities and notable differences compared to the assembly of monolayers on Au(111) surfaces have also been observed. A great deal of effort has been made to decipher the specific type of bonding to the GaAs surface. Thorough experiments with surface-sensitive techniques such as X-ray photoelectron spectroscopy (XPS) point toward the formation of As-S bonds on the surface. Other than GaAs, no other III-V semiconductor surfaces have been the subject of comprehensive monolayer studies. In particular, very few efforts have been made to understand the composition and structure of films composed of biomolecules on III-V semiconductors surfaces such as GaP, InP, and InAs. In this report, we examine the formation of SAMs on InP surfaces. Homogeneous and mixed monolayers were formed using four different synthetic peptide sequences and three thiol molecules, Table 1. The properties of the monolayers were characterized by contact angle measurements, ellipsometry, atomic force microscopy (AFM), and XPS. Our results show evidence for the formation of a covalent attachment via the sulfur when alkanethiols are used under conditions without rigorous control over humidity and oxygen. In the case of the peptide molecules, the XPS confirmed their presence on the surface and showed no evidence for either an In-S or a P-S bond. The films we created were not composed of multilayers and did not form thicker islands after they were examined by AFM. The thickness values obtained by ellipsometry measurements confirmed the AFM observations. Our studies also detailed that the mixed monolayers’ quality and composition were concentration-dependent. Our results suggest that mixed monolayers of peptides and alkanethiols can be used to functionalize InP in a rational and controllable fashion. Experimental Section Reagents and Materials. Fe-doped (4.5 × 108 cm-3) n-InP(100) wafers were obtained from Crystacomm (Mountain View, CA). 16-Mercaptohexadecanoic acid (MHA), 90%; 1-octade-
10.1021/jp066109w CCC: $37.00 © 2007 American Chemical Society Published on Web 02/13/2007
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TABLE 1: List of All Adsorbates adsorbates
adsorbates composition
TAT peptide 1 TAT peptide 2 TAT peptide 3 TAT peptide 4 1-octadecanethiol (ODT) 16-mercaptohexadecanoicacid (MHA) 6-mercapto-1-hexanol (MHL)
H2N-Cys-Arg-LysLys-Arg-Arg-Gln-Arg-Arg-OH H2N-Cys-Arg-Arg-Gln-Arg-Arg-LysLys-Arg-OH H2N-Cys-Arg-Lys Lys-Arg-Arg-Gln-Arg-Arg-Arg-OH H2N-Cys-Arg-Arg-Arg-Gln-Arg-Arg-Lys Lys-Arg-OH HS(CH2)17CH3 HS(CH2)15CO2H HS(CH2)6OH
canethiol (ODT), 98%; and 6-Mercapto-1-hexanol (MHL), 97% were purchased from Aldrich. All other solvents were purchased from Mallinckrodt Chemicals. The four peptide sequences (Table 1) containing an arginine-rich RNA-binding domain were synthesized by Bio-Synthesis (Lewisville, TX) where they were purified by high-performance liquid chromatography (HPLC) and were analyzed by MALDI-TOF. Surface Cleaning and Functionalization. Individual InP substrates (0.5 × 0.5 cm2) were consecutively cleaned by ultrasonication with three solvents: acetone, methanol, and isopropanol. Subsequently, each surface was immersed in concentrated HF solution (49%) for 10 min. The InP substrates were then rinsed with water and by isopropanol.16 The HF etch process removes the native oxide layer from the surface. An important part of the functionalization procedure involved the immediate placement of freshly cleaned InP into adsorbate solution. This resulted in a better modification with homogeneous and mixed monolyaers. (a) Homogeneous Monolayers. Solutions with a concentration of 1 mM were prepared in ethanol when MHA, ODT, and MHL were used. The freshly cleaned InP samples were immersed in each solution for 24-48 h. Subsequently, the samples were rinsed with ethanol and were dried under nitrogen gas. The adsorption of homogeneous monolayers using each peptide sequence was done at 4-8 °C using 1 mM solutions for 24 h. All peptide solutions were prepared in N,N-dimethyl-formamide (DMF). Following this treatment, the surfaces were rinsed several times with DMF to remove excess peptide. Each surface was washed multiple times with ultrapure water and was dried under nitrogen gas. (b) Mixed Monolayers. The mixed monolayers were prepared using mixed solutions of thiol and peptide in DMF. The twocomponent solutions were prepared from equal molar solutions using the following volume-to-volume ratios: 1:1, 1:5, and 1:10 (peptide:alkanethiol). Mixed monolayers were prepared using combinations of the four peptide sequences, ODT, and MHL. The thiols used were chosen to compare the effect of alkane chain and terminal functional group on the quality and properties of the mixed monolayers. Contact Angle Measurements. All contact angle measurements were performed using a Tantac, Inc Contact Angle Meter (Model CAM-PLUS MICRO). The static contact angles were read by the half-angle method. Each batch of surfaces was evaluated by putting a drop of water (∼1 µL) on a chosen substrate using a syringe equipped with a micrometer. In every case, the angle was measured within 30 s of the drop deposition. All surfaces were characterized by taking at least five measurements which were subsequently averaged. Atomic Force Microscopy (AFM) Characterization. AFM was used to characterize the topography of each surface. The instrument was Multi-Mode Nanoscope IIIa from Digital Instruments. The nanoscope software was used to analyze all AFM images. Single-beam-shaped tapping mode tips (Model # OTESPA7) were purchased from Veeco Instruments, CA. The spring constant was 42 N/m. The imaging was done at room
