Versatile Decoration of Glass Surfaces To Probe Individual Protein

Langmuir 2010, 26 (18) , 14707-14715. DOI: 10.1021/la103086b. Chun-lai Ren, Daniel Carvajal, Kenneth R. Shull and Igal Szleifer . Streptavidin−Bioti...
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Langmuir 2002, 18, 846-854

Versatile Decoration of Glass Surfaces To Probe Individual Protein-Protein Interactions and Cellular Adhesion Emilie Perret,† Andrew Leung,‡ Anne Morel,† He´le`ne Feracci,† and Pierre Nassoy*,§ Institut Curie, Unite´ Mixte de Recherches 144, 26 rue d’Ulm, 75248 Paris Cedex 05, France, University of British Columbia, Department of Pathology, Vancouver, British Columbia, V6T 1Z1 Canada, and Institut Curie, Unite´ Mixte de Recherches 168, 11 rue P. et M. Curie, 75248 Paris Cedex 05, France Received September 28, 2001. In Final Form: November 28, 2001 The capability to durably link biological macromolecules to solid supports is fundamental for the development of biosensors and many diagnostic techniques, as well as for the investigation of biomolecular interactions such as adhesion of cells onto biomimetic substrates. Here, we describe two simple and reproducible chemical procedures to decorate glass surfaces with specific ligands at a variable and controlled surface density. The first method uses the streptavidin-biotin complex for further immobilization of biotinylated proteins, while the second method performs a direct covalent attachment of proteins to glass. Both procedures were characterized by optical interferometry to measure molecular-layer thickness, fluorescence flow cytometry to evaluate surface density, and qualitative adhesion/aggregation assays to assay protein functionality. Both routes were first applied to streptavidin as a model protein, and extended to an homotypic calcium-dependent adhesive protein, namely E-cadherin. We mainly discuss key issues that must be addressed when control of the protein surface density and passivation of the surface against nonspecific adsorption are required.

Introduction Immobilization of proteins has been widely studied and used as a tool for revealing protein interaction and function in immunoassays, biosensors, or affinity chromatography. One of the main concerns has been the reduction of the biological activity of the proteins due to immobilizationinduced conformational changes. Numerous methods (from simple physisorption to very sophisticated linkages through synthesized cross-linkers) have been successfully implemented to produce functionalized surfaces with biologically active macromolecules, to allow a subsequent detection of specific recognition between the immobilized ligands and free receptors in solution.1 Following the advent of single-molecule experiments (using atomic force microscopy,2 optical tweezers,3 or a biomembrane force probe4), micropatterning techniques,5 and DNA or protein chips,6 new requirements have now to be considered. Measuring the adhesion strength of single protein-protein * To whom correspondence and request for materials should be addressed. E-mail: [email protected]. † Institut Curie, Unite ´ Mixte de Recherches 144. ‡ University of British Columbia. § Institut Curie, Unite ´ Mixte de Recherches 168. (1) (a) Hermanson, G. T.; Krishna Mallia, A.; Smith, P. K. In Immobilized Affinity Ligand Techniques; Academic Press: New York, 1992. (b) Patel, N.; Davies, M. C.; Hartshorne, M.; Heaton, R. J.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Langmuir 1997, 13, 6485. (c) Sarkar, N.; Bhattacharjee, S.; Sivaram, S. Langmuir 1997, 13, 4142. (d) Lu, H. B.; Campbell, C. T.; Castner, D. G. Langmuir 2000, 16, 1711. (2) (a) Florin, E.-L.; Moy, V. T.; Gaub, H. E. Science 1994, 264, 415. (b) Lee, G. U.; Kidwell, D. A.; Colton, R. J. Langmuir 1994, 10, 354. (3) Finer, J. T.; Simmons, R. M.; Spudich, J. A. Nature 1994, 368, 113. (4) (a) Evans, E.; Ritchie, K.; Merkel, R. Biophys. J. 1995, 68, 2580. (b) Merkel, R.; Nassoy, P.; Leung, A.; Ritchie, K.; Evans, E. Nature 1999, 397, 50. (5) (a) Mooney, J. F.; Hunt, A. J.; McIntosh, J. R.; Liberko, C. A.; Walba, D. M.; Rogers, C. T. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12287. (b) Harnett, C. K.; Satyalakshmi, K. M.; Craighead, H. G. Langmuir 2001, 17, 178. (c) Ostuni, E.; Chen, C. S.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17, 2828.

