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Cluster Properties of Peptides on (100) Semiconductor Surfaces Karsten Goede,*,† Marius Grundmann,† Kai Holland-Nell,‡ and Annette G. Beck-Sickinger‡ UniVersita¨t Leipzig, Institut fu¨r Experimentelle Physik II, Linne´ strasse 5, 04103 Leipzig, Germany, and UniVersita¨t Leipzig, Institut fu¨r Biochemie, Bru¨derstrasse 34, 04103 Leipzig, Germany ReceiVed February 23, 2006. In Final Form: June 12, 2006 Peptide adhesion on semiconductor surfaces is quantitatively investigated by atomic force microscopy. The selected peptides are shown to cluster at the surface, with the larger, higher, and softer clusters appearing on the surfaces with lower peptide adhesion. Average cluster diameters vary from 40 nm on GaAs (100) to 300 nm on Si (100). Direct adhesion of the peptides to the surface competes with forming molecular aggregates that offer an overall reduced surface contact.
Introduction Peptide adhesion to semiconductors has enjoyed considerable experimental research interest in the last few years.1-4 This is partly because such hybrid organic-inorganic interfaces provide an easily accessible model system for the fundamental process of molecular self-assembly. Furthermore, possible future applications in fields such as lithography-free, oligo-molecular (opto)electronics, and the coupling of smart biomolecules to established data-processing systems or ultra-susceptible sensors may lie ahead. Yet currently, it is not outright clear what drives microscopically the specificity of these assembly processes. Predicting the adhesion behavior of peptides with known sequences to given (inorganic semiconductor) surfaces is still a challenge. In particular, it is under discussion to which extent adhesion of a hetero-peptide can be explained by interpolating between adhesion properties of its single amino acid components.3 Other empirical results suggest that best-adhering peptides at least on GaAs (100) exhibit rather complex sequences.1 A recent empirical study has provided a large database of how well homopeptides and related ones can mediate the adhesion of yeast to II-VI semiconductors.4 Specific adhesion results2-4 differ remarkably with respect to best-adhering amino acids or properties of their preferred substrate surfaces. Another completely open question is the true nature of the peptide-substrate binding. With a simulation-supported microscopical adhesion model still lacking, suggested phenomenological explanations seem to suffer in validity when applied beyond special cases. The succession within a given set of amino acids and hence peptide conformation should also play a major role in determining the adhesion of a peptide, as has been shown empirically.2,4 Correlating empirical data with simulations of peptide conformations, which account for the surrounding solvent and the resultant adhesion to a specific substrate lattice, should enable progress here. Promising approaches toward a theoretical adhesion model are a phase transition-simulating chain-growth * Corresponding author. E-mail:
[email protected]; tel.: +49 341 97-32654; fax: +49 341 97-32668. † Institut fu ¨ r Experimentelle Physik II. ‡ Institut fu ¨ r Biochemie. (1) Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M. Nature 2000, 405, 665-668. (2) Goede, K.; Busch, P.; Grundmann, M. Nano Lett. 2004, 4 (11), 21152120. (3) Willett, R. L.; Baldwin, K. W.; West, K. W.; Pfeiffer, L. N. PNAS 2005, 102 (22), 7817-7822. (4) Peelle, B. R.; Krauland, E. M.; Wittrup, K. D.; Belcher, A. M. Langmuir 2005, 21 (15), 6929-6933.