temperature (24-27 °C) and humidity of 20-40%. Depending on the experimental conditions, the imaging was carried out with scan speeds of 2-3 Hz. The roughness of each surface was determined by acquiring topography images at five randomly chosen locations. The roughness of each image was calculated using the Nanoscope software (Version 5.12r5), and an average for a particular surface was computed. X-ray Photoelectron Spectroscopy (XPS). All data were acquired using a Kratos Axis ULTRA X-ray Photoelectron Spectrometer. This instrument model has a monochromatic Al KR X-ray source, small area extraction optics, a spherical capacitor electron energy analyzer, and a dual-channel plate position sensitive detector. All samples were initially analyzed by taking survey spectra from 0 to 1100 eV at constant pass energy of 160 eV. Subsequently, high-resolution spectra for In 3d, P 2p, O 1s, C 1s, N 1s, and S 2p were collected at pass energy of 40 eV. Curve fitting of the all XPS spectra was performed using Gaussian-Lorenzian line shapes and a commercial software package. All data were analyzed using the XPS peak software (Version 4.1). All spectra were referenced by setting the hydrocarbon C 1s binding energy to 285.0 eV to compensate for residual charging effects. All data was plotted on the basis of the intensity obtained from the instrument and is shown as collected to represent signal-to-noise ratios. Ellipsometry. Ellipsometric measurements were collected with a Gartner Scientific ellipsometer (Model L116S). The instrument uses a HeNe laser and operates at a wavelength of 6328 Å and an incident angle of 70°. The refractive index of all adsorbates was assumed to be 1.5. The software used to compute the thickness values was LGEMP and was purchased from the instrument manufacturer. The thickness of each film on a given surface was measured at 10 different locations. Subsequently, average values were computed and standard deviations are reported. Results and Discussion The formation of monolayers composed of alkanethiols on InP has been the subject of previous research.17-21 Past studies confirmed that it is possible to covalently attach molecules to the In atoms on the surface. Other compounds used in the modification of InP have included different kinds of sulfides.22 To date, no comprehensive studies have been done to understand how to modify InP surfaces with biomolecules. The discussion in this paper compares and contrasts the ability of alkanethiols to assemble on InP surfaces with that of small synthetic peptides. All peptides, Table 1, were synthesized to contain a cysteine at their N-terminus. The peptide sequences we used belong to the family of so-called cell-permeating peptides and are derived from the TAT protein.23 These peptides are known to have specific recognition properties toward RNA. In the discussion below, we elaborate on the results we obtained from the various characterization techniques capable of revealing the film morphology, thickness, and peptide bonding to the InP surfaces. All data used to construct the figures in this discussion section
3712 J. Phys. Chem. C, Vol. 111, No. 9, 2007 is also shown in tabular form in the Supporting Information. We have performed some preliminary experiments to assess the biological activity of the peptides after they are anchored on the InP substrates. These studies will be part of a separate report we plan to publish in the future. In this work, we also explore the possibility of forming mixed monolayers on InP using biomolecules and alkanethiols as the adsorbates. Mixed monolayers have been extensively studied on metal surfaces since they can be applied in biosensing and tissue engineering strategies.24 Mixed monolayers provide a fast, convenient, and cheap way of forming wettability gradients on surfaces.25 Before one can optimally utilize mixed monolayers on InP for biological applications, it is necessary to understand their composition and structure. The surface characterization detailed in this part of the paper allowed us to map the properties of both homogeneous and mixed SAMs on InP surfaces. Monolayer Formation, Surface Morphology Characterization, and Wettability Measurements. We began this study by forming homogeneous monolayers of each of the adsorbates we chose to study, Table 1. All peptide sequences were similar in length and were rich in arginine content. The abundance and placement of the arginine in each sequence is important for future biorecognition experiments. For the purposes of this study, which concentrates solely on the adsorbates’ ability to form monolayers on InP surfaces, all peptides are highly cationic. The alkanethiol adsorbates had different terminal groups and varied in the number of carbons in the alkane chain. After we formed homogeneous monolayers of each adsorbate, we examined the possibility to form mixed monolayers using the following combinations: ODT and each peptide sequence as well as MHL and each peptide sequence. These combinations were chosen because previous studies have pointed out that the properties of mixed monolayers are dependent on the differences among the chain length of each adsorbate, the terminal groups, and ratios of each one on the surface.25 We examined the properties of the mixed monolayers composed of each of the peptides sequences because we anticipate using them in future device modification schemes. The composition of the mixed solution does not directly equal the composition of the mixed SAMs on the surface.26 Therefore, one needs to prepare each mixed monolayer of interest and systematically characterize it to decipher its composition. The morphology