bonds using these ultrasensitive force probes requires a strong attachment of the sparse partner molecules on the tips or substrates, while avoiding any other nonspecific interaction. The fabrication of DNA or protein microarrays requires the oligonucleotide or protein surface density to be finely tuned in order to optimize the recognition efficiency.7 Finally, the behavior of cells on adhering or repelling substrates has been investigated so far by designing some patterned surfaces.8 However, it may be of interest to fabricate homogeneous surfaces with variable protein grafting densities. In all these experiments, the main common bottlenecks remain reproducibility and durability in the preparation of decorated surfaces. By durable, we mean strong linkages without nonspecific physisorption and without any risk of degradation in time. By reproducible, we mean a controlled density of anchored test ligands. Moreover, once linked, the ligand must retain its biological activity, which means that (1) the attached biomolecule must not be denatured or inactivated at the surface, (2) the attachment site must not interfere with the functional structure of the protein, and (3) the tethered biomolecule must be free to orient and move for specific recognition. Here, we describe two methods for decorating glass surfaces (slides and beads) with some biomolecules (proteins) at a tunable density. The first approach uses a sandwich-like construct based on the streptavidin-biotin complex. Biotin is a vitamin which binds to streptavidin with a very high affinity (Kd ) 10-15 M).9 We will show (6) (a) Martin, B. D.; Gaber, B. P.; Patterson, C. H.; Turner, D. C. Langmuir 1998, 14, 3971. (b) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760. (7) Shchepinov, M. S.; Case-Green, S. C.; Southern, E. M. Nucleic Acids Res. 1997, 25, 1155. (8) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425. (9) (a) Green, N. M. Adv. Protein Chem. 1975, 29, 85. (b) Livnah, O.; Bayer, E. A.; Wilcheck, M.; Sussman, J. L. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5076.

10.1021/la015601y CCC: $22.00 © 2002 American Chemical Society Published on Web 01/10/2002

Versatile Decoration of Glass Surfaces

Figure 1. Molecular structures of the mercaptosilane (MPTS) and the three cross-linkers: Mal-PEG3400-biotin, Mal-PEG5000methoxy, and VS-PEG3400-NHS.

how to vary the streptavidin density from close-packing down to zero while passivating the surface against nonspecific interaction. A glass substrate, which is decorated with biotin, and subsequently with streptavidin, could then serve as a suitable template for immobilization of other biotinylated proteins. The second route provides a direct covalent coupling of proteins to a preactivated glass surface. For the sake of simplicity and comparison of the two methods, streptavidin has been chosen as a model protein. The present study puts the emphasis on the way to dilute the protein surface density while keeping a low level of nonspecific background. The aim of the present paper is to show the feasibility and the possibility of the concept. After describing the implementation of the two immobilization methods, we will characterize the chemical treatment from thickness and surface density measurements and test the performance of derivatized beads by various functionality assays. Our approach was finally extended to another type of protein, namely an homotypic calcium-dependent adhesive protein called cadherin.10 By investigating the specificity of the interaction between cells expressing cadherins and homemade surfaces, we will show that this strategy can be successfully used to study the complex nature of molecular interactions between receptor and ligand at the scale of individual molecules as well as to gain insight into the fundamental mechanisms of cellular adhesion at the mesoscopic scale. Experimental Section Materials. (a) Substrates. Silica beads (Bangs Laboratories, 2.3 µm in diameter) and ordinary borosilicate cover glasses (Fisher Scientific Corp.) were used as templates for immobilization. (b) Reagents. All reagents were obtained commercially and used without further purification. All buffers (phosphate buffered saline solution (PBS) and carbonate-bicarbonate buffer (CB)) were made up in deionized water (Millipore, 18 MΩm‚cm). All solvents were high-performance liquid chromatography (HPLC) grade. Methanol, acetic acid, and anhydrous DMSO were purchased from Sigma-Aldrich, as was the functionalized silane which we used, (3-mercaptopropyl)trimethoxysilane (MPTS) (Figure 1). All biotinylated derivatives and cross-linkers were purchased from Shearwater Polymers: Mal-PEG3400-biotin, VSPEG3400-NHS, and Mal-PEG5000-methoxy (Figure 1). (c) Proteins. Streptavidin (Jackson ImmunoResearch Laboratories Inc.) was chosen as a model system. β-Casein (SigmaAldrich) was taken as a control for nonspecific interactions. Fluorescein-biotin was obtained from Molecular Probes and used to estimate the amount of streptavidin grafted by fluorescence techniques. (10) Takeichi, M. Science 1991, 251, 1451.

