Surface Characterization of Mixed Self-Assembled Monolayers

Mar 28, 2001 - The self-assembly of streptavidin onto biotinylated alkylthiolate monolayers on gold has served as an important model system for protei...
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Langmuir 2001, 17, 2807-2816

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Surface Characterization of Mixed Self-Assembled Monolayers Designed for Streptavidin Immobilization Kjell E. Nelson,† Lara Gamble,† Linda S. Jung,‡ Maximiliane S. Boeckl,‡ Esmaeel Naeemi,§ Stephen L. Golledge,† Tomikazu Sasaki,‡ David G. Castner,*,†,§ Charles T. Campbell,*,‡ and Patrick S. Stayton*,† Department of Bioengineering, Department of Chemistry, and Department of Chemical Engineering, University of Washington, Seattle, Washington 98195 Received August 2, 2000. In Final Form: February 8, 2001 The self-assembly of streptavidin onto biotinylated alkylthiolate monolayers on gold has served as an important model system for protein immobilization at surfaces. Here, we report a detailed study of the surface composition and structure of mixed self-assembled monolayers (SAMs) containing biotinylated and diluent alkylthiolates and their use to specifically immobilize streptavidin. X-ray photoelectron spectroscopy (XPS), angle-resolved XPS (ARXPS), near-edge X-ray absorption fine structure (NEXAFS), and surface plasmon resonance (SPR) have been used to characterize the films produced on gold from a range of binary mixtures of a biotinylated alkylthiol (BAT) and either a C16 methyl-terminated thiol (mercaptohexadecane, MHD) or a C11-oligo(ethylene glycol)-terminated (OEG) thiol in ethanol. The correlation between the solution mole fraction of BAT and its surface mole fraction (χBAT,sur) indicates that it adsorbs ∼4-fold faster than OEG but slightly slower than MHD. ARXPS analysis demonstrates that the biotin terminus of the BAT is exposed at the surface of mixed monolayers with χBAT,sur < 0.5 but is randomly distributed through BAT-rich films. Thus, the OEG diluent not only adds nonfouling properties but induces an improved concentration of biotin at the surface and reduces the exposure of the methylene segments of BAT. NEXAFS characterization demonstrates that pure OEG and mixed BAT/OEG SAMs do not show significant anisotropy in C-C bond orientation, in contrast to MHD and mixed BAT/MHD SAMs, whose aliphatic segments exhibit pseudo-crystalline packing. SPR measurements of streptavidin binding to and competitive dissociation from the different mixed SAMs indicate that streptavidin binds both specifically and nonspecifically to the BAT/MHD SAMs but purely specifically to BAT/OEG SAMs with χBAT,sur < 0.5. For BAT/OEG mixtures with χBAT,sur ) 0.1-0.5, specifically bound streptavidin coverages of ∼80% of the C(2,2,2) two-dimensional streptavidin crystalline density (∼280 ng/cm2) can be reproducibly achieved. These composite results clarify the relationship between the specificity of streptavidin recognition and the surface architecture and properties of the mixed SAMs.

Introduction The immobilization of proteins with maximum retention of activity and minimized nonspecific interactions is a key goal in the development of many diagnostic, affinity separation, and biomaterial technologies. One of the most important proteins in current diagnostic and separations technologies is streptavidin, and there has been considerable use of immobilized streptavidin for both commercial and laboratory purposes. Streptavidin’s four biotin binding sites and their dyad symmetry make this protein uniquely suited as an adapter for binding a second layer of biotinylated molecules. Knoll and co-workers have pioneered a technique for streptavidin immobilization that utilizes biotinylated alkylthiol self-assembled monolayers (SAMs) on gold,1-6 which provides a high degree of control over surface physical properties. This approach lends itself directly to sensitive in situ detection of protein adsorption * Authors to whom correspondence should be addressed. Patrick S. Stayton, Department of Bioengineering, Box 352125, University of Washington, Seattle, WA 98195-2125; Phone, (206) 685-8148; Fax, (206) 685-8256. David G. Castner, Department of Chemical Engineering, Box 351750, University of Washington, Seattle, WA 98195-1750; Phone, (206) 543-8094; Fax, (206) 543-3778. Charles T. Campbell, Department of Chemistry, Box 351700, University of Washington, Seattle, WA 98195-1700; Phone, (206) 616-6085; Fax, (206) 616-6250. † Department of Bioengineering. ‡ Department of Chemistry. § Department of Chemical Engineering. (1) Haussling, L.; Ringsdorf, H.; Schmitt, F. J.; Knoll, W. Langmuir 1991, 7, 1837-1840.