of the functionalized surfaces was initially characterized by AFM. The results from tapping-mode AFM measurements on each type of functionalized InP surface showed that they are smooth (see Supporting Information). The HF etched InP showed a higher surface roughness (rms value of 0.281 ( 0.059 nm) compared to that of clean GaAs (0.18 nm) and SiOx (0.17 nm) surfaces.27,28 The initial surface quality is known to have an influence on the quality and formation of monolayers when a solution adsorption method is used.29 Upon the adsorption of the three different alkanethiols (ODT, MHA, and MHL) as well as the TAT peptides from solution, the surface roughness did not change significantly. These fairly smooth surfaces (low rms values) suggest that reproducible monolayers can be formed on the InP surfaces. Furthermore, on the basis of the AFM images, one observes no clustering of the peptides on the surfaces. The AFM roughness measurements on the mixed SAMs indicate that the quality of the films was similar to the ones formed from homogeneous solutions. Furthermore, the heterogeneous surfaces showed no dominant features. However, slight changes in surface roughness, or contrast differences in phase images, are not enough to allow one to make quantitative conclusions regarding the amount of
Park and Ivanisevic peptide molecules on the surface. Overall, the AFM evaluation demonstrated the lack of peptide clustering on the surface and island formation when various adsorbates were used. Static contact angle measurements were carried out to observe the wettability of InP surfaces after each type of modification and are summarized in Figure 1. The cleaned InP surfaces are expected to change from hydrophobic to hydrophilic during the HF etching step. After cleaning, all surfaces had contact angles below 30°. From the contact angle on surfaces modified with homogeneous solutions, we concluded that one can generate high-quality monolayers on the cleaned InP regardless of the type of adsorbate used. The average contact angles for ODT, MHA, and MHL monolayers were 110°, 70°, and 37°, respectively. The hydrophobicity or hydrophilicity is expected to be dependent on the end functional groups. The results we obtained for the ODT and MHL monolayers are in agreement with previously studies.17 ODT monolayers are expected to form the most ordered films because of the interactions among the straight alkane chains.14,30 We had the most trouble getting reproducible results with MHA and speculate that the terminal groups can contribute to the formation of disordered monolayers.14,17 The choice of solvent can also influence the quality of the monolayers.31,32 Our qualitative observations show differences between monolayers formed in ethanol and DMF. We have not studied this dependence in enough depth and detailed characterization of the solvent dependence can result in a separate publication. In the case of all of the peptide adsorbates, the contact angles were measured to be ∼28°. This data provides evidence that the TAT peptides are attaching to the InP surface through a conformation that leaves the hydrophilic residue to point away from the surface.15,28,33 Two trends are observed in the contact angle measurements of mixed monolayers. In the case of ODT and peptide mixtures with different concentrations, there is a fairly smooth transition between extremely hydrophobic and extremely hydrophilic surfaces. When the concentration of ODT was increased, the surfaces were becoming more hydrophobic and vice versa. In all cases, when mixtures with different concentrations of MHL and peptide were used, the contact angles were more hydrophobic compared to the values obtained with homogeneous solutions of each of these adsorbates. The two trends we observe in Figure 1 are qualitatively the same as the ones reported on gold surfaces when mixtures of thiols with different functional groups and alkane chain lengths were used to form the monolayers.25 Our data shows that when two adsorbates with hydrophilic functional groups are utilized, and they differ considerably in length (MHL and the peptides), the mixed monolayers are more disordered than the ones that can form if homogeneous solutions are used. Our observations also indicate that when the mixtures are composed of adsorbates with two different types of terminal functional groups, hydrophobic (ODT) versus hydrophilic (peptide), the monolayers’ wettability was dependent on the concentration. Furthermore, the hydrophobicity of such monolayers can be more easily predicted. XPS Characterization. XPS was used to assess the chemical composition of the surfaces after the various adsorption steps. The technique allowed the chemical composition of the surfaces to be characterized using the elements’ characteristic binding energies. We began the characterization by taking survey spectra and looked for the presence of C 1s, In 3d, N 1s, O 1s, P 2p, and S 2p on all modified surfaces. In each case, we subsequently collected high-resolution XPS spectra to understand the structure of the various species on the surface. Figure 2 shows representative data from the following surfaces: (1) Clean InP; (2) InP
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Figure 1. Summary of the contact angle measurements and surface coverage data for all homogeneous and mixed monolayers. The coverage was calculated using the number of N atoms on the InP surface. The coverage for homogeneous monolayers composed of ODT, MHA, and MHL was calculated using the number of S atoms on the InP surface and is not plotted on each graph. See the discussion section for each value.
