pubs.acs.org/Langmuir © 2010 American Chemical Society
Chemical Modifications of Au/SiO2 Template Substrates for Patterned Biofunctional Surfaces Elisabeth Briand,† Vincent Humblot,‡ Jessem Landoulsi,‡ Sarunas Petronis,† Claire-Marie Pradier,‡ Bengt Kasemo,† and Sofia Svedhem*,† ‡
† Department of Applied Physics, Chalmers University of Technology, 412 96 G€ oteborg, Sweden, and Laboratoire de R eactivit e de Surface, UMR CNRS 7197, Universit e Pierre et Marie Curie - Paris VI, 75252 Paris Cedex 05, France
Received May 10, 2010. Revised Manuscript Received July 16, 2010 The aim of this work was to create patterned surfaces for localized and specific biochemical recognition. For this purpose, we have developed a protocol for orthogonal and material-selective surface modifications of microfabricated patterned surfaces composed of SiO2 areas (100 μm diameter) surrounded by Au. The SiO2 spots were chemically modified by a sequence of reactions (silanization using an amine-terminated silane (APTES), followed by amine coupling of a biotin analogue and biospecific recognition) to achieve efficient immobilization of streptavidin in a functional form. The surrounding Au was rendered inert to protein adsorption by modification by HS(CH2)10CONH(CH2)2(OCH2CH2)7OH (thiol-OEG). The surface modification protocol was developed by testing separately homogeneous SiO2 and Au surfaces, to obtain the two following results: (i) SiO2 surfaces which allowed the grafting of streptavidin, and subsequent immobilization of biotinylated antibodies, and (ii) Au surfaces showing almost no affinity for the same streptavidin and antibody solutions. The surface interactions were monitored by quartz crystal microbalance with dissipation monitoring (QCM-D), and chemical analyses were performed by polarization modulation-reflexion absorption infrared spectroscopy (PM-RAIRS) and X-ray photoelectron spectroscopy (XPS) to assess the validity of the initial orthogonal assembly of APTES and thiol-OEG. Eventually, microscopy imaging of the modified Au/SiO2 patterned substrates validated the specific binding of streptavidin on the SiO2/APTES areas, as well as the subsequent binding of biotinylated anti-rIgG and further detection of fluorescent rIgG on the functionalized SiO2 areas. These results demonstrate a successful protocol for the preparation of patterned biofunctional surfaces, based on microfabricated Au/SiO2 templates and supported by careful surface analysis. The strong immobilization of the biomolecules resulting from the described protocol is advantageous in particular for micropatterned substrates for cell-surface interactions.
Introduction An increasing number of applications, especially in biotechnology, requires surfaces with defined regions of different chemical functionality to achieve site-specific attachment of one species in some areas while minimizing unwanted surface interactions in other areas.1 Some strategies to achieve such patterned surfaces involves the modification of homogeneous substrates by microcontact printing techniques, through transfer of organic compounds on defined positions of the substrate,2,3 or by chemical modification of some parts of the surface, for example, via electron irradiation4 or extreme UV interference lithography,5 potentially followed by the replacement of the nonmodified molecules by another species. Another approach is based on the combination of different (inorganic) substrate materials for the creation of the patterned template surface, commonly via photolithography. In this case, the patterned inorganic surfaces are designed for orthogonal chemical modifications to obtain the desired final functionally patterned substrates, a procedure which takes advantage of the affinity of certain molecular groups toward *To whom correspondence should be addressed. (1) Schmidt, R. C.; Healy, K. E. J. Biomed. Mater. Res. 2009, 90A, 1252–1261. (2) James, L. W.; Amit, K.; Hans, A. B.; Enoch, K.; George, M. W. Nanotechnology 1996, 7, 452. (3) Ghosh, M.; Alves, C.; Tong, Z.; Tettey, K.; Konstantopoulos, K.; Stebe, K. J. Langmuir 2008, 24, 8134–8142. (4) Turchanin, A.; Tinazli, M.; El-Desawy, H.; Grossmann, M.; Schnietz, H. H.; Solak, R.; Tampe; G€olzh€auser, A. Adv. Mater. 2008, 20, 471–477. (5) Turchanin, M.; Schnietz, M.; El-Desawy, H.; Solak, C.; David; G€olzh€auser, A. Small 2007, 3, 2114–2119.
