Tailoring GaN Semiconductor Surfaces with Biomolecules - The

J. Phys. Chem. B 2008, 112, 8799–8805

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Tailoring GaN Semiconductor Surfaces with Biomolecules Elias Estephan,† Christian Larroque,‡ Fre´de´ric J. G. Cuisinier,§ Zolta´n Ba´lint,| and Csilla Gergely*,† Groupe d’Etude des Semi-conducteurs, UMR 5650, CNRS-UniVersite´ Montpellier II, 34095 Montpellier Cedex 5, France, Centre Re´gional de Lutte contre le Cancer Val d’Aurelle-Paul Lamarque, UniVersite´ Montpellier I, 34298 Montpellier, France, EA 4203, UFR Odontologie, UniVersite´ Montpellier I, 34193 Montpellier Cedex 5, France, and Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, 6726 Szeged, Hungary ReceiVed: May 9, 2008; ReVised Manuscript ReceiVed: June 6, 2008

Functionalization of semiconductors constitutes a crucial step in using these materials for various electronic, photonic, biomedical, and sensing applications. Within the various possible approaches, selection of materialbinding biomolecules from a random biological library, based on the natural recognition of proteins or peptides toward specific material, offers many advantages, most notably biocompatibility. Here we report on the selective functionalization of GaN, an important semiconductor that has found broad uses in the past decade due to its efficient electroluminescence and pronounced chemical stability. A 12-mer peptide (“GaN_probe”) with specific recognition for GaN has evolved. The subtle interplay of mostly nonpolar hydrophobic and some polar amino acidic residues defines the high affinity adhesion properties of the peptide. The interaction forces between the peptide and GaN are quantified, and the hydrophobic domain of the GaN_probe is identified as primordial for the binding specificity. These nanosized binding blocks are further used for controlled placement of biotin-streptavidin complexes on the GaN surface. Thus, the controlled grow of a new, patterned inorganic-organic hybrid material is achieved. Tailoring of GaN by biological molecules can lead to a new class of nanostructured semiconductor-based devices. Introduction Device functionality can be seriously limited by complex phenomena at the solid/liquid interface often resulting in a loss of activity of biological molecules due to unfolding upon immobilization.1 Therefore, complex chemistry like thiolylation2 and silanization3 are to be applied for efficient covalent coupling. Other approaches are based on the adsorption of functionalized self-assembled monolayers4 or polyelectrolyte multilayer films5 that act as an intermediary between the surface and protein. An alternative possibility is the selection of material-binding biomolecules from a random biological library, based on the natural recognition of proteins or peptides toward specific material.6,7 Functionalization is an important issue also in developing affinity-based optical biosensors. This class of sensors uses the biologically derived high selectivity for the detection of selective binding between target molecules and capture agents, like the ligand-receptor pair, antibody-antigen, nucleotides pairing, etc. The inconveniences of the existing optical biosensor methods are the required large sensing area as well as the substratedependent binding of molecules driven by unspecific interactions, which give rise to serious limitations in the detection accuracy and the detection limit. Miniaturization keeping at the * Corresponding author. Tel.: 0033467143248. Fax: 0033467143760. E-mail: [email protected]. † UMR 5650, CNRS-Universite ´ Montpellier II. ‡ Centre Re ´ gional de Lutte contre le Cancer Val d’Aurelle-Paul Lamarque, Universite´ Montpellier I. § UFR Odontologie, Universite ´ Montpellier I. | Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences.

same time a large sensing area by assuring high specificity of the sensing surface is the challenge of current research in biosensing. Therefore, our interest focused on developing new sensing substrates presenting high binding selectivity and sensitivity for biological molecules, as well as biocompatibility. The chosen substrate is the GaN (group III-nitride) semiconductor as it presents unique features for development of photonics-based devices due to the direct band gap electroluminescence in the blue and near-ultraviolet parts of its optical spectrum. Their transparency and their significant electro-optic, piezo-electric, and nonlinear coefficients mean that wide-bandgap III-nitride epitaxial structures provide an attractive potential base for the development of integrated optical devices and circuits for operation at wavelengths throughout the blue/violet part of the visible spectrum and a substantial part of the near-ultraviolet (UV) spectrum. The basic interest of using a GaN substrate is also due to its light emitting properties modified by exposure to a gas or liquid containing the molecule to be sensed (for chemical or biological agent identification).8 Applications of III-nitride semiconductors have been found in developing potentiometric anion sensors9 or in elaborating GaN-based heterostructures for sensor applications monitoring adsorption of ions, wetting by polar liquids, and exposure to gases.10 A GaN-based biosensor for detection of penicillin has also been reported.11 In most cases, surface functionalization has been performed by silanization. GaN is also a semiconductor that reveals an excellent chemical stability and biocompatibility12 rendering it very attractive for biomedical applications, too. Complex biological systems require a physiological environment and a sufficient degree of biocompatibility of the solid substrate. This already precludes the use