Langmuir, Vol. 18, No. 3, 2002 847 E-cadherin fragments were not commercially available. They were synthesized and purified according to the procedure described hereafter. The cDNA of full lengh mouse E-cadherin11 (kindly provided by L.Larue, Institut Curie, Orsay, France) was used as a template for PCR amplification of DNA coding for the first two extracellular domains EC1-2 fused with the hexahistidine tag in the C-terminal position. The following primers were used: 5′-CCC CAT ATG GAC TGG GTC ATC CCT C-3′ and 5′-CCG CTC GAG GAA GAC AGG AGC GTT GT-3′. The obtained fragments were cloned in the pET24a expression vector (Novagen) using the XhoI/NdeI site. For EC1-2 purification, TB (Terrific Broth) media cultures of 500 mL containing 50 µg/mL kanamycin were inoculated by transformed colonies picked up from agar plates. Cultures were grown at 37 °C until an optical density of 0.6-0.8 was reached. Expression was induced by the addition of 1.7 mM isopropylβ-D-thiogalactoside (IPTG). After 2 h, cells were harvested by centrifugation and stored at -70 °C. Cell pellets were resuspended in lysis buffer (4 M urea; 50 mM Na2HPO4 pH 7.8; 20 mM imidazole; 20 mM β-mercaptoethanol). The lysate was clarified by centrifugation, and the supernatant was incubated for 2 h at 4 °C with Ni2+-NTA (NitriloTriacetic Acid) agarose resin (Quiagen). The beads were extensively washed with lysis buffer and then stepwisely dialyzed against PBS. Cadherin fragments were eluted with 0.25 M imidazole. Following purification, the purity of the proteins was checked by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining. (d) Cells. We used biotinylated red blood cells to qualitatively assay the functionality of streptavidin-coated beads. Fresh red blood cells were obtained from donors and washed once with PBS 290 mOsm and three times with 0.1 M carbonatebicarbonate buffer (pH 8.5). Then, the cells were biotinylated by incubation in a 0.5 mM NHS-PEG3400-biotin solution made from CB buffer for 30 min. After three washes with Tris buffered saline (pH 7.5), the biotinylated cells were resuspended in PBS 290 mOsm plus 0.5 wt % bovine serum albumin (to prevent crenation and adhesion to the glass). We used rat basophilic leukemia (RBL) cells expressing E-cadherin to test the adhesive properties of the cadherin-coated bead. They were cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS), antibiotics, and 2 mM L-glutamine (GibcoBRL, Life Technologies) in a 37 °C incubator under an atmosphere of 6% CO2/94% air. To obtain clonal wild-type E-cadherin expressor cells, full length cDNA of mouse E-cadherin inserted in a pGK vector was introduced in RBL cells by electroporation. After a further 24 h in culture, the cells were split and hygromycin (Roche) was added to a final concentration of 1.2 mg/mL to select stable transformants. After 12-15 days, resistant colonies were isolated and examined for cell surface expression of E-cadherin by immunofluorescence and Western blotting. Several colonies were picked, and one was selected for the highest levels of E-cadherin. Surface Functionalization. (a) General Routes. We implemented two surface modification routes to decorate glass surfaces with proteins. The first one (method 1) is based on a sandwichlike construct using the streptavidin-biotin complex as a template for possible further immobilization of biotinylated proteins. The second approach (method 2) provides a direct covalent attachment of proteins. Both routes allow a fine control of the surface density of the protein and are displayed schematically in Figure 2. For the preparation of biotinylated surfaces (method 1), precleaned glass surfaces were first activated with a mercaptosilane, followed by reaction with maleimide (Mal) end-groups of long flexible biotinylated derivatives mixed with methoxyterminated analogues (if dilution is desired). Finally, streptavidin is coupled in a straightforward way by incubation. The preparation of covalently linked proteins to glass (method 2) is slightly different. To render the method as general as possible, we chose to perform a selective binding of proteins through their amino residues. Depending upon the protein, many different amino sites are likely to be reacting with the crosslinker. A random orientation of the linker to the protein is (11) Larue, L.; Antos, C.; Butz, S.; Huber, O.; Delmas, V.; Dominis, M.; Kemler, R. Development 1996, 122, 3185.