algorithm5 or an adopted hydrophobic-polar (HP) model.6 Different conformational phases5 of a given peptide might have a strong impact on its adhesion behavior since the display of the amino acid side chains to the surrounding solvent or the substrate is conformation dependent. In this paper, we want to draw attention to another intriguing aspect of peptides on semiconductors, namely, the formation of surface- and peptide sequence-specific clusters. This property has so far largely escaped research interest because of the dominance of luminescence detection of dye-tagged particles1,3,4 (providing insufficient spatial resolution) and because in some cases, the peptides have been fused to phages1 or yeast cells.4 The surface and sequence specificity of the peptide adhesion coefficient (PAC) (i.e., the surface fraction covered by peptides) has been investigated in ref 2 for three of the four peptide sequences that are used in this work. Materials and Methods Peptide Selection and Synthesis. Measurements presented in the current work mainly use the small peptide AQNPSDNNTHTH.7 It has been shown2 that its PAC on various (100) surfaces of classic inorganic semiconductors ranges from 25% on GaAs to 1% on Si under the same standard conditions. Peptides with derivated sequences are investigated to study the sequence specificity of the cluster formation effects. The peptides were synthesized by automated multiple solid-phase peptide synthesis (Syro, Multisyntech, Bochum, Germany) using the Wang resin to obtain a peptide acid (30 mg, resin loading 0.6 mmol/g) and by using the fluorenyl-9-methoxycarbonyl (Fmoc)/t-butyl strategy. Fmoc-amino acids (10-fold excess) were introduced by double coupling procedures (2 × 36 min) using in situ activation with diisopropylcarbodiimide and 1-hydroxybenzotriazol. The Fmoc removal was carried out with 40% piperidine in DMF for 3 min, 20% piperidine for 7 min, and finally 40% piperidine for 5 min. The peptides were cleaved by using a mixture of trifluoroacetic acid/thioanisol/thiocresol (90:5:5, v/v) for 3 h. Afterward, the peptides were precipitated from ice-cold diethyl ether, collected by centrifugation, and washed 4 times. Purification of the peptides was achieved by preparative HPLC on a RP C-18 column (Waters, 300 × 25 mm, 5 µm) with a gradient of 20-70% B in A (A ) 0.1% trifluoroacetic acid in water and B ) 0.08% trifluoroacetic acid in acetonitrile) over 45 min and a flow of 15 mL/min. All (5) Bachmann, M.; Janke, W. Phys. ReV. Lett. 2005, 91, 058102. Bachmann, M.; Janke, W. Phys. ReV. E 2006, 73, 020901 (R). (6) Bogner, T.; Degenhard, A.; Schmid, F. Phys. ReV. Lett. 2004, 93, 268108. (7) A: alanine; Q: glutamine; N: asparagines; P: praline; S: serine; D: aspartate; T: threonine; and H: histidine. Abbreviations according to the proposals of the IUPAC IUB commission for biochemical nomenclature, Eur. J. Biochem. 1984, 139, 9-37.
10.1021/la0605236 CCC: $33.50 © 2006 American Chemical Society Published on Web 08/12/2006
Peptide Properties on (100) Semiconductor Surfaces relevant fractions were collected and further analyzed (by MALDITOF mass spectrometry and RP-HPLC). The achieved purity of the peptide was g95%. Sample Preparation. All GaAs samples have been grown by metal-organic vapor phase epitaxy. The other samples have been prepared from commercially available substrates. (100) grown semiconductor sample pieces with an average surface sizes of 1 cm2 have been prepared. Native oxides and other particles covering the surfaces were removed by etching for 1 min in an ammonium fluoride solution (87.5% NH4F: H2O and 12.5% HF: H2O) followed by a water rinse and distilled water bath for 2 min. This etch does not attack the pure semiconductor surfaces. Si samples showed typical hydrophobic properties after etching, indicating a clean Si surface. We have investigated the clean semiconductor surfaces by AFM to estimate the cleanness and flatness of the respective surfaces. The maximum particle coverage was 0.2%, which is well below the PACs discussed in the text. After images were leveled by a firstorder plane fit when necessary to remove a sample tilt, the maximum roughness rms of the clean (100) sample surfaces was 0.37 nm, while for most samples, the roughness rms value was well below 0.30 nm. These values are typical for freshly etched surfaces of the investigated semiconductors. Suitable clean and flat sample pieces have been exposed to a diluted watery solution of the respective peptide within seconds after etching. The peptide concentration in this Tris-buffered saline (Carl Roth, Karlsruhe, Germany; pH 7.6) was 1 µg/mL, and the dwell was 2 h. This saline solution in distilled water was used to minimize peptide cluster lumping in solution. Samples have been subsequently exposed to a water rinse and distilled water bath to remove unbound particles. In this way, we can exclude the observed effects to result from the different semiconductor surface energies that might otherwise lead to surface-specific formation of loose peptide clusters. Peptide particles that still appear on the surface after the wash can be expected to have formed a strong binding to the surface when the semiconductor was still in solution. This sticking to the surface holds for the majority of clusters.2 Measurements. After a drying time of 6 