Adsorption and Stability of Streptavidin on Cluster-Assembled

Sep 27, 2008 - Scuola Europea di Medicina Molecolare, and Department of Experimental Oncology, European Institute of. Oncology, Campus IFOM-IEO, Via ...
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Langmuir 2008, 24, 11637-11644

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Adsorption and Stability of Streptavidin on Cluster-Assembled Nanostructured TiOx Films Luca Giorgetti,*,†,‡ Gero Bongiorno,† Alesssandro Podestà,† Giuseppe Berlanda,† Pasquale Emanuele Scopelliti,† Roberta Carbone,§ and Paolo Milani*,† CIMAINA and Dipartimento di Fisica, UniVersity of Milano, Via Celoria 16, 20133 Milano, Italy, SEMM, Scuola Europea di Medicina Molecolare, and Department of Experimental Oncology, European Institute of Oncology, Campus IFOM-IEO, Via Adamello 16, 20139 Milan, Italy, and Tethis srl, Via Russoli 3, 20143 Milano, Italy ReceiVed June 18, 2008. ReVised Manuscript ReceiVed August 4, 2008 The study of the adsorption of proteins on nanostructured surfaces is of fundamental importance to understand and control cell-surface interactions and, notably, cell adhesion and proliferation; it can also play a strategic role in the design and fabrication of nanostructured devices for postgenomic and proteomic applications. We have recently demonstrated that cluster-assembled nanostructured TiOx films produced by supersonic cluster beam deposition possess excellent biocompatibility and that these films can be functionalized with streptavidin, allowing the immobilization of biotinylated retroviral particles and the realization of living-cell microarrays for phenotype screening. Here we present a multitechnique investigation of the adsorption mechanisms of streptavidin on cluster-assembled TiOx films. We show that this nanostructured surface provides an optimal balance between adsorption efficacy and protein functionality. By using low-resolution protein arrays, we demonstrate that a layer of adsorbed streptavidin can be stably maintained on a cluster-assembled TiOx surface under cell culture conditions and that streptavidin retains its biological activity in the adsorbed layer. The adsorption mechanisms are investigated by atomic force microscopy in force spectroscopy mode and by valence-band photoemission spectroscopy, highlighting the potential role of the interaction of the exposed carboxyl groups on streptavidin with the titanium atoms of the nanostructured surface.

Introduction The immobilization of proteins on solid surfaces has a fundamental influence on cell adhesion and proliferation: the protein-material interface can be thus considered to be the playground where the mechanisms governing biocompatibility can be explored and where the strategies for the fabrication of effective protein and cell microarrays can be identified and tested.1,2 Protein-surface interaction is determined by the chemistry and morphology of the substrate in a complex way that is far from being understood: in particular, it is not clear how the interplay between parameters such as surface chemistry and surface topography influence the amount and conformation of adsorbed proteins.2-5 To elucidate the role of substrate topography and to fabricate biocompatible interfaces capable of mimicking the physiological conditions of the extracellular environment, a large number of studies have been devoted to the investigation of cell interactions with artificially produced nanostructures such as pits, pillars, grooves, dots, and random structures obtained by chemically or physically etching metallic, semiconducting, and polymeric surfaces.6,7 Particular efforts have been devoted to the topographical modification of titanium and titanium dioxide surfaces * Corresponding authors: [email protected], pmilani@ mi.infn.it. † University of Milano. ‡ SEMM and European Institute of Oncology. § Tethis srl. (1) Gallagher, W. M.; Lynch, I.; Allen, L. T.; Miller, I.; Penney, S. C.; O’Connor, D. P.; Pennington, S.; Keenan, A. K.; Dawson, K. A. Biomaterials 2006, 27, 5871–5882. (2) Lynch, I. Physica A 2007, 373. (3) Xu, L. C.; Siedlecki, C. A. Biomaterials 2007, 28, 3273–3283. (4) Rechendorff, K.; Hovgaard, M. B.; Foss, M.; Zhdanov, V. P.; Besenbacher, F. Langmuir 2006, 22, 10885–10888. (5) Han, M.; Sethuraman, A.; Kane, R. S.; Belfort, G. Langmuir 2003, 19, 9868–9872.

because these materials are among the most studied and most well characterized biomaterials.8 In parallel, different strategies for the functionalization of surfaces with molecular groups favoring protein adhesion have been proposed.9 Recently, we demonstrated that nanostructured TiOx (ns-TiOx) films obtained by supersonic cluster beam deposition have excellent biocompatibility by performing long-term experiments with a range of cancer and primary cells.10 Ns-TiOx films resulting from a random stacking of nanoparticles are characterized, on the nanoscale, by granularity and porosity mimicking those of extra-cellular matrix (ECM) structures.10,11 Their large nanoscale porosity, along with the abundance of adsorption sites and defects,12 makes cluster-assembled TiOx a promising candidate as a substrate for the adsorption and stable docking of proteins. By exploiting these properties, we employed ns-TiOx films as multifunctional substrates for macromolecule functionalization and cell culture in the context of a surface-mediated gene transduction protocol.11 In particular, we demonstrated the feasibility of a retroviral microarray technology in which biotinylated retroviruses were docked and localized onto the (6) Dalby, M. J.; Gadegaard, N.; Tare, R.; Andar, A.; Riehle, M. O.; Herzyk, P.; Wilkinson, C. D.; Oreffo, R. O. Nat. Mater. 2007, 6, 997–1003. (7) Shin, H. Biomaterials 2007, 28, 126–133. (8) Brunette, D. M.; Textor, M.; Thomsen, P. Titanium in Medicine: Material Science, Surface Science, Engineering, Biological Responses and Medical Applications. Springer: Berlin, 2001. (9) Mrksich, M.; Dike, L. E.; Tien, J.; Ingber, D. E.; Whitesides, G. M. Exp. Cell Res. 1997, 235, 305–313. (10) Carbone, R.; Marangi, I.; Zanardi, A.; Giorgetti, L.; Chierici, E.; Berlanda, G.; Podesta, A.; Fiorentini, F.; Bongiorno, G.; Piseri, P.; Pelicci, P. G.; Milani, P. Biomaterials 2006, 27, 3221–3229. (11) Carbone, R.; Giorgetti, L.; Zanardi, A.; Marangi, I.; Chierici, E.; Bongiorno, G.; Fiorentini, F.; Faretta, M.; Piseri, P.; Pelicci, P. G.; Milani, P. Biomaterials 2007, 28, 2244–2253. (12) Caruso, T.; Lenardi, C.; Agostino, R. G.; Amati, M.; Bongiorno, G.; Mazza, T.; Policicchio, A.; Formoso, V.; Maccallini, E.; Colavita, E.; Chiarello, G.; Finetti, P.; Sutara, F.; Skala, T.; Piseri, P.; Prince, K. C.; Milani, P. J. Chem. Phys. 2008, 128, 094704.