using surface plasmon resonance (SPR). The two biotin binding sites on each side of this protein should enable it to attach to such surfaces via either one or two biotin links, as is supported by kinetic measurements of biotininduced dissociation.7,8 The flexibility of SAMs and the variety of modes available to streptavidin for binding to biotinylated self-assembled monolayers make this an interesting model system for examining the relationship between surface composition and architecture and protein adsorption. The potential of SAMs for achieving a high degree of molecular control over surface composition and architecture has been extensively characterized.9-16 The SAMs (2) Muller, W.; Ringsdorf, H.; Rump, E.; Wildburg, G.; Zhang, X.; Angermaier, L.; Knoll, W.; Liley, M.; Spinke, J. Science 1993, 262, 17061708. (3) Herron, J. N.; Muller, W.; Paudler, M.; Riegler, H.; Ringsdorf, H.; Suci, P. A. Langmuir 1992, 8, 1413-1416. (4) Knoll, W.; Liley, M.; Piscevic, D.; Spinke, J.; Tarlov, M. J. Adv. Biophys. 1997, 34, 231-251. (5) Spinke, J.; Liley, M.; Schmitt, F. J.; Guder, H. J.; Angermaier, L.; Knoll, W. J. Chem. Phys. 1993, 9, 7012-7019. (6) Spinke, J.; Liley, M.; Guder, H.-J.; Angermaier, L.; Knoll, W. Langmuir 1993, 9, 1821-1825. (7) Pe´rez-Luna, V. H.; O’Brien, M. J.; Opperman, K. A.; Hampton, P. D.; Stayton, P. S.; Klumb, L.; Lopez, G. P. J. Am. Chem. Soc. 1999, 121, 6469-6478. (8) Jung, L. S.; Nelson, K. E.; Campbell, C. T.; Stayton, P. S.; Yee, S. S.; Perez-Luna, V.; Lopez, G. P. Sens. Actuators, B 1999, B54, 137144. (9) 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. (10) Bain, C. D.; Evall, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7164-7165.

10.1021/la001111e CCC: $20.00 © 2001 American Chemical Society Published on Web 03/28/2001

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Figure 1. Bond-line structures of the thiols used in this study. Note that the BAT molecule is slightly longer than similar biotinylated thiols used in previously published reports (refs 4-7).