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Figure 2. Schematic representation of the two surface modification routes. In both cases, glass is first silanized with the MPTS trifunctional silane. Method 1 uses the streptavidinbiotin construct. Biotinylation of the surface at a controlled density of biotin is performed by mixing two polymeric PEG homofunctional cross-linkers: one has a biotin end group, and the other one has a methoxy end which serves both for dilution of the biotin sites and for passivation of the surface against nonspecific adsorption of proteins. Subsequent incubation in a streptavidin solution provides the desired modified glass surfaces. Method 2 performs a direct covalent grafting of the protein to the surfaces. Streptavidin is first conjugated to an hetero-bifunctional, namely VS-PEG3400-NHS, via a reaction between the NHS group (N-hydroxysuccinimide ester) and available amino residues of the protein sequence. Then, the conjugated protein is allowed to react with the mercapto surface groups via the VS (vinylsulfone) termination. If desired, dlution with VS-PEG3400-COOH allows us to finely tune the protein surface density. therefore expected. However, as pointed out by other authors,12 the presence of a long and flexible spacer may provide sufficent distance and mobility for the protein to reorient properly and bind efficiently to its associated ligand. To do so, we chose a polymeric cross-linker bearing two functional end groups, namely N-hydroxysuccinimide (NHS) and vinylsulfone (VS). The solution of protein was first labeled with VS-PEG3400-NHS in aqueous buffer. The NHS group is amine-specific and thus reactive toward exposed amino residues in the peptidic sequence of the protein. The unreacted cross-linkers are then eliminated by size exclusion chromatography or, equivalently, by dialysis, so that proteins bearing the thiol-reactive VS group may react with surface mercapto groups of the glass substrates pretreated with MPTS. When the surface density of protein is desired to be reduced, vinylsulfone-tagged proteins are diluted with monofunctional VS-PEG3400-COOH (obtained from hydrolysis of VS-PEG3400-NHS in pure water) in the desired ratios, prior to reaction. The choice of a VS-terminated cross-linker was dictated by its greater stability against hydrolysis over that of the maleimide function.13 (12) (a) Hinterdorfer, P.; Baumgartner, W.; Gruber, H. J.; Schilcher, K.; Schindler, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 3477. (b) Hinterdorfer, P.; Kienberger, F.; Raab, A.; Gruber, H. J.; Baumgartner, W.; Kada, G.; Riener, C.; Wielert-Badt, S.; Borken, C.; Schindler, H. Single Mol. 2000, 1, 99.