functionalized with ODT; (3) InP functionalized with MHA; (4) InP functionalized with MHL; (5) InP functionalized with TAT peptide 1; and (6) InP functionalized with a mixture of TAT peptide 1 and ODT (equimolar solutions; 1:5; v:v). The XPS data were used to understand several key issues: (1) how much of the oxide is removed from the InP surface after it is etched; (2) do adsorbates attach to In or P sites; (3) do mixed monolayers bind differently to the surface; (4) what are the differences in chemical compositions of the surfaces when different concentrations were used to make the mixed monolayers; and (5) do surfur-containing groups oxidize on the surface. Below, we discuss the most important quantitative data gathered from deconvoluting the high-resolution XPS spectra. Several different batches of samples were prepared to verify the validity of our results. In all cases, the data was referenced to the C 1s peak at 285.00 eV. All broad peaks we collected were deconvoluted. The deconvolution was performed with commercially available software and revealed the presence of several different chemical species after modification of the surface. The deconvolution results are summarized in the Supporting Information. The clean InP sample contained a lot of carbon, Figure 2A, from hydrocarbon contamination that was impossible to eliminate during the time it took to transfer the samples to the XPS chamber. Nitrogen was present on the surface only after the adsorption steps that utilized peptides, Figure 2C. Deconvolution of the peaks in such cases revealed the presence of NH2, NH3+, and amides, which confirms the presence of amino acids on the surface. Comparison among the C 1s and N 1s data for homogeneous versus mixed monolayers reveals that the presence of amide species is concentration-
dependent, and therefore one can tune the composition of the surface simply by adjusting the concentration of the adsorbate solution. The high-resolution spectra in the In 3d and P 2p region can be used to monitor the amount of oxides on the InP surface.17 As the InP surface was modified, the intensities and areas of the In 3d and P 2p peaks decreased, which indicates that the absorbates prevented oxidation of the InP surfaces. Figure 2B shows a comparison among the In 3d spectra from surfaces with different adsorbates. In these spectra, the peak centered at 444.6 eV is attributed to In 3d5/2 and is due to In-P bonds. In principle, one can monitor the formation of In-S bonds from the In 3d5/2 spectra since the formation of these bonds is expected to cause a shift by 0.3-0.8 eV. In our data, the In 3d5/2 peak does shift to a higher binding energy after the immobilization of each adsorbate. The second peak in Figure 2B centered at 452 eV corresponds to In 3d3/2 and also shifts to a higher binding energy after each adsorption step. The data for the P 2p region presented in Figure 2E clearly shows a broad peak with a shoulder at 128-131 eV. The broad peak is due to P-In bonding and its portion with higher intensity and lower binding energy is due to P 2p3/2 and its less intense shoulder at higher binding energy is due to P 2p1/2. If there were bonds between P and S, one would observe a peak at 132 eV upon the adsorption of analytes. We did not observe P-S bonding. The P 2p region can also be used to monitor the formation of InPO4/InPO3. These species are expected to cause the appearance of a broad peak around 134 eV. No clear peaks above the noise level were observed at ∼134 eV after analysis of each data set. However, analysis of the O 1s region, Figure 2D, shows
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Figure 2. Representative high-resolution spectra for the following regions: (A) C 1s, (B) In 3d, (C) N 1s, (D) O 1s, (E) P 2p, and (F) S 2p. The numbers next to each spectra are used to denote the following surfaces: (1) Clean InP, (2) InP functionalized with ODT, (3) InP functionalized with MHA, (4) InP functionalized with MHL, (5) InP functionalized with TAT peptide 1, and (6) InP functionalized with a mixture of TAT peptide 1 and ODT (equimolar solutions, 1:5, v:v).