that have been studied range from highly ordered and densely packed arrays of aliphatic thiols13,14,17-19 to mixtures of thiols with different chain lengths 20-22 and/ or headgroup functionalities.16,23-26 These previous studies of mixed SAMs suggest that the composition and coverage of the film can be influenced by intermolecular interactions between the thiols, the solvent, and the surface during assembly.10,22,25-29 Moreover, departure from all-trans extended, pseudocrystalline arrays of thiols in SAMs may occur because of poor packing between thiolates within the film.30-35 As a consequence of these effects as well as differences in intrinsic adsorption rate constants for the thiols, the solution thiol ratio used during assembly may not be equivalent to the surface thiol ratio of the resulting (11) Dubois, L. H.; Nuzzo, R. G. Annu. Rev. Phys. Chem. 1992, 43, 437-463. (12) Grunze, M. Phys. Scr., T 1993, T49B, 711-717. (13) Laibinis, P. E.; Whitesides, G. M.; Allara, D. L.; Yu, T.-T.; Parikh, A. N.; Nuzzo, R. G. J. Am. Chem. Soc. 1991, 113, 7152-7167. (14) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112, 558-569. (15) Ulman, A. Chem. Rev. 1996, 96, 1533-1554. (16) Whitesides, G. M.; Laibinis, P. E. Langmuir 1990, 6, 87-96. (17) Nuzzo, R. G.; Zegarski, B. R.; Dubois, L. H. J. Am. Chem. Soc. 1987, 109, 733-740. (18) Bain, C. D.; Whitesides, G. M. Science 1988, 240, 62-63. (19) Cavalleri, O.; Hirstein, A.; Bucher, J. P.; Kern, K. Thin Solid Films 1996, 284-285, 392-395. (20) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7164-7175. (21) Fenter, P.; Eisenberger, P.; Liang, K. S. Phys. Rev. Lett. 1993, 70, 2447-2450. (22) Laibinis, P. E.; Nuzzo, R. G.; Whitesides, G. M. J. Phys. Chem. 1992, 96, 5097-5105. (23) Sigal, G. B.; Bamdad, C.; Barberis, A.; Strominger, J.; Whitesides, G. M. Anal. Chem. 1996, 68, 490-497. (24) Olbris, D. J.; Ulman, A.; Shnidman, Y. J. Chem. Phys. 1995, 102, 6865-6873. (25) Folkers, J. P.; Laibinis, P. E.; Whitesides, G. M. Langmuir 1992, 8, 1330-1341. (26) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12-20. (27) Allara, D. L. Biosens. Bioelectron. 1995, 10, 771-783. (28) DeBono, R. F.; Loucks, G. D.; Della Manna, D.; Krull, U. J. Can. J. Chem. 1996, 74, 677-688. (29) Hahner, G.; Kinzler, M.; Thummler, C.; Woll, C.; Grunze, M. J. Vac. Sci. Technol., A 1992, 10, 2758-2763. (30) Zhao, X. M.; Wilbur, J. L.; Whitesides, G. M. Langmuir 1996, 12, 3257-3264. (31) Schlenoff, J. B.; Li, M.; Ly, H. J. Am. Chem. Soc. 1995, 117, 12528-12536. (32) Delamarche, E.; Michel, B. Thin Solid Films 1996, 273, 54-60. (33) Delamarche, E.; Michel, B.; Biebuyck, H. A.; Gerber, C. Adv. Mater. 1996, 8, 719. (34) Camillone, N., III.; Chidsey, C. E. D.; Eisenberger, P.; Fenter, P.; Li, J.; Liang, K. S.; Liu, G. Y.; Scoles, G. J. Chem. Phys. 1993, 99, 744-747. (35) Camillone, N., III.; Leung, T. Y. B.; Scoles, G. Surf. Sci. 1997, 373, 333-349.

mixed SAM, and defects in surface order and thiol orientation may expose unwanted chemical groups at the surface-solution interface, resulting in undesirable or uncontrollable surface properties. Therefore, to properly interpret the protein binding characteristics of mixed SAMs, it is essential that their composition, concentration profile, and long-range lateral order be characterized. Our interest in engineering streptavidin variants that are optimally designed for binding36-38 and in immobilizing biotinylated molecules8,39,40 has led us to utilize these SAMs. The available literature suggests that a high degree of structural complexity exists in similar mixed biotinylated SAMs,5-7 but to our knowledge a detailed structural characterization of these SAMs is still not available. To fill this gap and to develop a fuller understanding of the molecular details of streptavidin adsorption to SAM surfaces, we have performed a series of detailed surface characterizations of two mixed SAMs that contain a biotinylated alkylthiolate (BAT) component and either a oligo(ethylene oxide)- or methyl-terminated alkylthiolate diluent (OEG or MHD, respectively) (Figure 1) that are similar to those used in the original reports.1,5-7 Here, we address how these different diluent alkylthiolates affect the structure of mixed biotinylated SAMs and their binding of streptavidin. Using X-ray photoelectron spectroscopy (XPS), angle-resolved XPS (ARXPS), and near-edge X-ray absorption fine structure (NEXAFS), we have determined the composition, thickness, composition depth profile, and lateral order of these binary SAMs. Using these data, we can directly correlate the interfacial properties of these mixed SAMs with their streptavidin binding dynamics and specificity as determined by SPR. Materials and Methods Materials. MHD was purchased from Pierce and was used as received. Synthesis details for BAT and OEG can be found in the Supporting Information to this text. Preparation of Mixed Self-Assembled Monolayers. Glass slides or silicon wafers were used as substrates and coated with (36) Chilkoti, A.; Boland, T.; Ratner, B. D.; Stayton, P. S. Biophys. J. 1995, 69, 2125-2130. (37) Chilkoti, A.; Tan, P. H.; Stayton, P. S. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 1754-1758. (38) Klumb, L. A.; Chu, V.; Stayton, P. S. Biochemistry 1998, 37, 7657-7663. (39) Jung, L. S.; Shumaker-Parry, J. S.; Campbell, C. T.; Yee, S. S.; Gelb, M. H. J. Am. Chem. Soc. 2000, 122, 4177-4184. (40) Shumaker-Parry, J. S.; Campbell, C. T.; Stormo, G. D.; Silbaq, F. S.; Aebersold, R. H. Probing Protein:DNA Interactions Using a Uniform Monolayer of DNA and Surface Plasmon Resonance. In SPIE Photonics West Conference, International Biomedical Optics Symposium, San Jose, CA, 2000.

