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QCM-D on Mica for Parallel QCM-DsAFM Studies Ralf P. Richter and Alain Brisson* Laboratoire d’Imagerie Mole´ culaire et Nano-Bio-Technologie, IECB, UMR-CNRS 5471, Universite´ Bordeaux I, 2 Rue Robert Escarpit, 33607 Pessac Cedex, France Received January 19, 2004. In Final Form: March 18, 2004 Quartz crystal microbalance with dissipation monitoring (QCM-D) has developed into a recognized method to study adsorption processes in liquid, such as the formation of supported lipid bilayers and protein adsorption. However, the large intrinsic roughness of currently used gold-coated or silica-coated QCM-D sensors limits parallel structural characterization by atomic force microscopy (AFM). We present a method for coating QCM-D sensors with thin mica sheets operating in liquid with high stability and sensitivity. We define criteria to objectively assess the reliability of the QCM-D measurements and demonstrate that the mica-coated sensors can be used to follow the formation of supported lipid membranes and subsequent protein adsorption. This method allows combining QCM-D and AFM investigations on identical supports, providing detailed physicochemical and structural characterization of model membranes.
Introduction Besides the potential application of supported lipid bilayers (SLBs) and two-dimensional (2D) ordered protein assemblies as building blocks in biofunctional surfaces, 1-4 the self-assembly mechanisms driving the formation of these structures are of fundamental scientific interest.5-12 From previous studies on the formation of SLBs by spreading of vesicles on hydrophilic supports5,6,9,12-23 and on the 2D crystallization of proteins on lipid membranes,3,7,10,24-26 a qualitative picture of structural inter* Corresponding author. E-mail:
[email protected]. (1) Sackmann, E. Science 1996, 271, 43-48. (2) Wetzer, B.; Pum, D.; Sleytr, U. B. J. Struct. Biol. 1997, 119, 123128. (3) Reviakine, I.; Bergsma-Schutter, W.; Brisson, A. J. Struct. Biol. 1998, 121, 356-361. (4) Reviakine, I.; Brisson, A. Langmuir 2001, 17, 8293-8299. (5) Seifert, U. Adv. Phys. 1997, 46, 13-137. (6) Reviakine, I.; Brisson, A. Langmuir 2000, 16, 1806-1815. (7) Reviakine, I.; Bergsma-Schutter, W.; Maze`res-Dubut, C.; Govorukhina, N.; Brisson, A. J. Struct. Biol. 2000, 131, 234-239. (8) Reviakine, I.; Bergsma-Schutter, A.; Morozov, A. N.; Brisson, A. Langmuir 2001, 17, 1680-1686. (9) Zhdanov, V. P.; Keller, C. A.; Glasma¨star, K.; Kasemo, B. J. Chem. Phys. 2000, 112, 900-909. (10) Zhdanov, V. P.; Ho¨o¨k, F.; Kasemo, B. Proteins: Struct., Funct., Genet. 2001, 43, 489-498. (11) Noro, M. G.; Bates, M. A.; Brisson, A.; Frenkel, D. Langmuir 2002, 18, 2988-2992. (12) Richter, R.; Mukhopadhay, A.; Brisson, A. Biophys. J. 2003, 85, 3035-3047. (13) Keller, C. A.; Glasma¨star, K.; Zhdanov, V. P.; Kasemo, B. Phys. Rev. Lett. 2000, 84, 5443-5446. (14) Keller, C. A.; Kasemo, B. Biophys. J. 1998, 75, 1397-1402. (15) Reimhult, E.; Ho¨o¨k, F.; Kasemo, B. Phys. Rev. E 2002, 66, 051905-1-4. (16) Reimhult, E.; Ho¨o¨k, F.; Kasemo, B. J. Chem. Phys. 2002, 117, 7401-7404. (17) Johnson, J. M.; Taekijp, H.; Chu, S.; Boxer, S. G. Biophys. J. 2002, 83, 3371-3379. (18) Cremer, P. S.; Boxer, S. G. J. Phys. Chem. B 1999, 103, 25542559. (19) Jass, J.; Tja¨rnhage, T.; Puu, G. Biophys. J. 2000, 79, 31533163. (20) Nollert, P.; Kiefer, H.; Ja¨hnig, F. Biophys. J. 1995, 69, 14471455. (21) Ra¨dler, J.; Strey, H.; Sackmann, E. Langmuir 1995, 11, 45394548. (22) Lipowsky, R.; Seifert, U. Mol. Cryst. Liq. Cryst. 1991, 202, 1725. (23) Zhdanov, V. P.; Kasemo, B. Langmuir 2001, 17, 3518-3521. (24) Yatcilla, M. T.; Robertson, C. R.; Gast, A. P. Langmuir 1998, 14, 497-503. (25) Wang, S.-W.; Robertson, C. R.; Gast, A. P. Langmuir 1999, 15, 1541-1548.