Perret et al. (b) Sample Preparation. (i) Cleaning. The cleaning procedure that we chose has been inspired from treatments used in metaloxide semiconductor technology,14 where washing is aimed to degrease the surface and refresh the superficial layers of native silicon oxide. Glass beads or cover slides were thus soaked in a (1/0.1/5) mixture of (ammonium hydroxide-hydrogen peroxidewater), at boiling temperature for 5 min. Next, substrates were rinsed in ultrapure water and stored, if needed, in deionized water. (ii) Silanization. After cleaning, surfaces were treated with a mercaptosilane: (3-mercaptopropyl)trimethoxysilane (MPTS). A schematic representation of the chemical structure appears in Figure 1. The commonly accepted scheme for silane coupling starts with an hydrolysis of the methoxy groups. Next, hydrolyzed silanes hydrogen-bind with surface silanols. Finally, they crosslink with silica and with neighboring molecules through a condensation reaction.15 The choice of a trifunctional silane was guided by our own experience and previous studies which reported lower resistance to hydrolysis of mono- and difunctional silanes.16 The most prevalent and easy-to-use protocol consists of performing the silanization in 94% acidic methanol (0.15 M acetic acid) + 4% water + 2% silane (as reported for example in ref 17). Water is used to speed up the hydrolysis, and acetic acid has a “regulation” role insofar as the condensation is supposed to be kept slow in a mildly acidic environment. Clean slides and beads were immersed in a freshly prepared silanization solution for 1 h at room temperature and then washed three times with methanol to remove unreacted materials. Finally, the materials were baked for 5 min in a clean drying oven at 120 °C to drive off any excess moisture and enhance covalent linking to silica. (iii) Biotinylation and Immobilization of Streptavidin (Method 1): Following silanization, the next step was to functionalize the carrier mercapto-surface with biotin. The selected biotinylated derivative is displayed in Figure 1. It is referred to as Mal-PEG3400biotin. The Mal (maleimide) group is mercapto-specific in defined conditions of pH. The PEG sequence is composed of 77 ethylene glycol (-CH2-CH2-O-) monomers (the superscript indicates the average molecular weight of the PEG3400 segment); its contour length is about 34 nm. When the surface density of biotin sites had to be reduced, the biotinylated linker was mixed with a passive linker. As a surface diluent, we chose Mal-PEG5000methoxy (see Figure 1) to match as close as possible the biotinylated linker length. The following procedure was used to prepare biotinylated surfaces. A mixture of Mal-PEG3400-biotin-Mal-PEG5000-methoxy at desired ratios) was dissolved in PBS buffer at pH 7.5. Microbeads or slides were soaked in the freshly prepared solution at room temperature for 1 h with gentle mixing, after which the surfaces were washed in distilled water. When desired, biotinylated glass beads or slides were incubated in a solution of streptavidin at 10 µg/mL in PBS for 1 h. The substrates were stored at 4 °C until use in PBS + streptavidin, to compensate any release of streptavidin. Prior to use, they were washed with fresh PBS. (iv) Covalent Attachment of Proteins to Glass (Method 2). To covalently link the protein to the preactivated substrate, the previous procedure had to be slightly modified. Instead of preparing a surface modified with functional groups which are reactive with amino acids of the protein, we first coupled the protein to an hetero-bifunctional cross-linker, VS-PEG3400-NHS, by incubation in carbonate-bicarbonate buffer (pH 8.5) for 1 h. The resulting conjugated proteins carrying a VS-terminated spacer were purified from unreacted PEGs using spin-column chromatography (MicroSpin G-25 columns from Amersham) or, equivalently, dialysis with Slide-A-Lyser dialysis cassettes (Pierce Chemicals). In parallel, a solution of VS-PEG3400-NHS in (13) Morpurgo, M.; Veronese, F. M.; Kachensky, D.; Harris, J. M. Bioconjugate Chem. 1996, 7, 363. (14) Suzuki, T.; Adachi, S. Jpn. J. Appl. Phys. 1994, 33, 2689. (15) (a) Plueddemann, E. P. Silane Coupling Agents; Plenum Press: New York and London, 1982. (b) Kessel, C. R.; Granick, S. Langmuir 1991, 7, 532. (16) Gautier, S.; Aime´, J. P.; Bouhacina, T.; Attias, A. J.; Desbat, B. Langmuir 1996, 12, 5126. (17) Lee, G. U.; Chrisey, L. A.; O’Ferrall, C. E.; Pillof, D. E.; Turner, N. H.; Colton, R. J. Isr. J. Chem. 1996, 36, 81.