678 DOI: 10.1021/la101858y
specific materials. This last strategy has been termed ‘‘orthogonal self-assembly” by Laibinis et al.,6 “selective molecular assembly patterning” (SMAP) by Michel et al.,7 and “substrate selective patterning” (SSP) by Bergkvist et al.8 Various chemical compounds and solid substrates have been used in such surface modification protocols, for example, TiO2/SiO2 templates modified (sequentially) with alkylphosphates and PLL-g-PEG,7,9 Au/ AlO3 templates functionalized with alkanethiols and alkane phosphates or carboxylic acids,6,10 and Au/SiO2 templates modified and with alkanethiol and poly(ethylene glycol) (PEG)-silanes.8 In this report, we have developed a protocol for biofunctional modification of Au/SiO2 templates by, first, amino silane (APTES) and, second, thiol-PEG. This choice was made to allow for a thermal treatment after the silanization to stabilize the structure of the silane,11-13 knowing that the alkylthiol layers are (6) Laibinis, P. E.; Hickman, J. J.; Wrighton, M. S.; Whitesides, G. M. Science 1989, 245, 845–847. (7) Michel, R.; Reviakine, I.; Sutherland, D.; Fokas, C.; Csucs, G.; Danuser, G.; Spencer, N. D.; Textor, M. Langmuir 2002, 18, 8580–8586. (8) Bergkvist, M.; Niamsiri, N.; Strickland, A. D.; Batt, C. A. Surf. Sci. 2008, 602, 2121–2127. (9) Michel, R.; Lussi, J. W.; Csucs, G.; Reviakine, I.; Danuser, G.; Ketterer, B.; Hubbell, J. A.; Textor, M.; Spencer, N. D. Langmuir 2002, 18, 3281–3287. (10) Burdinski, D.; Saalmink, M.; van den Berg, J. P. W. G.; van der Marel, C. Angew. Chem., Int. Ed. 2006, 45, 4355–4358. (11) Guo, Z.; Guilfoyle, R. A.; Thiel, A. J.; Wang, R.; Smith, L. M. Nucleic Acids Res. 1994, 22, 5456–5465. (12) Krasnoslobodtsev, V.; Smirnov, S. N. Langmuir 2002, 18, 3181. (13) Pasternack, R. M.; Rivillon Amy, S.; Chabal, Y. J. Langmuir 2008, 24, 12963–12971.
Published on Web 12/13/2010
Langmuir 2011, 27(2), 678–685
Briand et al.
Article
Material and Methods
Figure 1. Schematic representation of the chemical functionalization strategy for the Au/SiO2 patterned template surface in order to spatially control the immobilization of bioactive molecules (not to scale).
known to be unstable when submitted to thermal treatment and to decompose when heated above 350 K.14 Thus, starting from Si wafers processed by photolithography into Au-coated substrates with SiO2 spots (100 μm in diameter), we arrive at a simple procedure to obtain patterned surfaces exposing specifically functionalized areas surrounded by a surface resistant to protein adsorption. The surface modification protocol was designed to result in a protein resistant surface, with biotinylated spots onto which streptavidin can be bound, followed by another biotinylated biomolecule of choice (Figure 1), here biotinylated antibodies. The biotin/streptavidin system was chosen for its wellknown properties and the strong binding between the two compounds, and the general strategy developed in this work can be transposed easily to other applications involving other chemicals. The aim of this work is to demonstrate the spatially defined immobilization of biofunctional proteins in templated patterns. The strong binding of compounds to the template surfaces makes this protocol attractive for use in cell-substrate interaction studies, for example, addressing issues related to longterm stability of the pattern template when used for cell culture.15-18 Four different surface analytical techniques were used to characterize the successive steps of the presented surface modification protocol: polarization modulation-reflection absorption infrared spectroscopy (PM-RAIRS), x-ray photoelectron spectroscopy (XPS), quartz crystal microbalance with dissipation monitoring (QCM-D), and fluorescence microscopy. These various techniques offer complementary structural and functional information at the successive process steps. (14) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321–335. (15) Falconnet, D.; Csucs, G.; Grandin, H. M.; Textor, M. Biomaterials 2006, 27, 3044–3063. (16) Lensen, M. C.; Schulte, V. A.; Salber, J.; Diez, M.; Menges, F.; M€oller, M. Pure Appl. Chem. 2008, 80, 2479–2487. (17) Selhuber-Unkel, C.; Lopez-Garcia, M.; Kessler, H.; Spatz, J. Biophys. J. 2008, 95, 5424–5431. (18) Kilian, K. A.; Bugarija, B.; Lahn, B. T.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 4872–4877.