10.1021/jp804112y CCC: $40.75  2008 American Chemical Society Published on Web 06/26/2008

8800 J. Phys. Chem. B, Vol. 112, No. 29, 2008 of many materials for this purpose and/or requires the presence of specific buffer layers to increase the biocompatibility. Therefore, application of natural biomolecules like peptides during functionalization could be desirable in many cases, such as production of implantable optical sensors. Here, we report on surface modification of the GaN semiconductor by a specific 12-mer peptide produced by the combinatorial phage display technique, an original technology for identification and application of material-specific biomolecules. Due to their small dimensions and unique surface recognition properties, peptides could be further used for controlled placement of biomolecules on patterned surfaces. Our functionalization method could present an alternative solution also for the problems encountered in the actually existing sensing methods due to the unwanted unspecific interactions. Experimental Section Substrate Preparation. Wafers of undoped (n-type residual) GaN (0001) layer, grown by molecular-beam epitaxy on a sapphire substrate, were carefully cleaned by the successive treatment with trichloroethane, acetone, methanol, and rinsed deionized water and then dried with N2. In order to remove the native oxide, the samples were etched with dilute HCl (HCl:H2O ) 1:10) for 2 min, then immersed in deionized water for 1 min and nitrogen dried. The condition of the sample surface was monitored by Atomic Force Microscopy. Phage Display. The M13 bacteriophage library (Molecular Probes) was exposed to the GaN substrate in phosphate-buffered saline solution containing 0.1% TWEEN-20 (PBST). Following rocking for 1 h at room temperature, the surfaces were washed typically 10 times by PBST to rinse the unbound phages. In the next step, the bound phages were eluted from the surface under conditions that disrupt the interaction between the displayed peptide and the target: first a glycine-HCl (pH ) 2.2) solution was added for 10 min, and then the surface was transferred to a fresh tube and neutralized with Tris-HCL (pH ) 9.1). The eluted phages are infected into Escherichia coli ER2738 host bacterial cells and thereby amplified. After three rounds of biopanning, monoclonal phage populations may be selected and analyzed individually. Finally, 10 phages are recovered and amplified, followed by extraction and sequencing of their DNA that will define the sequence of the expressed peptide. Surface Functionalization. Once the sequences were determined, the specific peptide for the GaN semiconductor was synthesized. After surface preparation, a 5 × 5 mm sample of GaN (0001) was incubated in 100 µM of the specific peptide (diluted in PBST) for 2 h, followed by a thorough rinsing step to remove excess unbound peptide. Fourier Transform Infrared Spectroscopy in Attenuated Reflectance Mode. The FTIR-ATR spectra were obtained using a Nicolet, Avatar 330, spectrophotometer with a diamond ATR crystal. D2O was used as solvent instead of water because the amide I band of the protein is strongly affected by the appreciable absorption band of water at around 1643 cm-1 (O-H bending), whereas the corresponding vibration in D2O absorbs at around 1209 cm-1. When the spectrum of the peptide (1 mM) adsorbed on GaN was recorded, the GaN was placed with an upside down configuration on the top of the ATR crystal. Force Measurements by Atomic Force Microscopy. Relative binding strengths of the peptide were evaluated using MFP3D AFM from Asylum Research, Santa Barbara, California. Force measurements were taken at constant loading rates (vertical piezo velocity of 1 µm s-1). The spring constant of