Versatile Decoration of Glass Surfaces

Figure 3. (a) Schematic of a chemically modified bead sitting on a similarly modified cover glass. The gap distance between both surfaces is twice the thickness of the coating layer. (b) Reflection interference contrast image of a glass microbead above a cover glass. Analysis of the circular interference pattern was used to measure the distance between both glass surfaces (bead and cover glass). carbonate-bicarbonate buffer (pH 8.5) was left for hydrolysis of the NHS function into carboxylate. The VS-functionalized proteins and the VS-PEGs were then mixed at the desired molar ratios and incubated with mercapto-silanized glass in PBS for 3 h at room temperature and overnight at 4 °C. Surface Characterization Techniques. Several assays were employed to characterize the surface structures bound to glass and to test the functionality of bound proteins. (a) Measurements of Thickness. Thicknesses of surface layers were determined by optical measurements of the separation between the glass interfaces of a microsphere with/without a molecular coating and a cover glass substrate either with or without a molecular coating as well. With a microbead sedimented onto a cover glass under water, a reflection interference contrast microscope (RICM) was used to encode the distance between the glass interfaces of the bead and cover slip into a circular fringe pattern,18 as shown in Figure 3. The Newton “ring” pattern was produced by interference of strong reflections from the upper cover glass and microbead glass interfaces. In concentrated salt solutions, electrostatic repulsion was screened so that naked beads rested directly on bare glass slides with less than 1 nm separation, as shown by the lack of Brownian movements. Deviations from zero optical separation were due to the combined roughness of the surfaces and intercalated water (index nwater ) 1.33). The optical path length was governed by separation distance and total index of refraction. When coated with organic molecules, the RICM pattern involved an index of refraction contributed by several layers, which was bounded by the value 1.33 for water and ∼1.5 for silane, polymer, or protein at maximum density. However, the layers were certainly not maximally packed, so for simplicity, we have treated the multimolecular structures as a single layer with arbitrary refractive index equal to 1.4. As such, the maximum error in the index could not have exceeded 0.1, so the error in the optical phase thickness was negligible (∼0.07 or less). (18) (a) Radler, J.; Sackmann, E. Langmuir 1992, 8, 848. (b) Heinrich, V.; Ritchie, K.; Mohandas, N.; Evans, E. Biophys. J. 2001, 81, 1452. Note that the number of fringes increases with the size of the probe bead. However, the accuracy of the bead elevation measurements mainly comes from the fit of the first two sharp fringes. This could be easily achieved with 2 µm microbeads.

Langmuir, Vol. 18, No. 3, 2002 849 Using online video image analysis of the fringe pattern, the optical thicknesses of the intervening molecular layer(s) were determined with a resolution of ∼1 nm.4,18 Video images of each bead were averaged over 500 video frames to improve signal/ noise and thereby thickness resolution. Thicknesses were monitored at each step of the chemical procedure in order to examine the evolution of the coating structure. Over 30 to 50 beads were tested at each step in PBS. As a control for bead roughness, we measured elevations of naked beads above a clean cover glass. In the case of coated beads, the standard deviations in the height distribution were essentially equivalent to the mean roughness determined for naked beads, which indicated that the layers were quite homogeneous. (b) Measurements of Protein Surface Density by Flow Cytometry. Quantitative flow cytometry was performed to determine the surface density of immobilized proteins. The flow cytometric analysis used a Becton Dickinson FACScalibur flow cytometer equipped with an argon ion laser. The laser output is fixed at 488 nm. The CellQuest software package was used to determine the events of interest from forward and side scatter parameters. Size-gating allowed a discrimination between individual microbeads and doublets or aggregates. The mean fluorescence intensity (at 530 nm) of a single bead was obtained from the mean channel number of the fluorescence histograms of the gated population. Data were collected for 10 000 beads for each set of experiments and compensated for background fluorescence (autofluorescence of glass and nonspecific adsorption on inactivated beads) by subtracting the mean fluorescence intensity obtained for the control microspheres. The average fluorescence was then converted to the number of fluorophores after calibration. (c) Assays of Bead Functionality after Surface Modification. (i) Bead-to-Cell Sticking. The functionality of beads decorated with proteins (either streptavidin or cadherin) was qualitatively assayed by bringing such beads in contact with cells carrying surface counter-receptors (i.e. biotin or cadherin). Biotinylated red blood cells were first fixed to a streptavidinderivatized slide used as the bottom of a flow chamber. Then a large excess of streptavidin-decorated beads was incubated in the chamber filled with PBS 150 mOsm. After 30 min, the chamber was gently washed with fresh PBS in order to eliminate all sedimented beads. As a control, casein-decorated spheres were used instead of streptavidin beads. Similarly, RBL-E-cadherin cells grown on glass cover slips were incubated with glass beads coated with EC1-2 or casein for 1 h at 37 °C in DMEM. Cells were then extensively washed with PBS-Ca under a 10 mL pipet outflow and fixed with 4% paraformaldehyde in PBS-Ca. Cells were then observed and photographed, and cells with at least one bound bead attached were scored. Data were expressed as a percentage of cells with bound beads. Biotinylated red blood cells and cadherin-expressing RBL cells were imaged using an inverted microscope in bright field illumination. (ii) Bead Aggregation Assay. Bead aggregation experiments were carried out both in the presence of calcium and in the presence of EDTA (which chelates divalent cations) to test the bead binding activity through the calcium dependence of cadherins. Cadherin-derivatized beads (about 106 for each set of experiments) were thus resuspended in HMF (10 mM Hepes, magnesium-free, pH 7.4, 10 mM CaCl2) containing 0.05% casein or HCMF (10 mM Hepes, calcium magnesium-free, pH 7.4, 1 mM EDTA) + 0.05% casein in each well of a 24-well plastic plate. Similar tests were also performed on beads conjugated with casein (as an irrelevant protein). Plates were gently agitated at room temperature for 45 min. Bead suspensions were then randomly photographed under the microscope, and the number and sizes of bead aggregates were scored. The extent of bead aggregation was represented by the index NEDTA - NCa/NEDTA, where NCa is the number of bead aggregates with calcium at the end of the incubation time and NEDTA is the number of particles with EDTA. Each assay was repeated two times, and a minimum of 4500 beads were counted for each condition. (iii) Dissociation Rate Measurement. The dissociation kinetics of the streptavidin-biotin complex was monitored in an envi-