mediates and driving forces is emerging, whereas a quantitative characterization of the self-assembly kinetics is still largely lacking. These studies have also shown that combining information from various surface-sensitive methods is required to get a detailed description and understanding of such processes. However, a direct correlation of results obtained from different surfacesensitive methods is hampered by the fact that they use various types of support materials, which play a determining role in the adsorption process investigated. In particular, we have shown recently that the nature of the surface can influence the pathways of SLB formation as well as the naturesorder versus disordersof protein assemblies.6,7,12,27 In this study, we present a method to deposit mica sheets on QCM-D sensors for measurements in liquid. Our objective is to perform studies of adsorption processes, by QCM-D and AFM, allowing parallel physicochemical and structural analysis on identical supports. Quartz crystal microbalance with dissipation monitoring (QCM-D), a technique that provides information of bound mass, water content, and viscoelastic properties of surface-bound material,28 has become popular to investigate the formation of SLBs12-16 as well as interactions of SLBs with proteins and other biomolecules.29-35 Among materials currently used in surface-sensitive methods, which include glass, gold, graphite, mica, silica, (26) Oling, F.; Bergsma-Schutter, W.; Brisson, A. J. Struct. Biol. 2001, 133, 55-63. (27) Richter, R.; Brisson, A. Langmuir 2003, 19, 1632-1640. (28) Ho¨o¨k, F.; Larsson, C.; Fant, C. In Encyclopedia of Surface and Colloid Science; Somasundaran, P., Ed.; Marcel Dekker: New York, 2002; pp 774-791. (29) Glasma¨star, K.; Ho¨o¨k, F.; Kasemo, B. J. Colloid Interface Sci. 2002, 246, 40-47. (30) Kastl, K.; Ross, M.; Gerke, V.; Steinem, C. Biochemistry 2002, 41, 10087-10094. (31) Govorukhina, N.; Bergsma-Schutter, A.; Maze`res-Dubut, C.; Maze`res, S.; Drakopoulou, E.; Bystrykh, L.; Oling, F.; Mukhopadhyay, A.; Reviakine, I.; Lai Kee Him, J.; Brisson, A. In Annexins: Biological importance and annexin-related pathologies; Bandorowicz-Pikula, J., Ed.; Landes Bioscience/Eurekah.com: Georgetown, TX, 2003; pp 3755. (32) Larsson, C.; Rodahl, M.; Ho¨o¨k, F. Anal. Chem. 2003, 75, 50805087. (33) Richter, R.; Lai Kee Him, J.; Brisson, A. Mater. Today 2003, 6, 32-37. (34) Fant, C.; Elwing, H.; Ho¨o¨k, F. Biomacromolecules 2002, 3, 732741. (35) Ho¨o¨k, F.; Ray, A.; Norde´n, B.; Kasemo, B. Langmuir 2001, 17, 8305-8312.
10.1021/la049827n CCC: $27.50 © 2004 American Chemical Society Published on Web 04/30/2004
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silicon, or titanium oxide, mica is unique for its intrinsic hydrophilicity and because it is easily cleaved into large (cm2) atomically flat areas, making it an ideal surface for imaging by AFM.27,36,37 Previous attempts to develop micacoated sensors for QCM so far focused on applications in gaseous environment38-40 or, when applied in liquid, resulted in limited stability, preventing quantitative evaluation.41 The objectives of this work are (i) to demonstrate the feasibility of performing QCM-D experiments in liquids on mica with high stability and sensitivity, (ii) to evaluate how the reliability of mica-QCM-D can be ensured on the quantitative level, (iii) to demonstrate that mica-QCM-D allows to follow self-assembly processes such as SLBformation or protein 2D crystallization, and (iv) to demonstrate the potential of a combination of AFM and QCM-D on identical supports. Materials and Methods Materials. Dioleoylphosphatidylcholine (DOPC) and dioleoylphospatidylserine (DOPS) were purchased from Avanti Polar-Lipids (AL). Recombinant rat annexin A5 was overexpressed in Escherichia coli and purified as previously described.7 Other chemicals were purchased from Sigma. Ultrapure water with a resistivity of 18.2 MΩ was used (Maxima, USF ELGA, France). Muscovite mica disks of 12-mm diameter were purchased from Metafix (Montdidier, France). QCM-D sensor crystals (5 MHz) with gold electrodes, reactively sputter coated with 50 nm silicon oxide, were purchased from Q-SENSE (Gothenburg, Sweden). Low viscosity epoxy glue (EPOTEK 377) was purchased from Gentec Benelux (Waterloo, Belgium). A buffer solution made of 150 mM NaCl, 2 mM NaN3, and 10 mM HEPES, pH 7.4, was prepared in ultrapure water, and either 2 mM EDTA or CaCl2 was added as indicated in the text. Small unilamellar vesicles (SUVs) of desired lipid mixture were prepared by sonication as described earlier.12 Before use, vesicle suspensions were diluted at 0.1 mg/mL. Annexin A5 was used at a concentration of 20 µg/mL. Preparation of Mica-Coated QCM-D Sensors. Prior to the mica deposition, the QCM-D sensor crystals were rinsed in water and ethanol, blow-dried with nitrogen, and exposed to UV/ozone (BHK, CA) for 10 min.27 Mica disks were cleaved with Scotch tape, until a thin sheet (