Estephan et al. the tip was calibrated in the presence of PBS solution by the thermal fluctuation method and found to be about 18 pN nm-1. The ultrasoft AFM cantilever tips (Biolever-Olympus) were rinsed with copious amounts of Milli-Q water (18 MΩ cm) and then dried. In the next step, tip functionalization was performed: the AFM cantilever tips were incubated in 1 µg mL-1 biotinylated bovine serum albumin (BBSA; Sigma) solution in PBST, pH 7.0, at room temperature overnight, and then the tip was incubated for 30 min in 100 µg mL-1 streptavidin in PBST, and finally in BSA (1%) for 1 h to block the nonspecific binding sites. All the steps were separated by thorough rinsing. Biotinilated peptide was fixed on the tip prior to each measurement. Preparation of Patterned Surface. SiO2 masks were prepared on the GaN surface by the lift off method. This rather easy to use method involves only one masking step and the standard photolithography. A square shaped mask printed on a transparency was deposited on the GaN previously covered by a resin. Subsequently, the sample was exposed to UV light radiation, which renders the resin soluble according to the mask pattern. The deposit of the SiO2 was then performed by the plasma enhanced chemical vapor deposition method. Finally, the surface was cleaned of the resin with acetone, and a nested square pattern was etched containing 30 µm thick lines of GaN with 120 µm SiO2 spacing between each line. The height of the SiO2 deposition was 100 nm. Fluorescence Microscopy. Fluorescence images were captured with a Zeiss (Le Pecq, France) epifluorescence microscope equipped with a JAI (Glostrup, Denmark) charge-coupled device camera run by Metasystems (Altlussheim, Germany) image analysis software. For labeling, streptavidin FITC (excitation, 480 nm; emission, 540 nm, from Sigma Aldrich) was used. Results and Discussion 1. Elaboration of the Specific Peptide. Phage display is a powerful technology allowing the elaboration of peptides that interact with a desired target. The screening of phage display libraries is usually accomplished by biopanning processes based on an affinity selection: phage populations are exposed in turn to targets to selectively capture binding phages.13,14 Successive rounds of binding, washing, elution, and amplification ensure that the originally diverse phage population is increasingly enriched with phage with a certain propensity to bind to the target. Finally, monoclonal phage populations with desired specificities can be selected. As the genotype of each peptide phenotype is carried within phages, once the peptides of interest have been isolated, the sequence encoding them can be determined. Therefore, phage display has rapidly become a vital tool in studies aimed at identifying molecules that bind to a specific target and improving particular features of preexisting molecules with respect to, e.g., ultrafine specificity, affinity, and reaction rate characteristics. Phage displayed proteins/peptides can be screened against not only a wide range of biological targets but also inorganic ones.7 We used a M13 bacteriophage library to screen 1010 different 12-mer peptides against an undoped GaN (0001) layer (n-type residual), grown by molecular-beam epitaxy on a sapphire substrate. Native oxides were removed according to previously given procedures7 prior to adsorption of phages on the substrate. The screening procedure, based on affinity selection, was then performed: (i) phage particles in phosphate-buffered saline solution containing 0.1% TWEEN-20 (PBST) are exposed to the target GaN (0001) surface; (ii) nonbinding phages are removed by washing; and (iii) binding phages are eluted, infected into Escherichia coli ER2738 host bacteria, and thereby amplified. These biopanning rounds are then repeated, typically three times. In fact, the