that some small amount of oxides are present on the surface after the cleaning procedure and that they remain on the surface regardless of which type of adsorbate is used. The data in Figure 2F represents the S 2p region. When alkanethiols are utilized, S binds to In which results in peaks centered between 161 and 163 eV. We recorded no evidence for the formation of In-S bonds when homogeneous peptide solutions were adsorbed on the surface. The high-resolution XPS data shows no evidence for the formation of a covalent bond between the peptides and the surface. On the basis of the contact angle of the cleaned InP substrate, one expects it to be negatively charged and since all peptide adsorbates were cationic, the formation of ionic bonds with the surface is a possible mechanism of attachment. The low-contact angles measured after the peptide adsorption, which suggest that hydrophilic residues point away from the surface, and the high-tilt angles (see below) also support this mechanism. None of the homogeneous monolayers exhibited any S-O binding which results in a peak at ∼168 eV. The formation of S-O species in mixed monolayers was dependent on the concentration of the peptide. At low peptide concentrations, no S-O species were formed. This observation is expected since the mixed monolayers are more disordered than the homogeneous ones. Previous studies in the literature have indicated that disordered monolayers are more likely to be unstable over time and permit the formation of oxides on the surface.34 The XPS data can also be used to see if there is any relationship between the solution composition and the adsorbate film composition, Figure 3. After the deconvolution of the C 1s region, the two most important species one identifies are
C-C (284.9-285.0 eV) and amide C (288.2-288.9 eV). In the case of mixed monolayers composed of TAT peptides and ODT, one can increase the amount of C-C species in the films when the concentration of ODT is increased. This general trend is not observed when the films were prepared from mixtures of TAT peptides and MHL. This XPS data is consistent with the contact angle measurements described above. The composition of the mixed monolayers comprised of adsorbates with two different terminal groups (ODT vs peptide) can be more easily controlled by adjusting the concentration of each molecule in the mixture. Film Thickness Characterization and Surface Coverage Calculations. We employed two different techniques to determine the thickness of the films on the InP surface. The thickness was determined to understand the orientation of the molecules within the film when homogeneous and mixed monolayers were formed. In principle, Fourier transform-infrared reflection absorption spectroscopy (FT-IRRAS) is the technique of choice when one needs to understand the orientation of adsorbates on the surface.30,35,36 Our FT-IRRAS investigations showed only negative absorption bands when s-polarization was used which is indicative of low-absorption substrates.37 In general, we had trouble getting reproducible FT-IRRAS results. Therefore, we opted to understand the orientation of the molecules on the surface by first characterizing the thickness of the films through ellipsometric and XPS data. The ellipsometric thickness values are contrasted to the thickness extracted from XPS measurements in Figures 4-6. The values for the homogeneous films support the notion that a monolayer was formed on the surface. The ellipsometric thickness of the mixed monolayers showed
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Figure 3. Adsorbate film composition based on data extracted from the high-resolution C 1s region of the XPS data. The XPS data was deconvoluted to extract the percent of C-C and amide C species. The plots show the relationship between the solution composition used to prepare the monolayer and the deduced film composition.
it is easier to control the thickness of the mixed monolayers when the TAT peptides and ODT are used to adjust the concentration of the adsorbate solutions. The film thickness was also determined from the attenuation of the XPS intensities of the InP substrates. The thickness was obtained using the standard uniform overlayer model which is given by eq 1:39-41
[
( Lt )]
I ) I o 1 - exp -
Figure 4. Film thicknesses derived from (A) XPS data and (B) ellipsometry data for homogeneous monolayers composed of each adsorbate.
dependence on the ODT concentration and no dependence on the MHL concentration. These observations are consistent with the notion that more disordered films are formed when the shorter alkanethiol is used. Disordered films can be nonuniform at various surface locations, and this can be a major source of error in the ellipsometry measurements. Nonuniform substrates are known to affect the refractive index.38 In addition, the trends one observes in the thickness values obtained by ellipsometry and XPS measurements are similar. These trends indicate that
(1)
where I o is the intensity of the In 3d5/2 and P 2p peaks from the InP substrates before the adsorbates were immobilized, I is the intensity of the In 3d5/2 and P 2p peaks from the InP substrates after the adsorption procedure, t is the thickness of the absorbate film, and L is the electron attenuation length (EAL) for the In 3d5/2 and P 2p peaks. EAL values were obtained using NIST SRD-82 software on the basis of the kinetic energy (KE) of the electrons, photoionization asymmetry parameter (β), inelastic mean free path (IMFP), and transport mean free path (TMFP).42-44 The kinetic energy of the photoelectron is given by eq 2:39
KE (eV) ) 1486.6 - BE
(2)
where BE is the binding energy of the photoelectron and 1486.6 is the energy of the X-rays.45-47 All the calculated and estimated parameters that were used in the thickness calculation are given in the Supporting Information. The thicknesses obtained from the XPS data are plotted next to the ellipsometric thicknesses in Figures 4-6. The values for the thicknesses calculated using the intensities of In 3d5/2 differed from the ones calculated using
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Figure 5. Film thicknesses derived from (A-D) XPS data and (E-H) ellipsometry data for mixed monolayers composed of TAT peptides and ODT.