10.1021/la801910p CCC: $40.75  2008 American Chemical Society Published on Web 09/27/2008

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Synthesis of Cluster-Assembled TiOx Thin Films. Nanostructured TiOx films were deposited by a supersonic cluster beam deposition (SCBD) apparatus equipped with a pulsed microplasma cluster source (PMCS). The PMCS operation principle is based on the ablation of a titanium rod by a helium plasma jet, ignited by a

pulsed electric discharge.16 After ablation, TiOx ions thermalize with helium and condense to form clusters. The mixture of clusters and inert gas is then extracted in vacuum through a nozzle to form a seeded supersonic beam, which is collected on a set of standard glass microscope slides located in the beam trajectory. The clusters kinetic energy is low enough to avoid fragmentation, and hence a nanostructured film is grown.17 The rms roughness of ns-TiOx films can typically be controlled during deposition in the range of 2-40 nm, with corresponding specific areas (the ratio of the surface to the projected area) in the range of 1 to 2. It should be noted that AFM tends to underestimate specific area values because of the finite tip size (tip convolution) and the inability to access surface overhangs. Film thickness typically is in the range of 5-400 nm. When specifically indicated in the text, samples were thermally annealed in air using a muffle furnace. The specified temperature (either 100 or 200 °C) was reached through slow ramping and was maintained for 2 h. Adsorption of Streptavidin and Protein Arrays. To test the stability of streptavidin adsorbed on TiOx, we spotted 30 nL droplets of Cy3-labeled streptavidin (FluoroLink Cy3-labeled streptavidin from Amersham Biosciences) on top of four cluster-assembled TiOxcoated glass slides in an 8 × 6 array format with of a BioJet 3000 Plus spotter from BioDot. Four identical arrays were printed contemporarily to ensure intersample uniformity. We tested different concentrations of fluorescent streptavidin diluted in buffer solution to optimize the stability of the layer (data not shown); the reported time-course assay was performed with 10 µg/mL streptavidin. After overnight incubation at 4 °C to allow binding saturation and washing with PBS/Tween 0.1%, the slides were placed in DMEM culture medium containing 10% serum at 37 °C for different periods of time (0, 8, 24, and 48 h), and the fluorescence signal from adsorbed molecules was detected with a microarray fluorescence scanner (Gene-Pix 4000B, Axon Instruments) and analyzed with Gene-Pix Pro 5.0 software in order to assess the release of streptavidin from the substrate. For the functional assay of adsorbed streptavidin, we spotted three 8 × 3 replicate arrays of streptavidin and BSA at three different concentrations (10 and 100 µg/mL and 1 mg/mL). After overnight incubation, the slide was washed twice in PBS/Tween 0.1% and then rinsed in Milli-Q water and centrifuged at 800 rpm for 3 min at 4 °C. Half of the spots were also spotted with 20 nL of biotinylated IgG (Biotinylated monoclonal anti-flag BioM2 from mouse, Sigma) diluted at 150 µg/mL in 2% BSA; the remaining spots were spotted with nonbiotinylated anti-flag (Sigma) at the same concentration. The two antibodies were incubated for 1 h at room temperature, then washed twice with PBS/Tween 0.1%, then rinsed with Milli-Q water. Cy5-labeled anti-mouse (Jackson Immune Research Laboratories) was spotted on top of the preceding features and then incubated for 1 h at room temperature; the slide was then washed according to the same procedure. Blank areas of the slide were spotted only with primary and/or with secondary antibodies as a control and were used for background normalization. Force Spectroscopy Characterization. An atomic force microscope (AFM) in force spectroscopy mode allows us to gain insight into the interaction mechanisms of biomolecules with surfaces in an aqueous environment by directly measuring interaction forces.18-20 The technique consists of recording the AFM cantilever deflection as a function of the relative tip-surface distance during repeated approach-retraction cycles. Periodically, the tip makes contact with the surface and then detaches from it. If functional groups are present on the tip, then specific adhesion events can take place during contact, causing the tip to stay in contact for part of the retraction branch of the curve until the elastic force of the cantilever overcomes the adhesive force. Quantitative measurement of this adhesion force

(13) Freitag, S.; LeTrong, I.; Klumb, L.; Stayton, P. S.; Stenkamp, R. E. Protein Sci. 1997, 6, 1157–1166. (14) Hughes, C.; Galea-Lauri, J.; Farzaneh, F.; Darling, D. Mol. Ther. 2001, 3, 623–630. (15) Mateo, C.; Grazu, V.; Pessela, B. C.; Montes, T.; Palomo, J. M.; Torres, R.; Lopez-Gallego, F.; Fernandez-Lafuente, R.; Guisan, J. M. Biochem. Soc. Trans. 2007, 35, 1593–1601.