Tailoring GaN Semiconductor Surfaces amplified phage population constitutes a secondary library that is greatly enriched in phage displaying peptides that bind to the target. If the biopanning steps are repeated, the phage population becomes less and less diverse as it becomes more and more enriched in the limited number of variants with binding capacity. After three rounds of biopanning, monoclonal phages were selected and analyzed individually. Finally, 10 phages were recovered and amplified, followed by extraction and sequencing of their DNA that will define the sequence of the expressed peptide. Several sequences have been identified such as: MSGDIISLAPTG, GPFFPKSLTTTS, NAPLSHIPENPR, SISAMPAPANSS, and SVSVGMKPSPRP. As the abundance of the last sequence was 60%, hence revealing a high affinity for the test surface, the sequence SVSVGMKPSPRP was selected as the specific peptide for the GaN (0001) surface, denoted “GaN_probe” in the following. This sequence presents a hydrophobic first half and a hydrophilic second half (as defined by Kyte and Doolittle15). We can note that the first SVSVGM hydrophobic part is followed by a positively charged lysine (K) residue then a sequence containing three proline (P) residues. Even if due to the short sequence of the peptide and the presence of the three proline residues, the existence of both helical and β-sheet structures can be excluded,16,17 and one might speculate that the presence of these amino acid side chains enhances the peptide’s binding capacities compared to the helical situation. Proline is unique among the naturally occurring amino acids in that the side chain wraps around to form a covalent bond with the backbone, severely restricting the backbone conformation of neighboring residues.18 Proline prevents the formation of hydrogen bonds and confers a high structural constraint to the peptide that could facilitate its adhesion onto the surface. It is still controversial to elucidate the peptide/semiconductor interface. Previous work has reported that the adhesion of an AQNPSDNNTHTH peptide to certain semiconductors is driven by substrate electronegativity and the acidity of amino acid side chains.19 We note that the SVSVGMKPSPRP peptide we have elaborated for the GaN is composed of seven nonpolar hydrophobic (2V, G, M, 3P), three neutral (3S), and two basic (K, R) amino acids; therefore, we can expect that other types of interactions might also contribute to the good adhesion of this peptide toward GaN, as will be discussed in the Section 3 below. 2. Characterization of the Functionalized GaN (0001). Once the sequence of the GaN_probe peptide was determined, the peptide was synthesized (Millegen, France) then presented on a GaN(0001) substrate. Fourier transform infrared spectroscopy in attenuated total reflection mode (FTIR-ATR) and atomic force microscopy (AFM) experiments have been employed to study and understand the GaN/peptide interface and how the selected peptide undergoes material recognition. 2.1. Secondary Structure of the Adsorbed Peptides. The structure of the peptide in adsorbed form was first checked by FTIR-ATR measurements. Two types of experiments have been performed. First the spectrum of 1 mM GaN_probe peptide in deuterated PBST has been recorded when a drop of peptide solution has been deposited directly on the ATR crystal, which is made of diamond (Figure 1A). Second, the spectrum of the same peptide adsorbed on the GaN surface has been registered (Figure 1B), also in ATR configuration. The first difference to be noted is that the spectrum due to the peptide adsorbed on the GaN is increasing considerably (×10), suggesting a high binding affinity of the peptide for the GaN compared to the diamond ATR crystal. Indeed, when the peptide was deposited directly on the ATR crystal, a very weak and almost featureless

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Figure 1. (A) FTIR-ATR spectra of the GaN specific peptide (1 mM) solution in PBST (in D2O) in the ATR cell, (B) when adsorbed on GaN and deposited on the ATR crystal. (C) Decomposition of the amide I band of the spectra presented in Figure 1B after smoothing and localization of minima of the second derivative that are used as starting values in the band decomposition.

spectra was recorded (Figure 1A), where only various vibration frequencies characteristic of the different amino acids of the peptide solution can be noticed (Table 1). As expected and discussed above, the unbound peptide presents no secondary structure, as no amide I band has been observed in the range of 1600-1700 cm-1. However, the peptide can be still identified due to the vibrations specific to the amino acid residuals (1475, 1735 cm-1). Contrary to the spectra of the “peptide in solution” (Figure 1A), behind the same vibrations typical to the peptide, the spectra of “peptide adsorbed on GaN” reveal a well-defined amide I band evidencing the appearance of secondary structures

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TABLE 1: Typical Vibration Frequencies for the Amino Acids of the Specific Peptide in the FTIR-ATR Absorption Spectrum from 500 to 4500 cm-1 20 λ (cm-1)