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Table 1. Thicknesses of the Successive Layers Obtained in the Preparation of Streptavidin Coated Microspheresa layer thickness ( standard deviation (nm)

treatment base-washing base-washing + MPTS aqueous silanization base-washing + MPTS aqueous silanization + Mal-PEG3400-biotin base-washing + MPTS aqueous silanization + Mal-PEG3400-biotin + 0.1 µg/mL streptavidin in PBS 290 mOsm base-washing + MPTS aqueous silanization + streptavidin-PEG3400-VS a

0.7 ( 2.6 13.6 ( 5.1 21.9 ( 5.7 26.5 ( 5.1 24.4 ( 5.3

These values are derived from the microsphere-cover glass separation using a reflection interference contrast microscope.

ronment of free biotin in excess to evaluate how durable is the linkage, in view of using these surfaces as tunable protein density templates. The experimental setup is as follows. Assembled around an epi-illuminated inverted microscope (Diavert, Leitz, Wetzlar, Germany), a laser microfluorescence was used to quantitate bead fluorescence. An area of about 10 µm2 in the central region of a microbead was excited with the 488 nm line of an argon ion laser (Innova 99 Coherent, Palo Alto, CA). Using a photomultiplier, the fluorescence intensity was collected and averaged over the size of a 2 µm bead. The reduction of fluorescence intensity over time for microbeads labeled with fluorescent biotin was used to monitor the dissociation kinetics of ligand/counter in the presence of an excess of ligand in solution. The streptavidinated beads were first bound with fluorescein-biotin at saturation and then incubated in a 2.5 mM PEG3400-biotin solution in PBS to monitor the competitive off-rate. Beads were washed three times in pure buffer before measurement of fluorescence, and at least 30 different beads were examined at each condition. Fluorescence intensities were recorded after incubation periods of a few minutes to several days. To avoid artifacts due to the possible fluctuations of the laser power over days, freshly prepared beads were examined as reference.

Results Monitoring Reaction Steps by Thickness Measurements. Functionalization of silica surfaces is expected to produce detectable layers on the glass surface. The step-by-step construction of the desired coating was monitored by measuring the apparent height of microbeads over a naked cover glass. Table 1 summarizes the bead heights ( standard deviations of the distribution for all the different treatments applied to achieve bead decoration with streptavidin. With regard to the silanization step, a thickness increase of 13 nm was found, which undoubtedly was due to multilayer coverages, since the length of the MPTS molecule in its all-trans conformation is expected to be about 1 nm. Also, the latter value was weakly affected by the immersion time. The reaction times were varied from 1 to 24 h, and all of the thicknesses fell in the range between 10 and 15 nm, indicating that the kinetics of the reaction was very fast. The grafting of Mal-PEG3400-biotin (method 1) produced an increase of the layer thickness of about 8.3 nm. This value is comparable with the layer thickness of a polymer chain in good solvent conditions in a “mushroom” regime: (xπ/2)N0.6a )5.9 nm (where N is the number of CH2CH2O units, N ) 77, and a ) 3.5 Å is the molecular size of a unit).19 In any case, it is well below the length of a fully stretched chain (27 nm). In other words, spontaneous grafting of the PEG-maleimide derivatives to the mercaptosilane layer did not lead to a dense brush but to a layer which was only hardly stretched compared to a dilute layer made of “non-interacting mushrooms”.20 After streptavidin incubation (method 1), a gain of 4.6 nm in thickness was observed, which is compatible with (19) De Gennes, P. G. Macromolecules 1980, 13, 1069.