h in air, the sample surfaces were investigated by AFM. We used a Dimension 3000 AFM in combination with a Nanoscope IIIa (Digital Instruments, now Veeco, Woodbury, NY) operating in tapping mode. The AFM probe was n+ silicon with a 123 µm cantilever and a spring constant of 59 N/m driven near its resonance frequency of 380 kHz. Scan rates were of the order of 5.0-0.15 µm/s, depending on the image size of 10-0.5 µm. Very similar images have been obtained with other probes (226 µm, 188 kHz, 45 N/m). PACs have been obtained by performing a grain analysis for each image using the SPIP program (version 1.9223, Image Metrology A/S, Lyngby, Denmark). Images were leveled using a first-order plane fit when necessary to remove a sample tilt. By doing so, it was possible to set the minimum detection height in the grain analysis to 0 nm above the average surface height, thus allowing each cluster to be detected. In the following, errors denote the standard deviation from the mean of eight equivalent measurements (and grain analyses), four of which have been performed on a second, equivalently prepared sample. Because of the relatively small cluster sizes on GaAs and GaP, 1 µm2 areas were analyzed on those samples, while for the other semiconductors, (10 µm)2 areas were used. However, no small clusters were overlooked as has been carefully checked on smaller analysis areas. Analyses of topography and phase images were carried out separately for the respective images of the same measurement. The phase-lag signal, which is recorded simultaneously with the topography information, can be used to map surfaces with different stiffness of the constituting substances.8 Energy dissipation during tip-sample interactions9 with its various causes10,11 translates into phase changes of the oscillating tip during scanning. Therefore, phase-lag imaging is well-suited for (8) Magonov, S. N.; Heaton, M. G. Am. Lab. 1998, 30, 9-15. (9) Cleveland, P.; Anczykowski, B.; Schmid, A. E.; Elings, V. B. Appl. Phys. Lett. 1998, 72, 2613-2615. (10) Bar, G.; Thomann, Y.; Brandsch, R.; Canthow, H.-J.; Whamgho, M. H. Langmuir 1997, 13, 3807-3812. (11) Raghavan, D.; Gu, X.; Van Landingham, M.; Nguyen, T. Langmuir 2000, 16, 9448-9459.
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Figure 1. Typical AFM topography images of (100) surfaces of GaAs (a) and Si (b), respectively. Both surfaces are partly covered by clusters of the peptide AQNPSDNNTHTH. (a) 1 µm2 surface area, height contrast 0.9 nm, and 454 clusters. (b) 100 µm2 surface area, height contrast 3 nm, and 18 clusters. detecting soft peptide clusters on hard semiconductor surfaces,2 hence providing qualitative information on the cluster softness and thus the material density within a cluster.
Results and Discussion Typical AFM topography images for peptide-exposed (100) surfaces of GaAs and Si, respectively, are depicted in Figure 1: the peptide AQNPSDNNTHTH adheres very well to GaAs and barely to Si. Figure 2 quantitatively shows the substrate dependence of cluster ensemble properties on various (100) semiconductor surfaces, as derived from the grain analysis of the respective AFM images. The PAC results in Figure 2a recall the overall adhesion result:2 the peptide AQNPSDNNTHTH adheres very well to medium-polar surfaces (like GaAs) and barely to nonpolar surfaces (like Ge and Si). This adhesion specificity very likely stems from the interplay between the substrate surface and the amino acid side chains (dominated by polar functional, hydrophilic groups in this peptide).1,2 Reading the figure from top to bottom, the number of clusters per area (Figure 2b) decreases even more drastically than the adhesion itself. Obviously, the clusters become laterally larger12 (Figure 2d) and higher (Figure 2e) the lower their respective PAC on a substrate. The share of small, oligomolecular clusters (defined as being smaller than 150 nm in diameter) also decreases for substrates with a lower PAC (Figure 2c). A typical GaAs sample has collected 3 × 10-5 mm3 peptide clusters per cm2 surface, while for other samples than GaAs, this amount is much lower. This figure can be derived from the PAC on GaAs (25%, Figure 2a) under the described conditions and the height of an average cluster on this surface (1.2 nm, Figure 2e) under the approximation that maximum and average height are equal for any one cluster. Assuming that the clusters have the same material density (very roughly 0.2 g/cm3) as the initial peptide powder used for preparing the solution, this corresponds to ∼6 ng per cm2 surface area or ∼0.02% of the peptide material within a typical 1 µg/mL, 30 mL solution bath. Accounting for the molecular weight of the peptide (1353.3 g/mol) and Avogadro’s number, this leads to ∼40 000 peptide molecules per µm2 GaAs surface and thus (Figure 2b) ∼100 peptide molecules per average cluster on GaAs. Elsewhere, a density of 20 000 peptides per µm2 has been reported as a typical value in the case of strong adhesion.3 Since a volume of ∼1500 nm3 can be assumed for a typical cluster on one of our GaAs samples (see Figure 2d,e), the very reasonable value of 15 nm3 can be deduced as the volume of a single peptide molecule. (12) The quantity diameter is defined here as 2 × sqrt (cluster area divided by PI), which evaluates the true diameter of a circle.