(16) Barborini, E.; Piseri, P.; Milani, P. J. Phys. D: Appl. Phys. 1999, 32, L105–L109. (17) Kholmanov, I. N.; Barborini, E.; Vinati, S.; Piseri, P.; Podesta, A.; Ducati, C.; Lenardi, C.; Milani, P. Nanotechnology 2003, 14, 1168–1173. (18) Leckband, D.; Israelachvili, J. Q. ReV. Biophys. 2001, 34, 105–267. (19) Willemsen, O. H.; Snel, M. M.; Cambi, A.; Greve, J.; De Grooth, B. G.; Figdor, C. G. Biophys. J. 2000, 79, 3267–3281. (20) Butt, H. J.; Cappella, B.; Kappl, M. Surf. Sci. Rep. 2005, 59, 1–152.

surface via a layer of streptavidin adsorbed on cluster-assembled TiOx. Streptavidin is a homotetrameric protein with a very high affinity (Ka ≈ 1013 M-1) for biotin:13 each monomer is able to bind one biotin molecule, hence a streptavidin molecule can arrange up to four bound biotin-conjugated moieties (e.g., biotinylated retroviruses, as demonstrated in Hughes et al.14). Besides being commonly employed in a variety of biological assays, streptavidin is often used to functionalize surfaces, where biotinylated molecules can be subsequently bound (e.g., microwell plastic plates for ELISA assays). Successful functionalization of a surface with streptavidin, as well as with any other protein in general, relies on a delicate equilibrium between adsorption efficacy (i.e., number of adsorbed proteins per unit area) and the preservation of folding, which is the condition for preserving protein functionality. Functionalization can be achieved either via nonspecific protein adsorption or by chemically linking the protein to the surface.15 Whereas nonspecifically adsorbing proteins on a surface (e.g., when setting up an ELISA assay in a microwell plate) is a practical procedure and does not require any preliminary surface treatment, it nevertheless compromises a priori the possibility of producing precise protein patterns on the surface. Not secondarily, higher amounts of protein can be generally adsorbed on hydrophobic surfaces, which in turn promotes the interaction of buried, nonpolar protein residues with the result of destabilizing the protein folded conformation and compromising its functionality. However, linking the protein to the surface via chemical bonding is more likely to result in the maintenance of a properly folded configuration but generally requires a chemical modification of the surface. This latter procedure might compromise the original properties of the material to be functionalized, notably its biocompatibility and therefore its applicability to living-cell-based assays. Here we report the characterization of the interaction of streptavidin with nanostructured cluster-assembled TiOx surfaces both as-deposited and thermally annealed. We show that streptavidin is efficiently adsorbed while maintaining its functionality toward biotinylated species with no need for any preliminary chemical treatment of the surface and thus that the streptavidin functionalization of ns-TiOx represents the optimal equilibrium between ease of procedure and maintenance of biological activity. We employed a low-resolution protein microarray technique to characterize the spatial localization, the stability, and the conformation of adsorbed streptavidin. Details of the streptavidin-ns-TiOx surface interaction have been investigated by photoelectron spectroscopy and atomic force microscopy (AFM) in force spectroscopy mode to highlight the role of the chemical interaction of functional groups exposed on the streptavidin surface and the undercoordinated titanium atoms that are abundantly present on the ns-TiOx surface.

Materials and Methods

StreptaVidin on Nanostructured TiOx Films allows us to characterize the strength of specific bonds that can be formed upon contact. A Multimode Nanoscope IV atomic force microscope (Veeco Instruments) was used to measure the interaction force between functionalized tips and nanostructured TiOx films in a liquid environment. Interaction forces were extracted from force-distance curves, as explained in detail in refs 18 and 20. Cantilever deflection was converted into force multiplying the raw deflection signal (V) by the deflection sensitivity (nm/V) and by the cantilever force constant (nN/nm). Each cantilever used in the experiments was calibrated by means of the Sader method,21 which allows us to measure the force constant of the cantilever to 10% accuracy. The deflection sensitivity was measured as the slope of a force curve in the contact region. Two types of functionalized cantilevers were used. For the direct experiment (i.e., streptavidin vs ns-TiOx), we used probes of the PT.GS.AU.SA series from Novascan Technologies. These probes have streptavidin-functionalized SiO2 spheres (radius 300 nm) attached at the end of rectangular cantilevers with a nominal spring constant of 0.03 N/m. For the control experiment (i.e., COOH vs ns-TiOx), we used probes of the CT.PEG.COOH series from Novascan Technologies, where a COOH group is bound to a conventional AFM tip mounted on a rectangular cantilever via a poly(ethylene glycol) (PEG) linker, with a typical length of 17 nm. The force constant of these cantilevers is typically 0.1 N/m, and the radius of curvature is 10-20 nm. All force measurements were performed in a droplet of phosphate-buffered saline (PBS) using a quartz fluid cell. The fluid cell was sonicated for 15 min in ethanol and then rinsed thoroughly with HPLC-grade water before use. The typical approaching rate was 10 nm/s. Force curves (256) were recorded in each force experiment, and then the tip was moved to a different location on the ns-TiOx film surface and the experiment was repeated. Force values were extracted from force curves using homemade routines developed in the Matlab environment. Surface Photoelectron Spectroscopy. Characterization of the interaction of water and acetic acid with cluster-assembled TiOx films has been performed by means of ultraviolet photoemission spectroscopy (UPS) at the material science beamline of the Elettra Synchrotron Radiation facility (Trieste, Italy), a bending magnet beamline with a tuning range from 45 to 900 eV equipped with a plane grating monochromator based on the SX-700 concept.22 The UHV experimental chamber, with a base pressure of 1 × 10-10 mbar, was equipped with a 150 mm mean radius hemispherical electron energy analyzer (Phoibos MCD 150, Specs) with multichannel detection. The photoelectron spectra, taken from samples at room temperature, were recorded at normal emission. The energy of the photon beam was set to 133 eV. To promote the desorption of atmospheric contaminants from the surface, films were mildly annealed in situ (1 × 10-9 mbar) at temperatures in the range of 150-200 °C for 2 h. This thermal treatment process does not modify the film morphology, but it may slightly increase the density of oxygen vacancies (and therefore the content of Ti 3d defects).