987

1091

vibration C-H C-O C-O-C C-C-C O-C-C S-O SdO

1457

1735

2553

2935

3432

C-H2 N-H O-H C-H3 N-H2 C-H3 NH2 S-H C-H CH3-O COOH CH2-O O-H C-O

when peptides are adsorbed (Figure 1B). The amide I region is very complex, but a curve fitting procedure can be applied to estimate quantitatively the area of several overlapping component bands assignable to different secondary structure elements. The amide I band was deconvoluted by fitting it with Lorentzianshaped components after smoothing and localization of minima which correspond to the positions of peaks within the band (Figure 1C). Vibrations typical for intermolecular β-sheet structures were identified at 1699 ((4) and 1686 ((3) cm-1 and at 1639 ((1) cm-1 for the intramolecular ones with the following weights: 44.55%, 19.34%, and 6.59%, respectively. The absorption at 1671 ((3) cm-1 is assigned to beta turns, with a weight of 22.05%. A band at 1651 ((2) cm-1 with a weight of 7.49% was also deconvoluted. This wavenumber is usually assigned to R-helices in proteins or longer polypeptides. The half-width for all peaks is less than 20 cm-1, which means that the secondary structure of the adsorbed peptide is well defined and dominated by β-sheet structures (71%) and beta turns (22%). Indeed, it is known that even if proline confers a strong constraint there is also a small but significant amount of flexibility in the proline ring coupled to the backbone conformation,18 allowing the formation of secondary structures. However, here we have to note that due to the rather high peptide concentration (1 mM) used to exceed the detection limit of the spectrograph, aggregation of peptides cannot be excluded. Our results demonstrate that the initially structureless peptide (when in solution) can undergo folding when adsorbed on GaN. 2.2. Morphology of the Functionalized GaN. For morphological studies of the functionalized GaN (0001) surface, AFM images in liquid were recorded in contact mode. Functionalization means exposing the surface to a peptide solution and letting it adsorb for 2 h, followed by a thorough rinsing. The high quality of an etched, clean GaN (0001) surface (Figure 2A) with a low roughness (in terms of root-mean-square, rms) of 0.12 ( 0.04 nm might allow a better resolution imaging when the AFM is operated in contact mode, then in tapping mode. When functionalized with its specific peptide at a concentration of 100 µM (Figure 2B), the formation of a very thin peptide layer is observed on the GaN surface, as the initial morphological features (0.5 nm steps corresponding to the lattice constant) of the bare GaN sample are still visible. Functionalizing lowers the initial roughness of the GaN from a value of 0.12 ( 0.04 nm to half of it, 0.07 ( 0.02 nm, confirming the formation of a thin homogeneous peptide layer on the GaN surface. Some traces of the scanning AFM tip can be seen on the image due to the contact mode, also enabling us to identify the peptide layer. When incubated with a more concentrated, 1 mM peptide (same as for the FTIR-ATR experiments), a thicker peptide layer and some aggregation were observed on the GaN surface (Figure 2C). The roughness increases to 0.27 ( 0.8 nm, confirming the appearance of inhomogeneities in the peptide layer. Finally, the reversibility of the functionalization has been

addressed by incubating the functionalized sample in 0.01 M sodium dodecyl sulfate (SDS) solution, then in 0.1 M HCl followed by a thorough rinsing, trying to remove the adsorbed peptide from the surface. Peptide aggregation and cluster formation was observed, resulting in an increased roughness value of 0.58 ( 0.09 nm (Figure 2D). The binding strength of the GaN_probe peptide seems really important, and we can state that functionalization is irreversible. Stability in time of the functionalized GaN samples has been also studied by checking the surface morphology when samples were kept in air for two days. The unmodified AFM images (not shown) evidence the stability of the peptide adhesion onto the GaN (0001), thus of the adsorbed species too. 3. Specificity of the GaN_Probe Peptide. The interaction forces between the GaN_probe peptide and GaN (0001) were measured by means of AFM in force mode. Force spectroscopy by AFM has been used extensively for measuring molecular binding forces for molecule pairs like streptavidin-biotin and antigen-antibody but also adhesion forces between proteins and surfaces.21,22 Monitoring of the unbinding process of adsorbed molecules under external stress leads to quantification of adhesion forces.23 The GaN_probe peptide was fixed (by streptavidin-biotin binding) on the previously functionalized gold-coated and ultrasoft AFM cantilever following the protocol described by Moy et al. (Figure 3).24 For this, the GaN_probe peptide was biotinilated at the C-terminus through a GGGSK spacer linker, thus the sequence of the GaN_probe became SVSVGMKPSPRPGGGSK-Biotin. The biotinilated peptide was fixed on the functionalized AFM tips prior to force measurement (Figure 3). The spring constant of the modified AFM tip was determined by the thermal noise method25 prior to each measurement and found to be about 18 ( 4 pN nm-1. The peptide-coated tip was then brought in contact (for 1 s) with the bare GaN (0001) surface, and the adhesion forces were measured at a constant retraction rate of 1 µm s-1. All the measurements have been performed in liquid. A typical force is presented in Figure 3A, and it was very reproducible for about 80-100 approach/retraction cycles. As the radius of curvature of the V-shaped Si3N4 tip is typically 25 nm, we can state that during an approach there is one molecule of streptavidin (with two biotin binding sites), thus two peptides that interact with the GaN (0001) surface. The histogram of the measured forces reveals a Gaussian shape with a mean force of around 300 pN (Figure 3B), proving that the interaction between the GaN-probe peptide and GaN (0001) surface has a mean value of 150 ( 10 pN. Compared to the strength of the H bonds of ∼17 pN26 and the high biotin-streptavidin strength ranging from 250 to 320 pN according to experimental conditions24,27,28 (highly specific and the strongest noncovalent bond), we can conclude on the specificity of the interaction between the GaN_probe peptide and the GaN of a strength of 150 ( 10 pN and state that it is a force situated in the range of weak antigen-antibody forces27 and of hydrophobic strengths. Indeed, we have already noted that the GaN_probe peptide reveals a hydrophobic and hydrophilic half-domain. To test the effect of the two different domains, two new classes of functionalized tips were produced: one with the hydrophobic, the other with the hydrophilic parts of the GaN_probe peptide. The hydrophobic (SVSVGM) and the hydrophilic parts (KPSPRP) of the peptides were synthesized, completed with some glycines (G) to have the same length as the initial GaN_probe peptide, then biotinilated via a linker to be fixed on the modified AFM tip. We have chosen to complete the sequence with glycines because they are the smallest amino acids and they do not interfere in the affinity of