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Figure 2. Ensemble-related properties of AQNPSDNNTHTH peptide clusters on various (100) surfaces of semiconductors. Average properties of the cluster ensemble are shown in light gray (a-c) and individual cluster properties averaged over all clusters in the investigated ensemble are shown in gray (d-f).
is mainly governed by the respective mutual attraction between peptide and substrate and that there is no major causal phase-lag cluster size dependence. If Hooke’s law holds for the case of pressing soft peptide cluster balls by the AFM tip on hard surfaces, a phase-lag independent of the cluster size is expected. This holds as long as the spring constant (i.e., the cluster material compliance) is independent of size and that the cluster is not so flat that it can be completely compressed by the tip, eventually the tip feeling the hard substrate. This expectation of a size independent phaselag is experimentally considered in more detail in the following. While Figure 2c-e deals with surface-specific properties averaged over all clusters in an ensemble, Figure 3 displays individual cluster properties of two typical ensembles on the GaAs (Figure 3a,b) and Si (Figure 3c,d) (100) surfaces, respectively. These two ensembles are also shown in the AFM images of Figure 1. The cluster height increases for both substrates roughly linearly with the cluster diameter. For Si, the phase-lag of a cluster seems to be indeed independent of its size. For GaAs, however, the picture is different: a clear phase-lag increase with size for small clusters weakens for bigger ones. We have empirically found this behavior to be very typical for GaAs and missing typical for low-PAC surfaces such as Si. Large clusters (diameter >40 nm) may thus approximately be described by Hooke’s law. Such weak or nonexistent size phase-lag dependencies within the same ensemble (Figure 3a,c) confirm that the much more pronounced phase-lag increase on different substrates (Figure 2f) for decreasing PAC (Figure 2a) is largely caused by the changing material softness within the clusters and not due to the increasing cluster size (Figure 2d). This system dependent softness may reflect the peptide-material density within a cluster and possibly existing differences in intramolecular conformation or intermolecular stacking. Direct adhesion to the surface and adhering to an already existing peptide cluster seem to be the two energetically competing alternatives for a peptide molecule that is just entering the peptide-substrate system from the solution. Obviously, the outcome of this contest crucially depends on the components of the considered system. The sequence specificity of the cluster formation becomes obvious when peptides with closely related sequences2 to the original sequence AQNPSDNNTHTH are investigated. This is visualized in Figure 4. Two other sequences, AQAPSDAATHTH and AQNPSDNNTATA, have been synthesized by exchanging the amino acids N and H for A, respectively. The last sequence TNHDHSNAPTNQ is a randomly permutated sequence of the original one. Clearly, creating small (big) peptide clusters is not a general property of GaAs (Si) but depends on a large extent on how well the peptide under consideration adheres. For high PACs, the cluster diameter generally tends to be smaller than for low PACs. This would be in agreement with the previous reasoning. However, it appears that no simple reciprocal dependence between PAC and cluster diameter can be derived here. The presence of clusters in the solution away from the substrate can be excluded as source of the described clustering effects because of their surface specificity and the high solubility13 of these short peptides with their dominance of hydrophilic side chains1,2 and their very low concentration14 in the sample
The phase-lag signal is shown in Figure 2f to increase with decreasing PAC. This means that on average, the big clusters on the low-PAC surfaces are more compressed by the probing tip than the smaller clusters on the high-PAC surfaces. In the following, it will be shown that this phase-lag PAC dependence
(13) The peptide dissolves immediately when added to water, even in the starting solution whose peptide concentration is about 40 times higher than those of the actual sample preparing solution. (14) Accounting for the molecular weight of the peptide (1353.3 g/mol), the peptide concentration in the solution of 1 µg/mL corresponds to 4.5 × 1011 peptide molecules per mL of water or a dilution of 7.4 × 1010 (water molecules per peptide molecule). The solution is then 0.75 nM.
Peptide Properties on (100) Semiconductor Surfaces
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Figure 3. Log-log plots of the phase-lag (a and c) and height (b and d) as a function of the cluster diameter for the two typical cluster ensembles shown in Figure 1. (a and b) 454 AQNPSDNNTHTH clusters on 1 µm2 (100) surface of GaAs. (c and d) ∼17 AQNPSDNNTHTH clusters on 100 µm2 (100) surface of Si. For panels a and b and c and d, respectively, topography and phase-lag have been recorded simultaneously and separately analyzed, which explains the analysis mismatch in cluster size between topography and phase-lag images. Nominal size separations for small clusters in panel b are due to the fixed number of probe points per measurement line.