Figure 6. Film thicknesses derived from (A-D) XPS data and (E-H) ellipsometry data for mixed monolayers composed of TAT peptides and MHL.
the intensities of P 2p. The P 2p peaks experience weaker attenuation.48 The high-resolution data we collected showed evidence for the formation of bonds between the In atoms and the alkanethiol adsorbates we used. Therefore, we chose to
utilize the thickness values calculated using the intensities of the In 3d5/2 peaks in our further analysis. The adsorbate film coverage, which is presented together with the contact angles in Figure 1, was calculated using a previously
Monolayers on Indium Phosphide Surfaces published procedure that has been validated for DNA films and peptide films.40,41 The coverage of the peptides was calculated using the number of N atoms on the InP substrates. The coverage of the alkanethiols was calculated using the number of S atoms on the InP surface and is not shown on the plots. The coverage for ODT, MHA, and MHL was 4.57 × 1013 molecules/cm2, 2.68 × 1013 molecules/cm2, and 2.46 × 1013 molecules/cm2, respectively. The coverage of alkanethiols on gold (4.65 × 1014 molecules/cm2)34 is higher than what we obtained on the InP (100) surface. The results also show that the homogeneous films of peptides on the InP (100) form very nonuniform monolayers. In addition, the data indicates that the peptide coverage can be increased and controlled by adjusting the concentration of the long alkanethiol (ODT) when mixed solutions are used. Rational control over the coverage of peptides on the surface with the short alkanethiol (MHL) and mixed solutions was difficult to accomplish and reproduce. This is presumably because of the inability of MHL to form very well ordered monolayers because of its length and polar end functional group. Figure 1 also allows us to contrast the contact angle data with the coverage of peptides on the surface. In the case of mixed monolayers composed of TAT peptides and ODT, when the coverage of peptide on the surface decreases the contact angle is higher. This observation is consistent with our earlier observation that one can control the coverage of the peptides on the surface by adjusting the concentration of each component in the mixed solution. In the case of films composed of TAT peptides and MHL, the trends observed in the contact angles and coverage data are similar and support the notion that the monolayers are fairly disordered. The XPS thickness values we calculated can be used to obtain the tilt angle of the adsorbates with respect to the surface normal. We chose to use the XPS thickness for these calculations as opposed to the ellipsometric thickness since the XPS analysis relies on a minimal set of assumptions for biomolecular films40 and the ellipsometry results were greatly affected by the nonuniformity of the film. We assumed that the lengths of ODT, MHA, and MHL were 28.5 Å, 25.2 Å, and 13 Å, respectively. The tilt angles for ODT, MHA, and MHL were calculated to be 46.5°, 61.3°, and 82.9°, respectively. The tilt angle for ODT is in agreement with previously published data on a similar alkanethiol structure adsorbed on InP (100).18 The lengths of TAT peptide 1, 2, 3, and 4 were assumed to be 44.5 Å, 44.5 Å, 49 Å, and 49 Å, respectively. The tilt angle for TAT peptide 1, 2, 3, and 4 were calculated to be 84.9°, 84.8°, 84.5°, and 82.7°, respectively. The estimates for the tilt angles are in agreement with the rest of the data discussed so far. Most of this evidence, with the exception of the ODT films, showed that the monolayers we formed were fairly disordered. The data we report here suggests that adsorbates generally tend to have higher tilt angles on the III-V semiconductor surfaces when compared to values in the 30° range for metal surfaces such as gold and silver.30 Also, the tilt angle of alkanethiols on another III-V material, GaAs (100), has been reported to be 57°.49 In our laboratory, we have done extensive studies to understand the structure and quality of monolayers composed on alkanethiols and TAT peptides on GaAs surfaces.15,50,51 In comparison with the results we report here on InP surfaces, our previous work on GaAs has shown that one can utilize various peptide sequences and form monolayers with similar surface chemistry and homogeneity. One can also observe evidence for the covalent attachment of the biomolecules to the GaAs surface when the S 2p region was examined. This is in contrast to the XPS data we obtained when peptides were adsorbed on InP