Results and Discussion Streptavidin Adsorption. Figure 1 shows surface topographies of the same ns-TiOx film (lateral scale 2 µm) acquired with the AFM as-deposited (A) and after annealing at 200 °C for 2 h (B). The surface morphology of ns-TiOx films consists of a fine raster of nanometer-sized grains, the smallest among them (with a diameter below 5 nm) being the primeval clusters produced in the cluster source and the others resulting from the aggregation or coalescence of clusters at the surface during deposition. The inset in Figure 1A shows a magnified view of a 250 nm × 250 nm area, which highlights the granular and porous structure of this material. Pore size below 1 nm is expected from AFM images. (21) Sader, J. E.; Chon, J. W. M.; Mulvaney, P. ReV. Sci. Instrum. 1999, 70, 3967–3969. (22) Vasina, R.; Kolarik, V.; Dolezel, P.; Mynar, M.; Vondracek, M.; Chab, V.; Slezak, J.; Comicioli, C.; Prince, K. C. Nucl. Instrum. Methods Phys. Res., Sect. A 2001, 467, 561–564.

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Figure 1. AFM surface topographies of the same ns-TiOx film (A) asdeposited and (B) after thermal annealing at 200 °C for 2 h. The lateral scale is 2 µm. The rms roughness is 4.6 nm for both images. The inset in panel A shows a magnified view (lateral scale 250 nm) of the film surface. The images have been acquired in a dry nitrogen atmosphere in tapping mode.

Neither the surface roughness nor the specific area of the film shown in Figure 1 was modified by the thermal treatment, with their values being 4.6 and 1.20 nm, respectively. We monitored the time-course stability of arrays of fluorescently labeled streptavidin (10 µg/mL), which were “printed” on cluster-assembled TiOx-coated microscope slides by means of a noncontact microarray spotter as described in the Materials and Methods section (Figure 2A). The printed slides were stored in cell-culture medium for up to 48 h to simulate culture conditions, and the average fluorescence signal of the arrays were recorded at four different time points with a microarray scanner. Because all slides were acquired with the same gain factor, the amount of fluorescence is proportional to the number of fluorescent molecules on the surface. After 48 h of incubation, the signal was found to be approximately 60% of the initial value (Figure 2B), showing that a significant fraction of the streptavidin molecules were still adsorbed on the cluster-assembled surface, indicating the high stability of the adsorbed species. We also checked that the spot morphology was precisely maintained as a requirement for the precise immobilization of biotinylated molecules. To assess the conformation of the adsorbed streptavidin layer, we probed the affinity of streptavidin molecules for a biotintagged antibody. Nonlabeled streptavidin was spotted on a clusterassembled TiOx-coated slide at increasing concentrations in an 8 × 3 array format in triplicate (Figure 2C). On top of one group of streptavidin spots we spotted a biotinylated anti-flag antibody; on the remaining spots, we spotted nonbiotinylated anti-flag as a negative control. After incubation, we washed the slide to remove the unbound antibodies, and we spotted a fluorescent secondary antibody recognizing both primary antibodies, either biotinylated or nonbiotinylated. The same secondary antibody was spotted on top of the entire array and incubated for 1 h; the slide was then washed, rinsed, and dried before detecting fluorescence. The signal from the nonbiotinylated antibodies was used as a measure of nonspecific interactions and was subtracted from the signal from the biotinylated antibody. To provide additional internal control, the same staining scheme was applied to nearby 8 × 3 arrays of bovine serum albumin (BSA). As expected, on the BSA spots the fluorescence signal was found to be constant, irrespective of the amount of underlying protein, whereas the signal from the biotinylated antibody on streptavidin increased significantly with increasing streptavidin concentration, reflecting the specific interaction of biotin with properly folded adsorbed streptavidin domains (Figure 2D). The nonbiotinylated antibody gave a constant, similar signal both on streptavidin and BSA, irrespectively of the concentration of the underlying protein, reflecting the nonspecific interactions between the antibody and

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Figure 2. (A) Low-resolution array of streptavidin-Cy3 spotted at 0.01 mg/mL on an ns-TiOx-coated glass slide and incubated in DMEM medium supplemented with 10% FCS at up to 48 h at 37 °C in the presence of 5% CO2. The fluorescence from identical copies of the array was quantified immediately after spotting and after 8, 24, and 48 h. Representative rows from the various arrays at the moment of acquisition are shown. (B) Fluorescence emitted from the arrays in panel A plotted as a function of incubation time, normalized to T0. Data points represent average values over 48 spots ( the standard deviation. (C) Nonlabeled streptavidin (ST) and BSA spotted onto cluster-assembled TiOx slides at increasing concentrations and adsorbed molecules probed either with anti-flag or biotinylated anti-Flag. A Cy5-conjugated secondary antibody was used to detect the antibody. (D) Fluorescence emitted by the spots in panel C quantified after subtracting the nonspecific background. Dark-gray bars, streptavidin; light-gray bars, BSA. The amount of immobilized biotinylated anti-flag increases only when streptavidin (and not BSA) is adsorbed on the surface, showing that adsorbed streptavidin maintains its functionality. (E) Detail of the specific and nonspecific contributions in the array of panel D. The average signals from biotinylated (B) and nonbiotinylated (NB) antibody on 0.1 mg/mL streptavidin and BSA are shown. Data in panel D are obtained by subtracting the NB contribution to the B contribution for each protein.