Figure 4. Cluster diameters for four different peptides on (100) surfaces of GaAs and Si, respectively. For each peptide, its sequence in short notation and the sequential change as compared to the original sequence is given below the diagram. Numbers in brackets denote the respective peptide adhesion coefficient on that surface, mostly from ref 2.
preparation solution (∼106 times lower than typical aggregationrelevant concentrations of relatively small peptides in experiment15 or simulation16). Furthermore, this cluster development is not the result of the surface-specific hydrophilicity that governs size and form of the evaporating water droplets during the sample drying: in that case, the clusters should be concentrated at certain surface spots and mechanically removable by washing. Yet, the majority (typically 66-75%) of clusters was empirically found2 to be distributed across the surface and to remain sticking to the surface, regardless of a washing process. Instead, we believe formation of the clusters takes place at the substrate surface either in the solution near the substrate or during the drying (15) Blondelle, S. E.; Forood, B.; Houghten; R. A.; Pe´rez-Paya´, E. Biochemistry 1997, 36 (27), 8393-8400. (16) Nguyen, H. D.; Hall, C. K. Biophys. J. 2004, 87, 4122-4134. Nguyen, H. D.; Hall, C. K. PNAS 2004, 101 (46), 16180-16185.
process, driven by the interplay of the (polar) peptide side chains with each other and with the more or less polar semiconductor surface. Although the persuasive assumption is at hand that on surfaces with a low PAC the peptide molecules prefer sticking to each other and thus forming bigger clusters rather than sticking to the surface atoms, the subtleties of this process are still to be revealed. We note that the described cluster appearance cannot be detected when the peptides are fused to cells1,4 or when the peptides are (without cell attachment) indirectly detected by recording the luminescence of an attached dye.3 Microscopical luminescence images (not shown) of our samples covered by dye-tagged peptides also do not allow for estimating the respective cluster sizes. Measurements of molecular circular dichroism (CD) spectra17 (not shown) reveal that under ambient temperature conditions, the vast majority of peptide molecules in a watery solution (concentration 10-4 M) exhibits a random coil structure. This applies for all four investigated sequences (see Figure 4). The existence of both helical and β-sheet structures can be excluded. This result also holds when the solution is made up of distilled water and 30% helix-supportive trifluorethanol. One might speculate that this higher flexibility of the amino acid side chains as compared to the helical situation enhances the peptide’s binding capacities. First conformation simulations18 of the peptides employed in this work predict chances to experimentally study the impact of conformational phase transitions (from random coil structures to helical ones) on the adhesion behavior when these peptides in low concentrated aqueous solution are cooled below sequence-specific temperatures. (17) Fasman, G. D. Circular Dichroism and the Conformational Analysis of Biomolecules; Plenum Press: New York, 1996. (18) Go¨koglu, G.; Bachmann, M.; Celik, T.; Janke, W. Structural Properties of Small Semiconductor-Binding Synthetic Peptides, Phys. ReV. E 2006, in press. Mohanty, S.; Irba¨ck, A. (Lunds Universided), private communication.
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Conclusion In summary, we have shown that exposing inorganic semiconductors to aqueous solutions of selected peptides can lead to self-assembled peptide cluster formation on their surfaces. The quantitative properties of these cluster ensembles are sequence and surface-specific, with the larger, higher, and softer clusters generally appearing on the surfaces with lower peptide adhesion. A qualitative explanation of these phenomena has been presented by arguing that for any peptide molecule, direct adhesion to the surface competes with forming molecular aggregates that offer an overall reduced surface contact. Such clustering should be considered in research directed toward hybrid organic-inorganic devices, be it as an obstacle or as a utilizable natural process.
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Acknowledgment. We thank V. Gottschalch for providing the semiconductor substrates and H. Herrnberger for help with the etching procedure, both at the Institut fu¨r Anorganische Chemie of Universita¨t Leipzig. We are grateful to F. Kremer of the Institut fu¨r Experimentelle Physik I of Universita¨t Leipzig for supplying us with the AFM. We are grateful to D. Haines at the Institut fu¨r Biochemie of Universita¨t Leipzig for synthesizing the peptides. We also thank M. Bachmann at the Institut fu¨r Theoretische Physik of Universita¨t Leipzig and G. Go¨koglu at Haceteppe U ¨ niversitesi Ankara for helpful discussions and performing first simulations of relevant peptide foldings and phase transitions. LA0605236