a streptavidin monomolecular layer (4.5 nm being the “height” of a streptavidin molecule9a). In an attempt to optimize the conditions of streptavidin immobilization, we found out that the salinity of the solution had little (if any) influence on the structure of the streptavidin layer which is built-up. From PBS 290 mOsm to deionized water, the thickness of the additional layer was on the order of 4-4.6 nm. Moreover, we checked that the incubation time (5 min to 2 h range) had no significant effect on the thickness of the final structured layer. When streptavidin was preliminarily conjugated to VSPEG-NHS and covalently grafted to glass (method 2), the measured thickness was on the order of 22 nm, which is slightly less than the one obtained with the first method. This decrease could be assigned to an incomplete coverage of the surface. We, however, observed that a subsequent incubation in a solution of free streptavidin did not lead to any additional layer due to nonspecific adsorption. The surface was therefore passivated against adsorption. Determination of Protein Site Densities. (a) Calibration. Flow cytometry was used to quantify the surface density of immobilized proteins bound to fluorescent ligands. To get an absolute estimate of the protein density on the bead surface, a preliminary calibration of the fluorescence intensity per bead as a function of the number of fluorophores is required. To do so, we proceeded in two steps. First, the total binding capacity of decorated microspheres was determined by a differential fluorimetric assay: the amount of fluorescein-biotin left free in solution after incubation with a known number of streptavidinderivatized beads (the concentration of the bead suspension being determined by cytometry) was measured by spectrofluorimetry. We found that 1 mg of beads prepared using 100% Mal-PEG-biotin and coated with streptavidin could bind 0.13 nmol of fluorescein-biotin. Knowing the size (2.3 µm in diameter) of the microspheres and assuming that one immobilized streptavidin can only bind two fluorescein-biotin molecules (for steric reasons), one may derive that the surface density in streptavidin is on the order of 3.3 × 104 molecules/µm2 (or, equivalently, 1 streptavidin/30 nm2). Calibration of fluorescence intensity as a function of streptavidin surface density was then performed by incubating streptavidin beads with mixtures of biotin and fluorescein-biotin in different ratios. Representative fluorescence histograms are shown in Figure 4a for different fractions in fluorescein-biotin. As expected, the fluorescence intensity increases with the fraction of fluorescent biotin. However, as seen in Figure 4b, there is no direct proportionality between the mean fluorescence intensity and the molar percentage in fluorescein-biotin (or, equivalently, the corresponding surface density in (20) Note that the weight of a 2 µm bead in water (or the equivalent pressure for a 0.1 µm2 footprint) is at least 6 orders of magnitude weaker that the repulsive pressure between two polymer brushes (see: De Gennes, P. G. Advances in Colloid and Interface Science 1987, 27, 189). We may therefore consider that gravity does not affect thickness measurements.

Versatile Decoration of Glass Surfaces

Figure 4. (a) Representative flow cytometry histograms of typical distributions of fluorescence intensity (along x axis) measured for the populations of beads saturated with streptavidin and revealed with different mixtures of fluorescein-biotin/ native biotin. (b) Calibration curve of the mean fluorescence intensity as a function of the bulk fraction in fluorescein-biotin. The dashed line shows the deviation of this curve from linearity at concentrations higher than 3%. (c) Flow cytometric analysis binding of fluorescein-biotin to streptavidin-coated beads prepared following method 1 (PEG-biotin-streptavidin construct) and method 2 (covalent grafting of streptavidin). Data are plotted as normalized mean channel fluorescence versus bulk percentage of “active sites” (meaning biotin groups or vinylsulfone-pegylated streptavidinssee text for details). Curve a corresponds to beads prepared according to route 1. Curve b corresponds to beads prepared according to route 2. The dashed line is the calibration curve shown in part b.

streptavidin as revealed by fluorescent biotin) over the whole range of concentration. In a low concentration regime in fluorescein-biotin (