Surface Optimization for Immobilizing Streptavidin ∼2 nm Cr and from ∼50 to ∼100 nm Au (Alfa Aesar, 99.9999% purity) by electron beam evaporation. SPR studies were conducted on glass microscope slides (Corning) coated with ∼2 nm Cr and ∼50 nm Au. Thiol mixtures of BAT and either MHD or OEG were made in reagent grade, dried, deoxygenated ethanol (McCormick) at the indicated ratios keeping the total sulfhydryl concentration constant at 10-4 M. The gold substrates were immersed in the thiol solution for 2-4 days at room temperature and then rinsed with ethanol or, for hydrophobic surfaces, first rinsed in hexanes and then ethanol and dried under Ar or N2. The SAMs were either used immediately or stored under N2. XPS Measurements. XPS measurements were made at the University of Washington NESAC/BIO surface analysis facility using a monochromatic Al KR X-ray source as described previously.41 Briefly, initial survey scans (0-1000 eV binding energy) were followed by detailed scans for carbon, gold, nitrogen, oxygen, and sulfur (150 eV detector pass energy, 20 eV windows, centered at 285, 84, 400, 532, and 161 eV binding energy, respectively). High-resolution sulfur spectra were collected essentially as described above except that the detector pass energy used was 50 eV, and the high-resolution carbon spectra were collected with a detector pass energy of either 50 or 25 eV. Angle-resolved XPS spectra were collected using the instrument parameters described above at takeoff angles θ of 0, 39, 55, 68, and 80° between the detector lens axis and the sample surface normal, with a 12° aperture placed over the analyzer lens. The number of scans taken at different angles was adjusted to optimize the signalto-noise ratio while minimizing X-ray induced sample damage to the OEG thiolates, which were found to be susceptible to beam damage as indicated by a decrease in oxygen percentage in survey scans taken before and after composition analysis (data not shown). To provide a clean Au reference signal for XPS thickness measurements, a freshly prepared gold surface was sputtered in the XPS analytical chamber for 5 min under Xe (3 × 10-7 Torr) with an accelerating voltage of 1 keV. The C1s signal was measured after the first 3 min of sputtering and then every minute thereafter until the carbon signal no longer decreased upon further etching. Unconstrained least-squares fitting of the XPS thickness data to the equation derived in the text (eq 6, below) using a χ2 minimization algorithm with NtotD, NtotB, VD, and VB as fitting parameters and the surface mole fraction of BAT (χB,sur) as the dependent variable is shown and described in detail in the text. Near-Edge X-ray Absorption Fine Structure. The nearedge X-ray absorption fine structure experiments were done at the National Synchrotron Light Source U7A beamline located at Brookhaven National Laboratory. This beamline uses a monochromator and 30/30 slits which gave a full-width at halfmaximum resolution of ∼0.15 eV at the carbon K-edge (∼285 eV). The monochromator energy scale was calibrated by setting the peak for the C1s f σ* transition in the graphite carbon K-edge NEXAFS spectrum to 285.35 eV.42 All NEXAFS spectra were normalized by the photocurrent from a gold-coated, 90% transmission grid placed in the incident X-ray beam. Partial electron yield (PEY) was measured by a channeltron with the cone negatively biased (-100 to -150 eV). Rotation of the sample changed the angle (φ) between the X-ray beam and the sample surface. Normal incidence of the X-ray beam on the surface corresponds to a polar angle of 90°, and glancing incidence of the X-ray beam corresponds to a polar angle of 20°. This definition of the polar angle for NEXAFS should not be confused with the definition of the XPS photoelectron takeoff angle. The electric field vector (E) is perpendicular to the X-ray beam, such that for normal incidence the E vector of the X-ray beam lies parallel to the surface. SPR Measurements. The SPR system used here has been described and characterized previously.43 Briefly, the SPR measurements of protein adsorption were performed by indexmatching the SAM functionalized sample slide to the coupling prism under a Plexiglas flow cell essentially consisting of a silicone (41) Castner, D. G.; Hinds, K.; Grainger, D. W. Langmuir 1996, 12, 5083-5086. (42) Morar, J. F.; Himpsel, F. J.; Hollinger, G.; Jordon, J. L.; Hughes, G.; McFeely, F. R. Phys. Rev. Lett. B 1986, 33, 1346-1349. (43) Jung, L. S.; Campbell, C. T.; Chinowsky, T. M.; Mar, M. N.; Yee, S. S. Langmuir 1998, 14, 5636-5648.