the adsorbed protein (Figure 2E); the same holds for the biotinylated antibody on BSA. This provides evidence that streptavidin molecules maintain a properly folded conformation once adsorbed on the ns-TiOx surface and that the surface efficiently binds biotinylated moieties. Thermal annealing at moderate temperatures of the clusterassembled films improves the streptavidin adsorption performance, but it has no effect on the biocompatibility of ns-TiOx10 and does not change the surface morphology as confirmed by AFM analysis (Figure 1). As shown in Figure 3A,B, when increasing the surface annealing temperature up to 200 °C we observed a monotonic increase in the amount of fluorescently labeled streptavidin adsorbed on the surface over a wide range of concentration. Strikingly, the surface that was annealed at 200 °C performed comparably to a commercially available slide (CodeLink activated slide from GE) that relies on succinimidyl ester-based coupling chemistry (Figure 3C). As another reference, we compared cluster-assembled TiOx with a nanostructured TiOx film that was produced by plasma sputtering, and we found that the cluster-assembled surface adsorbed approximately 8 times more streptavidin than did the sputtered one. The considerably higher performance of cluster-assembled TiOx may be ascribed to its much higher nanoscale porosity and surface area as well as to the high number of exposed and undercoordinated Ti atoms

that are presented by the nanostructured surface as a result of the high surface curvature of the topmost nanoparticles (see below and ref 10). The observed increase in streptavidin adsorption upon thermal annealing may be interpreted as a consequence of an increase in solvent accessibility to the pores and cavities of the surface as a result of the increasing surface hydrophilicity (notice the increase in spot size in Figure 3B), which in turn is associated with increased surface oxidation (Figure 8A). Force Spectroscopy Characterization. We have used streptavidin-functionalized AFM probes, as described in the Materials and Methods section, to study the interaction of streptavidin with ns-TiOx films in a liquid environment at physiological salt concentration (phosphate-buffered saline). Operation in force spectroscopy mode can allow the detection of single binding events between functional groups on the tip and active sites on the surface and the characterization of the strength of specific bonds that can be formed upon contact via the quantitative measurement of the adhesion force.19 We have collected several hundred force-distance curves for a probe-surface contact time of 0.2 s, at different surface positions, on both as-deposited and annealed TiOx films with different tips. The vertical distance between the flat part of the curve (no-interaction region) and the bottom of each well represents the adhesion force, or the strength of that particular

StreptaVidin on Nanostructured TiOx Films

Figure 3. (A) Low-resolution arrays of streptavidin-Cy3 were spotted on ns-TiOx-coated slides at the indicated concentrations, and the fluorescence intensity was quantified after 12 h in the culture medium. The surface was either not treated after deposition (2), annealed in air at 100 °C for 2 h ((), or annealed in air at 200 °C for 2 h (9). Data points represent average values over 24 spots ( the standard deviation. The data were fitted with Langmuir isotherms. (B) Representative spots from the arrays in panel A. The concentration is 5 mg/mL. (C) Performance of cluster-assembled TiOx with 5 mg/mL streptavidin-Cy3 tested against a commercial microarray slide (CodeLink from Amersham) and sputtered TiOx.

bond (inset of Figure 4A). Whether this bond represents a single bond (a single streptavidin molecule bound to the TiOx surface) or multiple bonds in parallel can be inferred by the statistical analysis of the measured adhesion forces. Figure 4A shows the probability distribution of measured adhesion forces extracted by the force curves, (i.e., the number of counts divided by the total number of curves showing at least one unbinding event). We notice that larger adhesion forces are more probable on the annealed than on the as-deposited ns-TiOx. Moreover, the probability of having at least one unbinding event in a single force-distance curve (calculated as the ratio of the number of curves containing at least one adhesion feature to the total number of curves) is also larger on annealed than on the as-deposited ns-TiOx (66 vs 48%). Focusing on the forces measured in single experiments, we observe that the adhesion force histograms exhibit equally spaced, repeated peaks. The data shown in Figure 4B were collected during a single force-distance experiment on a cluster-assembled TiOx sample that was annealed at 200 °C. A multi-Gaussian fit of the histogram allows to calculate an average separation of the peaks (excluding the first) of 56 ( 8 pN, which falls in the expected range for biomolecular interactions.18,23 The data shown in Figure 4 strongly support the hypothesis that the measured adhesion forces are the result of the rupture of multiple bonds in parallel (i.e., multiple single-molecule bonds (23) Zlatanova, J.; Lindsay, S. M.; Leuba, S. H. Prog. Biophys. Mol. Biol. 2000, 74, 37–61.

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Figure 4. Forces measured by AFM in force spectroscopy mode between streptavidin-coated beads and ns-TiOx films. (A) Probability distributions of the detachment forces measured on as-deposited and annealed nsTiOx films. The histogram collects all of the forces measured in different locations on the films using different tips. The inset shows a typical force-distance curve recorded by AFM. Detachment events (two in this case) are clearly recognizable as well as with respect to the long-distance baseline. The depth of the wells along the force axis (indicated by the arrows) represents the strength of the bonds. (B) Histogram of detachment forces measured in a single experiment on an annealed ns-TiOx film. Force peaks (except the first) are separated on average by 56 ( 8 pN, as revealed by a multi-Gaussian fit.

established between streptavidin molecules sparsely attached to the AFM probe and the TiOx surface). An effective contact area of 5000-8000 nm2 can be expected from spherical tips with a diameter of 600 nm. This area corresponds to a radius of 40-50 nm, which is the radius of a circular section of the probe located 5 nm away from the apex (a reasonable size for a protein). Hence, several streptavidin molecules can interact with the film surface simultaneously. The average peak separation that we observed can thus be interpreted as the strength of the single molecular bond between streptavidin and ns-TiOx. To confirm the hypothesis that multiple molecular bonds form in parallel during forcedistance measurements, we repeated the experiment on 200 °C annealed samples allowing for larger tip-surface interaction times (Figure 5). Contact times were 0.2, 1.2, and 2.2 s. With increasing interaction time, orientation changes of the protein with respect to the nanoscale environment may take place. This in turn might enhance the binding probability and therefore the occurrence of multiple bonds. Figure 5 shows that the average adhesion force shifts toward higher values when the interaction time increases, with a qualitative transition occurring between 1.2 and 2.2 s. For times equal to or below 1.2 s, the measured forces are distributed continuously in a range that gets broader when the interaction time increases. At 2.2 s, most of the detachment events that occurred at intermediate forces occur at larger forces of around 1.1 nN. A closer view of the shape of force-distance curves gives insight into the nature of the bonds that are formed. The inset of Figure 5 shows a comparison of