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Figure 2. AFM height micrographs (1 µm ×1 µm, respectively, 5 µm × 5 µm areas) taken in contact mode of (A) bare GaN (0001) sample imaged in air, (B) GaN functionalized with his specific peptide at a concentration of 100 µM imaged in PBST solution, (C) GaN functionalized with his specific peptide at 1 mM (same concentration as for the FTIR-ATR) imaged in PBST solution, and (D) GaN after a cleaning treatment with SDS and HCl imaged in PBST solution.

Figure 3. Functionalization of AFM cantilever tips: biotin-streptavidin-biotinilated peptide binding. (A) Typical force-distance curve: as the tip approaches the surface (line I), binding between two peptides and bare GaN results in an adhesion that is measured as a rupture force when the tip is retracted from the surface (line II). (B) Histogram of the rupture forces measured between two GaN specific peptides and the bare GaN (0001) surface.

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Figure 4. (A) Histogram of rupture forces between two hydrophobic domains of the GaN specific peptides and the bare GaN (0001) surface. (B) Histogram of rupture forces between two hydrophilic domains of the GaN specific peptides and the bare GaN (0001) surface.

Figure 5. (A) Optical microscopic image of a masked GaN sample. (B) Fluorescent microscopy of the masked GaN sample exposed to its specific biotinylated peptide GaN_probe labeled with FITC.

the peptide to adhere to the surface and they give flexibility in the coupling between the peptide and biotin moiety.29 The forces measured between the bare GaN and the hydrophobic and hydrophilic peptides (Figure 4A and 4B, respectively) reveal a random distribution, and it is evident that the interactions within the whole peptide and the GaN (Figure 3B) are statistically higher and specific compared to the half-parts. However, a mean value of about 250 and 150 pN can be derived from the two distributions that certainly contain the contribution of two peptides fixed on the AFM tip as discussed already above. Thus, a mean value of 125 pN is obtained for the interaction between the hydrophobic domain of the peptide and the GaN surface, and a smaller value of 75 pN is deduced for the force between the hydrophilic domain of the peptide and the GaN. Thus, we can speculate that, even if for the specificity of the GaN_probe peptide-GaN binding the whole sequence is needed, the hydrophobic domain of the GaN peptide is essential for the binding specificity. Contrary, by presenting the GaN_probe against an InAs (100) surface, no adhesion force has been found (data not shown). Control experiments have been done, and no specific force was measured when tips modified either with only biotin or with streptavidin were brought in contact with the GaN surface. 4. GaN Patterning by Selective Localization of BiotinStreptavidin. Toward a biofunctionalization of the inert, inorganic, semiconductor-based structures, these peptides may find use as natural binding sites for the controlled placement of the biological molecules to be studied. The final product will then be a semiconductor-based substrate revealing an array of a single variety of molecules, therefore presenting all the advantages to constitute a biologically derived high selectivitybased optical biosensor. The main role of the linker molecule GaN_probe is 2-fold: (i) to maintain a sufficiently low density of electronic defects at the semiconductor surface, which otherwise would have a negative impact on the electronic properties of the heterostructure and (ii) to provide a high density of docking sites for the specific attachment of biomolecules. A