Langmuir 2011, 27(2), 678–685
Chemicals. 3-(Aminopropyl)triethoxysilane (APTES), biotinamidohexanoyl-6-aminohexanoic acid N-hydroxysuccinimide ester (biotin-NHS), D-biotin, mouse monoclonal anti-rabbit IgG biotin conjugate (biotinylated anti-rIgG), FITC-conjugated rabbit IgG (FITC-rIgG), and phosphate buffered saline (PBS) (10 mM, pH 7.4) tablets were purchased from Sigma-Aldrich, Cy5-labeled streptavidin was obtained from GE Healthcare, and HS(CH2)10CONH(CH2)2(OCH2CH2)7OH (thiol-OEG) was from Polypure SA (Oslo, Norway). Solvents were of analytical grade. Water was filtered and deionized using a Milli-Q unit. Substrates. For PM-RAIRS and XPS measurements, Si wafers were coated with 200 nm of Au by e-beam evaporation at a rate of 1 A˚/s using an AVAC HVC600 general purpose multimaterial evaporator. The coated wafer was then cut into samples of 1 cm 1 cm. For QCM-D measurements, commercially available crystals with gold electrodes (Q-Sense, Sweden) were used either without further modification or after a subsequent coating of 5 nm of Ti (as an adhesion layer) and 50 nm of SiO2 by e-beam evaporation (AVAC HVC600) at a deposition rate of 1-2 A˚/s. Auger experiments on the modified quartz crystals (data not shown) confirmed the presence of SiO2 Patterned surfaces were prepared as follows: A double-sidepolished 300 Si wafer was coated with photoresist (Shipley 1813) by spin coating (2000 rpm for 1 min). The resist then was soft-baked on a hot-plate for 2 min at 110°C and exposed to UV light (400 nm wavelength, 10 mW/cm2 intensity, 10 s exposure time) through the photolithographic mask using a Karl S€ uss MJB2 aligner. The wafer was then dipped into a bath of MICROPOSIT MF-319 developer for 1 min to remove the illuminated photoresist areas. A 5 nm adhesion layer of Ti and 70 nm of Au were then evaporated on top of the substrate and the unexposed photoresist. Finally, the remaining photoresist with a top Ti/Au layer was removed by liftoff technique in a bath of acetone under ultrasonic agitation, leaving the patterned Au layer on the substrate. The different origin of the SiO2 layer on the QCM crystals (physical vapor deposition) and the Si wafers (spontaneously formed oxide layer) was not expected to change its reactivity toward silanes. The only significant differences between the two SiO2 layers would concern the roughness of the substrate, which we do not take into account in this study. Chemical Modification. Cleaning. Immediately before use, the surfaces were cleaned twice by treatment in a UV-ozone chamber (15 min), followed by ultrasonication (5 min) in each of acetone, isopropanol, and water. The samples were then dried under a stream of nitrogen. Silanization. The silanization protocol followed was the one described by Guo et al.11 A solution of APTES at a concentration of 2% v/v was prepared in a mixture of acetone and water (95/5). The silanes were allowed to hydrolyze for 2 h, and then the substrates were introduced into the solution for 30 min. After thorough rinsing with acetone, the surfaces were dried and annealed at 110 °C for 2 h. To modify the NH2 terminated silane self-assembled monolayer (SAM), a solution of biotin-NHS in PBS at 1 mg/mL was deposited on the surfaces for 1 h, followed by extensive rinsing with water. Thiolation. A 1 mM solution of thiol-OEG was prepared in ethanol. The samples were dipped into the thiol solution for at least 18 h before extensive rinsing with ethanol. Biospecifically Patterned Surfaces. After cleaning of the substrates, they were functionalized by silanization followed by thiolation (as described above). After thorough rinsing by ethanol and drying under a stream of nitrogen, 100 μL of each of the following five solutions in PBS was successively placed on top of the substrates: (i) biotin-NHS 1 mg/mL for 1 h, (ii) Cy5 labeled streptavidin 20 mg/mL solution for 1 h, (iii) biotinylated antirIgG 100 mg/L for 1 h, and (iv) FITC labeled rIgG 40 mg/L for 1 h. After each immersion, the surfaces were thoroughly rinsed DOI: 10.1021/la101858y
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with PBS and water. All the concentrations used in this work are in the range of what is usually used in the elaboration of immunosensors.
Experimental Techniques.
PM-RAIRS Measurements.