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Figure 5. Probability distributions of the detachment forces measured by AFM as a function of the interaction time between the probe and the surface. The streptavidin-coated beads were kept in contact with the surface before retraction for t ) 0.2, 1.2, and 2.2 s. As the contact time t increases, the probability of observing a larger detachment force increases. Between 1.2 and 2.2 s, a qualitative change in adsorption takes place: almost all forces are grouped around 1.1 nN. The inset shows two typical force curves acquired at t ) 0.2 and 2.2 s. The shape and intensity of the force curves reveal that for short interaction times mostly single molecular bonds are formed whereas for long times single detachment events at high forces take place, originating from many parallel bonds.

typical force-distance curves acquired on annealed ns-TiOx at 0.2 and 2.2 s. Whereas at short times several detachment events, at low force, can be observed, at long times only one detachment event, at much higher force, is typically observed. We interpret these results as further confirmation that multiple molecular bonds form in parallel when the streptavidin-coated probe is in contact with the ns-TiOx surface. For small interaction times, only a few bonds in parallel can form, with multiple bonds being proportionally less probable than single or few bonds. At intermediate times, the same trend is observed, with the longer contact time allowing for multiple bonds of higher order to form. An interaction time of 2.2 s seems to be enough to allow the saturation of all binding sites distributed across the effective contact area. This accounts for the well-peaked structure observed at 1.1 nN. This value corresponds, assuming that the single molecular bond strength is ∼60 pN, to roughly 18 molecular bonds in parallel. Assuming an effective interaction area of 5000-8000 nm2 and a protein diameter of 5 nm, we obtain a functionalization linear density of roughly 1 streptavidin molecule every 15-20 nm, which is quite reasonable. We performed control experiments to exclude a leading contribution to the observed adhesion events from nonspecific physical interactions arising from those portions of the streptavidin-coated probe that could be not covered with streptavidin molecules. We treated a streptavidin-coated probe with a PBS solution containing the protease trypsin to degrade proteins attached to the probe and expose the underlying succinimidyl coating, which is used as an interlayer in the probe-functionalization process. We chose to use trypsin because it is a highly effective protease and is expected to generate peptides with an average size of about 8-10 amino acids.24 The tips were washed in PBS/Tween 0.2% after trypsin treatment. Experiments were carried out at the three previously defined interaction times (0.2, 1.2, and 2.2 s). No detachment events were found in force curves acquired at different contact times after trypsin treatment. Curves (24) Thiede, B.; Hohenwarter, W.; Krah, A.; Mattow, J.; Schmid, M.; Schmidt, F.; Jungblut, P. R. Methods 2005, 35, 237–247.

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Figure 6. Typical force-distance curve acquired on an ns-TiOx film annealed at 200 °C for 2 h after the treatment of streptavidin attached to the AFM probe with trypsin, followed by washing in PBS/Tween 0.2%. The interaction time was 2 s. The curve does not show any adhesion events.

do not exhibit any adhesion feature, as shown in Figure 6. The curve in Figure 6, which is representative of the totality of acquired curves, was recorded on an ns-TiOx film annealed at 200 °C for 2 h using a streptavidin-coated probe that was treated with trypsin and then thoroughly washed with PBS/Tween 0.2%. The interaction time was 2 s. Under these conditions (annealing of ns-TiOx and a long interaction time), adhesion events are more favorable. Despite this, the curve is flat, indicating the absence of interactions between the film surface and the probe with all of the streptavidin molecules likely removed and the succinimdyl coating exposed. The curve in Figure 6 can be compared with the curves shown in Figures 4A and 5, wherein one or more adhesion events are clearly visible. These findings confirm the conjecture that the succinimdyl coating does not contribute substantially to the measured adhesion forces. We then decided to investigate further the origin of the 60 pN rupture events that we observed using streptavidin-coated probes. The interactions of even a single streptavidin molecule with the nanostructured TiOx surface are likely to be extremely complicated and may include equivalently important van der Waals, electrostatic, and entropic (hydrophobic) contributions. Nevertheless, motivated by the excellent performances of the cluster-assembled TiOx surface with respect to commercial, covalent linkage-based surfaces, we decided to challenge the intriguing hypothesis that a molecular bond may form between the TiOx surface and some functional groups on the surface of the protein. It is well known that carboxylic acids adsorb dissociatively on TiOx surfaces, a process that involves the formation of a coordination bond between the oxygen atoms of the carboxyl group and titanium atoms exposed by the surface. This has been demonstrated by a number of techniques, both in vacuum and in solution and using a variety of model carboxyl-terminated compounds and TiOx surfaces,25-30 and is supported by theoretical studies.31-33 (25) Fukui, K.; Onishi, H.; Iwasawa, Y. Chem. Phys. Lett. 1997, 280, 296– 301. (26) Guo, Q.; Cocks, I.; Williams, E. M. J. Chem. Phys. 1997, 106, 2924– 2931. (27) Strehle, M. A.; Rosch, P.; Petry, R.; Hauck, A.; Thull, R.; Kiefer, W.; Popp, J. Phys. Chem. Chem. Phys. 2004, 6, 5232–5236. (28) Vogel, E.; Meuer, P.; Kiefer, W.; Urlaub, R.; Thull, R. J. Mol. Struct. 1999, 483, 241–244. (29) Zhang, Q. L.; Du, L. C.; Weng, Y. X.; Wang, L.; Chen, H. Y.; Li, J. Q. J. Phys. Chem. B 2004, 108, 15077–15083. (30) Schnadt, J.; O’Shea, J. N.; Patthey, L.; Schiessling, J.; Krempasky, J.; Shi, M.; Martensson, N.; Bruhwiler, P. A. Surf. Sci. 2003, 544, 74–86. (31) Foster, A. S.; Nieminen, R. M. J. Chem. Phys. 2004, 121, 9039–9042.