J. Phys. Chem. C, Vol. 111, No. 9, 2007 3717 surfaces. In addition, the quality of the monolayers we have previously formed on GaAs is different compared to the ones we describe in this paper. Peptide, as well as alkanethiol monolayers, exhibit crystalline or well-ordered packing on the GaAs surfaces. Conclusions In summary, we present a detailed characterization of the properties of homogeneous and mixed monolayers formed from solutions of alkanethiols and cysteine-terminated peptides. The properties of these SAMs on InP(100) surfaces exhibit some notable differences when compared to similar systems on metal surfaces such as gold. Evidence for bonding between In-S was collected for all alkanethiols we examined. No evidence that supports the formation of covalent bonds between the peptides and the surface was gathered. The contact angles before and after adsorption, as well as the high-tilt angles, support the notion that the cationic peptides can interact with the surface via ionic bonds. The composition of the mixed monolayers can be controlled through the concentration of the solutions used during the adsorption process. The wettability of all films can be rationalized on the basis of the composition of each solution used to form the monolayers. Film thickness measurement by two different methods confirmed the monolayer formation. These measurements were utilized to compute the tilt angle of the adsorbates with respect to the surface normal. The tilt angles of the adsorbates were higher than those reported for SAMs on gold. The coverage of the adsorbates on the surface was lower than similar molecules on metal surfaces. The films composed of peptides were fairly nonuniform and disordered. Acknowledgment. This work was supported by NASA under award No. NCC 2-1363 and NSF (CHE-0614132). Any opinions, findings, and conclusions expressed in this material are those of the authors and do not necessarily reflect the views of the National Aeronautics and Space Administration. The authors acknowledge experimental help from Dr. Richard Haasch (UIUC) to carry out the XPS characterization. All XPS experiments were performed at the Center for Microanalysis of Materials, UIUC, which is partially supported by U.S. Department of Energy under grant DEFG02-96-ER45439. Supporting Information Available: Tables summarizing the AFM data, XPS deconvolution data, thickness, and coverage data as well as all the calculated and estimated parameters used in the XPS calculations. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Long, J. R.; Oyler, N.; Drobny, G. P.; Stayton, P. S. J. Am. Chem. Soc. 2002, 124, 6297. (2) Shi, L.; Paoli, V. D.; Rosenzweig, N.; Rosenzweig, Z. J. Am. Chem. Soc. 2006, 128, 10378. (3) Wagner, G. J.; Wark, A. W.; Lee, H. J.; Codner, E.; Saeki, T.; Fang, S.; Corn, R. M. Anal. Chem. 2004, 76, 5677. (4) Yu, L.; Banerjee, I. A.; Matsui, H. J. Am. Chem. Soc. 2003, 125, 14837. (5) Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M. Nature 2000, 405, 665. (6) Peelle, B. R.; Krauland, E. M.; Wittrup, K. D.; Belcher, A. M. Langmuir 2005, 21, 6929. (7) Goede, K.; Busch, P.; Grundmann, M. Nano Lett. 2004, 4, 2115. (8) Goede, K.; Grundmann, M.; Holland-Nell, K.; Beck-Sickinger, A. G. Langmuir 2006, 22, 8104. (9) Seker, F.; Meeker, K.; Kuech, T. F.; Ellis, A. B. Chem. ReV. 2000, 100, 2505. (10) Barriere, A. S.; Desbat, B.; Mombelli, B.; Tournay, V. Appl. Surf. Sci. 1993, 64, 225.