The Fourier transform infrared (FT-IR) instrument used in this work was a commercial NICOLET 5700 Nexus spectrometer. The external beam was focused on the sample with a mirror at an optimal incident angle of 85°. A ZnSe grid polarizer and a ZnSe photoelastic modulator, modulating the incident beam between p and s polarizations (HINDS Instruments, PEM 90, modulation frequency =37 kHz), were placed prior to the sample. The light reflected at the sample was then focused on a nitrogen-cooled MCT detector. The sum and difference interferograms were processed and Fourier-transformed to yield the differential reflectivity ΔR/R=(Rp - Rs)/(Rp þ Rs) which is the PM-RAIRS signal. A total of 128 scans were recorded at 8 cm-1 resolution for each spectrum. X-ray Photoelectron Spectroscopy. XPS analyses were performed using a SPECS (Phoibos MCD 150) X-ray photoelectron spectrometer (SPECS, Germany) equipped with a nonmonochromatized magnesium X-ray source (hν = 1253.6 eV) powered at 10 mA and 15 kV, and a Phoibos 150 hemispherical energy analyzer. The resulting analyzed area was 5 mm in diameter. A pass energy of 20 eV was used for the survey scan and 10 eV for narrow scans. The samples were fixed on the support using double-sided adhesive tape, and no charge stabilization device was used. The pressure in the analysis chamber during measurement was around 10-10 Torr or less. The photoelectron collection angle between the normal to the sample surface and the analyzer axis was 0°. The following sequence of spectra was recorded: survey spectrum, O 1s, C 1s, Au 4f, N 1s, S 2p, and Si 2p. The binding energy scale was set by fixing the C 1s component due to carbon only bound to carbon and hydrogen at 284.8 eV. The data treatment was performed with the Casa XPS software (Casa Software Ltd., U.K.). Unless stated otherwise, the peaks were decomposed using a linear baseline, and a component shape defined by the product of a Gauss and Lorentz function in a 70:30 ratio, respectively. Molar concentration ratios were calculated using peak areas normalized according to Scofield factors.19 QCM-D Measurements. QCM-D measurements were conducted using a commercial instrument (QCM-D E4, Q-sense AB, Sweden) at a temperature of 22 ( 0.1 °C. The device has been described in detail elsewhere.20,21 Briefly, oscillations of AT cut quartz crystals at the resonant frequency (here 5 MHz) or at one of its overtones (15, 25, 35, 45, 55, 65 MHz) are obtained when applying AC voltage. The drive circuit is then open-circuited, and the exponential decay of the oscillation amplitude is monitored. The dissipation, D, is defined as the fraction of energy of the oscillation that is dissipated during one period of oscillation. Upon adsorption of material on the crystal surface, the resonance frequency of the crystal (Δf) decreases and the dissipation shift (ΔD) reflects the viscoelastic properties of the adlayer. Solutions were injected into the measurement cell using a peristaltic pump (Ismatec IPC-N 4) with a flow rate of 100 μL/min. All frequency shifts were normalized with the overtone number. Prior to the protein adsorption, a PBS buffer solution was injected to establish a stable baseline. Fluorescence Microscopy. Experiments were carried out using a fluorescence microscope (BX61, Olympus, Germany) with a 20 water immersion objective (UMPLFLN-20XW). The samples were illuminated by a Hg lamp and analyzed using two wide bandpass fluorescence filters (U-MWIB2 and U-MWG2, Olympus, Germany). The images were processed with analysis software (Soft Imaging, Olympus, Germany) in multiple fluorescence mode. (19) Scofield, J. H. J. Electron Spectrosc. Relat. Phenom. 1976, 8, 129–137. (20) H€oo€k, F.; Kasemo, B.; Nylander, T.; Fant, C.; Sott, K.; Elwing, H. Anal. Chem. 2001, 73, 5796–5804. (21) Rodahl, M.; H€oo€k, F.; Kasemo, B. Anal. Chem. 1996, 68, 2219–2227.
680 DOI: 10.1021/la101858y
Results The aim of this work was to fabricate patterned biofunctional surfaces, and we have chosen to demonstrate spatially defined immunorecognition reactions through the site-specific immobilization of a biotinylated antibody. The surface modification protocol was based on a patterned inorganic SiO2/Au template, which was modified by a sequence of chemical reactions, such that the antibodies were specifically immobilized onto SiO2 whereas Au was modified to resist protein adsorption. Immobilization of Biotinylated Antibodies to SiO2 Surfaces. As a first step in the development of surfaces with antibody patterns, SiO2 films deposited onto QCM-D sensors were modified with APTES followed by the covalent grafting of biotinNHS. The further binding of streptavidin to this surface was monitored in situ by QCM-D, and the specificity of the interaction was tested by probing the adsorption of streptavidin which had been presaturated with biotin.22 The QCM-D frequency and dissipation shifts (Δf and ΔD) were monitored for the two experiments, and the results are displayed in Figure 2. The level of nonspecific binding of the presaturated streptavidin on the APTES and biotin-NHS modified surface was very low, about -2 Hz (note that negative frequency shifts correspond to mass uptake). In contrast, when a solution of nonsaturated streptavidin was in contact with the biotinylated surface (Figure 2), a frequency shift of -24 ( 1 Hz was recorded. The Δf value obtained by QCM-D for the layer of streptavidin was similar to previous studies23-26 and suggested a full coverage of streptavidin (i.e., a streptavidin monolayer) on the biotinylated surface. The recorded frequency shift corresponded to a coverage of ∼400 ng/cm2, according to the Sauerbrey equation.21,27 The low level of dissipation (