StreptaVidin on Nanostructured TiOx Films

Nonetheless, little attention has been given so far to the possible interactions of protein-exposed carboxyl groups with TiOx surfaces in an aqueous evironment. Aspartic acid and glutamic acid carry carboxyl-terminated side chains, which tend to be deprotonated at pH 7 and contribute a negative charge to the overall charge of a protein. Being polar, those amino acids are commonly found on the solvent-exposed surfaces of most proteins. Streptavidin in particular bears eight aspartic acids and five glutamic acids per monomer. To test whether the detachment events that we observed with streptavidin-coated tips could be ascribed to carboxyl-surface covalent bonding, we probed the interaction between an AFM tip functionalized with tethered carboxylic acids and the surface of cluster-assembled TiOx. Force-distance experiments on annealed and as-deposited ns-TiOx films were performed using COOH-terminated tips. A PEG linker (average length 17 nm) was used to tether the COOH group to the AFM tip. An elastic PEG spacer is often used in force spectroscopy experiments20,34 because its presence assures that the interesting detachment events occur at a predefined distance from the surface, allowing us to rule out from the forcecurve ensemble those curves exhibiting nonspecific detachment events. We could extract only 5 molecular unbinding events on as-deposited ns-TiOx out of about 500 force curves. On annealed ns-TiOx films, we found 30 unbinding events out of 500 force curves. The apparent adsorption probability of 6% should be corrected by a geometrical factor accounting for the larger effective contact area of streptavidin-coated beads with respect to that of COOH-functionalized tips. This factor is roughly 10, which makes the adsorption probability of COOH on ns-TiOx comparable to that of streptavidin on ns-TiOx. (The factor of ∼10 comes from the ratio of effective contact areas of streptavidincoated beads and of COOH-functionalized tips, i.e,. the squared ratio of effective radii. For streptavidin-coated beads, we take the radius to be equal to 50 nm; for COOH-functionalized tips, we take it to be equal to the average PEG spacer length, 17 nm.) All detachment events found in force curves reveal the typical shape characteristic of stretching an elastic PEG molecule (Figure 7, inset). The distance at which the rupture of the bond takes place is in the expected range (5-25 nm, with typical values between 15 and 20 nm). Thus, despite the poor statistics, the attribution of the observed detachment features in the forcedistance curves to the interaction between COOH groups and the ns-TiOx surface is certain. Figure 7 shows the histogram of measured detachment forces. A bimodal distribution was found, with a large mode at low force (26 ( 11 pN) and a marked mode at 68 ( 10 pN. This latter value is in good agreement with the repeated unbinding force separation that was measured using streptavidin-coated probes against ns-TiOx (Figure 4B). The mode at lower force may originate in noncovalent binding of COOH groups to ns-TiOx (these groups are more mobile with respect to those of streptavidin directly bound to the spherical probes) or to the binding of the PEG chains to the ns-TiOx surface (the fraction of PEG spacers terminated by a COOH group is supposed to be small). These results strongly suggest that carboxylterminated protein residues exposed at the surface of streptavidin molecules may play a major role in the adsorption of streptavidin to ns-TiOx surfaces. These considerations might be naturally extended to other proteins and other oxide surfaces.35 (32) Ojamae, L.; Aulin, C.; Pedersen, H.; Kall, P. O. J. Colloid Interface Sci. 2006, 296, 71–78. (33) Redfern, P. C.; Zapol, P.; Curtiss, L. A.; Rajh, T.; Thurnauer, M. C. J. Phys. Chem. B 2003, 107, 11419–11427. (34) Ratto, T. V.; Langry, K. C.; Rudd, R. E.; Balhorn, R. L.; Allen, M. J.; McElfresh, M. W. Biophys. J. 2004, 86, 2430–2437. (35) Rezwan, K.; Meier, L. P.; Gauckler, L. J. Biomaterials 2005, 26, 4351– 4357.

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Figure 7. Cumulative histogram of detachment forces measured by AFM on as-deposited and annealed ns-TiOx films using an AFM tip functionalized with COOH groups tethered to PEG linkers. The distribution shows a low-force mode at about 26 ( 11 pN and a marked mode at 68 ( 10 pN. The inset shows a typical force curve, where three single-molecule detachment events can be recognized. Here, the force is plotted as a function of the actual tip-surface distance. (See ref 20 for details.) The three peaks in the curve are typical fingerprints of the breaking of bonds occurring at the end of an elongated, pulled, elastic spacer. The distance at which bond rupture takes place is in the expected range.