dense interfacial layer of small organic molecules is necessary to passivate electronic surface states and to protect the semiconductor from solvent molecules and ions, since biomolecules are much larger than the surface unit cell of semiconductor substrates. Biological molecules in direct contact with an unprotected solid substrate will be subjected to denaturation and loss of their specific biochemical functionality. As a proof of concept, we have employed fluorescence microscopy to demonstrate the selective attachment of the biotinilated specific peptide (SVSVGMKPSPRPGGGSK-Biotin) to GaN, in close vicinity to a surface of different chemical and structural composition. For this purpose, a nested square pattern was etched onto a GaN wafer (Figure 5A). First a mask was prepared on the GaN surface by the lift off method, and then the deposition of SiO2 was performed by the plasma-enhanced chemical vapor deposition method. The obtained pattern is formed by 30 µm thick GaN lines separated by 120 µm SiO2 spacing. The nested pattern was exposed to 20 µM of biotinylated peptide in PBST, rinsed, then incubated with bovine serum albumine, 1% (BSA), to close unspecific binding sites, and finally labeled with fluorescent streptavidinFITC (Figure 5B).The obtained surface is observed under a fluorescent microscope to image the fluorescently labeled peptides. The tagged peptides are seen as green lines in (Figure 5B), corresponding to GaN lines labeled by the GaN_probe peptide. The SiO2 regions do not reveal any fluorescence, thus this part of the pattern remained unbound by the specific peptide. Control experiments were performed, where the exposure to the specific peptide step was missing and no fluorescence was observed. These control experiments validated the success of the selective localization of streptavidin on theGaN (0001) surface by means of the GaN-specific peptide. Conclusions The sequence of a peptide having specific recognition ability for the GaN (0001) surface was for the first time determined,

Tailoring GaN Semiconductor Surfaces and the parameters responsible for surface recognition were studied. The GaN_probe peptide shows a high affinity for GaN compared to other materials (diamond, SiO2, InAs) and is intended to be used to functionalize GaN for mediating attachment of biomolecules. The peptide formed a thin homogeneous layer when adsorbed irreversibly on the surface. At high concentrations, secondary structures were observed in the adsorbed layer. The interaction forces responsible for the peptide recognition were quantified (150 ( 10 pN) and found to be in the range of hydrophobic strengths. For specificity, the whole sequence of peptide is needed; however, a primordial role can be attributed to the hydrophobic domain of the peptide. The peptide was further used for selective localization of streptavidin via biotin binding. Hence, the controlled grow of a new, patterned inorganic-organic hybrid material was achieved. Our successful selective biofunctionalization by means of peptides can be considered as a breakthrough toward elaborating oriented biomolecule-covered nanopatterned semiconductor substrates encompassing development of new biosensors with optoelectronic applications. Acknowledgment. This work was supported by the Phoremost European Network of Excellence, Project Nr. 511616: NanoPhotonics to Realise Molecular Scale Technologies. We are grateful for P. Martineau (CRLC Val d’Aurelle, Montpellier, France) for his useful hints in performing phage display. We thank G. Varo (Institute of Biophysics, BRC, Szeged, Hungary) for kindly making available the MFP3D-AFM (Asylum Research) in his laboratory for force measurements, and L. Zimanyi (Institute of Biophysics, BRC, Szeged, Hungary) for carefully reading and lecturing the manuscript. We also thank S. Rouffenach (GES, UMR 5650, Universite´ Montpellier 2, France) for providing the GaN wafer. References and Notes (1) Norde, W. AdV. Colloid Interface Sci. 1986, 25, 267. (2) Neves-Petersen, M. T.; Snabe, T.; Klitgaard, S.; Duroux, M.; Petersen, S. B. Protein Sci. 2006, 15, 343.

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