3718 J. Phys. Chem. C, Vol. 111, No. 9, 2007 (11) Hollinger, G.; Bergignat, E. J. Vac. Sci. Technol., A 1985, 3, 2082. (12) Fu, Q.; Negro, E.; Chen, G.; Law, D. C.; Li, C. H.; Hicks, R. F.; Raghavachari, K. Phys. ReV. B 2002, 65, 075318. (13) Steward, M. P.; Maya, F.; Kosynkin, D. V.; Dirk, S. M.; Stapleton, J. J.; McGuiness, C. L.; Allara, D. L.; Tour, J. M. J. Am. Chem. Soc. 2003, 126, 370. (14) McGuiness, C.; Shaporenko, A.; Mars, C.; Uppili, S.; Zharnikov, M.; Allara, D. L. J. Am. Chem. Soc. 2006, 128, 5231. (15) Cho, Y.; Ivanisevic, A. J. Phys. Chem. B 2005, 109, 12731. (16) Schvartzman, M.; Sidorov, V.; Ritter, D.; Paz, Y. J. Vac. Sci. Technol., B 2003, 21 (1), 148. (17) Lim, H.; Carraro, C.; Maboudian, R. Langmuir 2004, 20, 743. (18) Yamamoto, H.; Butera, R. A.; Gu, Y.; Waldeck, D. H. Langmuir 1999, 15, 8640. (19) Gu, Y.; Lin, Z.; Smenthowski, V. S.; Waldeck, D. H. Langmuir 1995, 11, 1849. (20) Gu, Y.; Waldeck, D. H. J. Phys. Chem. B 1998, 102, 9015. (21) Zerullar, D.; Chasse, T. Langmuir 2002, 18, 5392. (22) Han, I. K.; Kim, E. K.; Lee, J. I.; Kim, S. H.; Kang, K. N. J. Appl. Phys. 1997, 81, 6986. (23) Long, K. S.; Crothers, D. M. Biochemistry 1995, 34, 8885. (24) Wijesundara, M. B. J.; Fuoco, E.; Hanley, L. Langmuir 2001, 17, 5721. (25) Folkers, J. P.; Laibinis, P. E.; Whitesides, G. M. Langmuir 1992, 8, 1330. (26) Roberts, C.; Chen, C. S.; Mrksich, M.; Martichonok, V.; Ingber, D. E.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 6548. (27) Brewer, N. J.; Leggett, G. J. Langmuir 2004, 20, 4109. (28) Cho, Y.; Ivanisevic, A. J. Phys. Chem. B 2005, 109, 6225. (29) Admiec, M.; Talik, E.; Gladki, A. Appl. Surf. Sci. 2006, 252, 3481. (30) Nuzzo, R.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112, 558. (31) Bain, C. D.; Evall, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7155.
Park and Ivanisevic (32) Kang, J. F.; Liao, S.; Jordan, R.; Ulman, A. J. Am. Chem. Soc. 1998, 120, 9662. (33) Cho, Y.; Ivanisevic, A. J. Phys. Chem. B 2004, 108, 15223. (34) Heister, K.; Frey, S.; Golzhauser, A.; Ulman, A.; Zharnikov, M. J. Phys. Chem. B 1999, 103, 11098. (35) Pluchery, O.; Chabal, Y. J.; Opila, R. L. J. Appl. Phys. 2003, 94, 2707. (36) Dluhy, R. A. J. Phys. Chem. 1986, 90, 1373. (37) Mielczarski, J. A.; Yoon, R. H. J. Phys. Chem. 1989, 93, 2034. (38) Surface and Thin Film Analysis; Bubert, H., Jenett, H., Eds.; WileyVCH Verlag GmbH: Weinheim, Federal Republic of Germany, 2002. (39) Sofia, S. J.; Premnath, V.; Merrill, E. W. Macromolecules 1998, 31, 5059. (40) Petrovykh, D. Y.; Kimura-Suda, H.; Tarlov, M. J.; Whitman, L. J. Langmuir 2004, 20, 429. (41) Popat, K. C.; Swan, E. E. L.; Desai, T. A. Langmuir 2005, 21, 7061. (42) Tanuma, S.; Powell, C. J.; Penn, D. R. Surf. Interface Anal. 2005, 37, 1. (43) Laibinis, P. E.; Bain, C. D.; Whitesides, G. M. J. Phys. Chem. 1991, 95, 7017. (44) NIST Electron Effective Attenuation Length Database, 1.1 ed.; National Institute Standard Technology: Gaithersburg, MD, 2001. (45) Tanuma, S.; Powell, C. J.; Penn, D. R. Surf. Interface Anal. 1994, 21, 165. (46) Schweppe, J.; Deslattes, R. D.; Mooney, T.; Powell, C. J. J. Electron Spectrosc. Relat. Phenom. 1994, 67, 463. (47) Scofield, J. H. J. Electron Spectrosc. Relat. Phenom. 1976, 8, 129. (48) Jablonski, A.; Powell, C. J. Surf. Sci. Rep. 2002, 47, 35. (49) Sheen, C. W.; Shi, J. X.; Maartensson, J.; Parikh, A. N.; Allara, D. L. J. Am. Chem. Soc. 1992, 114, 1514. (50) Cho, Y.; Ivanisevic, A. Langmuir 2006, 22, 1768. (51) Cho, Y.; Ivanisevic, A. Langmuir 2006, 22, 8670.