Ultraviolet Photoelectron Spectroscopy Characterization. We characterized the valence-band electronic structure of clusterassembled TiOx by ex situ photoelectron spectroscopy. Figure 8 reports a collection of valence band (VB) photoelectron spectra that we collected under different conditions. In Figure 8A, we show the VB spectra of an as-deposited film and of a film that was annealed at 200 °C in air. The states about 1 eV below the Fermi level (which is placed at 0 eV binding energy, BE) are attributed to Ti 3d states whereas the VB states in the range of 6-8 eV are related to O 2p orbitals.36,37 The features situated below the valence band at 11 and 13 eV BE can be attributed to residual atmospheric contamination (see Materials and Methods for a detailed description of sample preparation).38 As reported in our previous work,10,12 the surface of as-deposited clusterassembled TiOx is characterized by a remarkable abundance of Ti 3d defect states in the band gap related to surface oxygen vacancies and undercoordinated titanium atoms.39 These states are known to favor the dissociative adsorption of water molecules and to play a role in the chemisorption of a variety of organic molecules.36,37,40,41 After annealing in air at 200 °C, an almost complete oxidation of the surface takes place, signaled by the disappearance of the Ti 3d defect states, whereas the residual atmospheric contaminants are still present as a consequence of the very high reactivity of the surface. Oxidation (and possibly hydroxylation due to water chemisorption during annealing, which is unfortunately impossible to detect because of the strong signal from contaminants) is associated with an increase in surface hydrophilicity, as can be noticed in Figure 3B. This in turn may enhance the accessibility of the surface nanoscale pores and cavities to the solvent and accounts for the observed increase in protein adsorption shown in Figure 3A,B. We also characterized the reactivity of the cluster-assembled TiOx toward water and carboxylic groups by ex situ photoelectron (36) Diebold, U. Surf. Sci. Rep. 2003, 48, 53–229. (37) Henderson, M. A. Surf. Sci. Rep. 2002, 46, 5–308. (38) Cocks, I. D.; Guo, Q.; Patel, R.; Williams, E. M.; Roman, E.; deSegovia, J. L. Surf. Sci. 1997, 377, 135–139. (39) Sanjines, R.; Tang, H.; Berger, H.; Gozzo, F.; Margaritondo, G.; Levy, F. J. Appl. Phys. 1994, 75, 2945–2951. (40) de la Garza, L.; Saponjic, Z. V.; Dimitrijevic, N. M.; Thurnauer, M. C.; Rajh, T. J. Phys. Chem. B 2006, 110, 680–686. (41) Dimitrijevic, N. M.; Saponjic, Z. V.; Rabatic, B. M.; Rajh, T. J. Am. Chem. Soc. 2005, 127, 1344–1345.

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Figure 8. (A) UPS spectra of an as-deposited TiOx film (-) and a film that was annealed in air at 200 °C (---). (B) UPS spectra of an as-deposited TiOx film (-) and a film that was annealed in UHV at 150 °C (---). C. UPS spectra (photon energy 133 eV) obtained from an ns-TiOx film annealed at 150 °C under UHV conditions before (-) and after (---) exposure to 1 ML of water molecules. (D) UPS spectra (photon energy 133 eV) performed on a cluster-assembled titanium oxide film annealed at 200 °C under UHV conditions before (-) and after (---) exposure to 100 L of acetic acid molecules.

spectroscopy. We prepared the samples by annealing in UHV at 150 °C in order to desorb atmospheric contaminants and to be able to eventually resolve newly formed molecular orbitals below the O 2p states. As expected, UHV annealing promotes the reduction of the surface and a strong decrease in contamination (Figure 8B). UHV-annealed samples were exposed to water and acetic acid, which we chose as a model carboxyl-containing molecule. We observe (Figure 8C) that the intensity of the Ti 3d peak does not change after exposure to 1 megalangmuir (ML) of water molecules at room temperature, indicating that the interaction with water molecules does not change the oxidation state of the nanostructured surface.36 We also observe the rising of the spectral features related to both molecular water (1b2 states placed at about 13.5 eV and 3a1 states placed at 9.5 eV) and hydroxyl groups coming from dissociative water adsorption (3σ states of OH- placed at about 11 eV).37,42,43 These results provide evidence of complex behavior for the interaction of the samples with water as it is adsorbed in both molecular and dissociated forms. When the hydrated surface was exposed to 100 langmuir of acetic acid, we observed a substantial absence of variation in the intensity of the Ti 3d peak (Figure 8D). New intense features appeared outside the valence band at about 11 and 13 eV, which have already been reported in previous work on the interaction of single-crystal TiOx surfaces with carboxylic acids.38,43 These spectral features have been attributed to the dissociative adsorption of acetic acid to surface Ti atoms through the breaking of the O-H bond36,38 and the formation of acetate complexes, a process that does not induce the oxidization of the sample surface.36 This confirms that the surface of ns-TiOx promotes the chemisorption of carboxylic groups to the abundant superficial and/or undercoordinated titanium atoms. Because the carboxyl groups on aspartic and glutamic acid residues are expected to be as reactive (42) Di Valentin, C.; Tilocca, A.; Selloni, A.; Beck, T. J.; Klust, A.; Batzill, M.; Losovyj, Y.; Diebold, U. J. Am. Chem. Soc. 2005, 127, 9895–9903. (43) Wang, L. Q.; Ferris, K. F.; Shultz, A. N.; Baer, D. R.; Engelhard, M. H. Surf. Sci. 1997, 380, 352–364.

as those on acetic acid, these observations are a further indication of the role of a molecular bond between acidic protein side chains and the surface in protein adsorption on cluster-assembled TiOx.44

Conclusions We have characterized the performance of cluster-assembled TiOx in terms of the adsorption efficacy, functionality, and stability of streptavidin. By combining protein microarray methodology with AFM and photoelectron spectroscopy, we have shown that a layer of functionally integer (properly folded) streptavidin molecules can be maintained on biologically relevant timescales (>48 h) on cluster-assembled TiOx, allowing biotinylated moieties to be stably immobilized on the surface in precise patterns. Thermal annealing of the nanostructured films at moderate temperature enhances the amount of adsorbed protein while maintaining the biocompatibility of the surface. We showed that the strength of the interactions of single streptavidin molecules with the surface is consistent with the hypothesis that a covalent bond may be formed between protein acidic (carboxy-terminated) side chains and superficial and undercoordinated titanium atoms on the surface. Ex situ photoelectron spectroscopy characterization of ns-TiOx surfaces exposed to a model carboxylic acid (acetic acid) provide further confirmation of this picture. Acknowledgment. We acknowledge the help of Marzia De Marni in setting up the array experiment in Figure 3. This work has been partially supported by AIRC under OGCG grant Development and Integration of High-Throughput Technologies for the Functional Genomics of Cancer, by Fondazione Cariplo under grant Sviluppo di Sistemi di Cultura Cellulare su Materiali Biocompatibili Nanostrutturali per lo Studio di Patologie a Scopo Eziologico e Teraupetico and by grant FIRB RBNE03B8KK-08 from the Italian Ministry of the University and Scientific Research. LA801910P (44) Qiu, T. Z.; Barteau, M. A. J. Colloid Interface Sci. 2006, 303, 229–235.