Langmuir, Vol. 17, No. 9, 2001 2809 Table 1. XPS Data at θ ) 55° of Surface Elemental Percentages (Not Including Au) of Mixed BAT/MHD SAMs as a Function of Solution Mole Fraction of BATa χBAT,sol

%C

%N

%O

%S

bulk MHDb 0 0.2 0.4 0.6 0.8 1 bulk BATb

94.12 95.9 ( 1.0 92.8 ( 2.3 88.3 ( 0.7 80.8 ( 1.1 74.2 ( 0.6 72.4 ( 1.4 74.47

0.0 0.0 ( 0.0 0.4 ( 0.5 2.9 ( 0.6 5.4 ( 0.6 7.9 ( 0.3 9.3 ( 0.6 8.51

0.0 1.0 ( 0.7 4.2 ( 1.9 6.3 ( 0.6 11.2 ( 0.8 15.0 ( 0.3 15.4 ( 1.5 12.77

5.88 2.9 ( 0.2 2.6 ( 0.3 2.5 ( 0.3 2.7 ( 0.3 2.9 ( 0.3 2.8 ( 0.5 4.26

a Errors noted are (1 standard deviation (n g 3). b Based on pure, bulk compound.

Table 2. XPS Data at θ ) 55° of Surface Elemental Percentages (Not Including Au) of a Representative Series of Mixed BAT/OEG SAMs as a Function of Solution Mole Fraction of BATa χBAT,sol OEGb

bulk 0 0.05 0.10 0.20 0.40 0.50 1.00 bulk BATb

%C

%N

%O

%S

76.0 75.9 78.7 76.1 76.4 76.9 73.7 74.5 74.47

0 0 1.6 2.4 5.0 5.1 8.8 9.6 8.51

20.0 21.3 17.4 18.4 15.7 14.8 13.6 12.9 12.77

4.0 2.9 2.4 3.1 2.8 3.3 4.0 2.9 4.26

a Values are (0.5 atom % (determined by the stability and reproducibility of the instrument). b Based on pure, bulk compound.

gasket that creates an 80 µL channel. One milliliter samples of degassed buffer solution (0.15 M phosphate-buffered saline (PBS) at pH 7.4) were delivered to the sensor surface at a rate of 10 mL/min. Once the baseline resonance wavelength stabilized, the buffer above the sample was rapidly exchanged with PBS (1.25 mL at 10.0 mL/min) containing 0.1 mg/mL recombinant core streptavidin. After injection, the flow was stopped and the protein sample was allowed to remain in contact with the surface. A shift in the coupling wavelength was indicative of protein binding to the surface as discussed in ref 43. The absolute wavelength shift of the SPR minimum was converted into protein coverage in ng/cm2 according to the formalism presented in ref 8. The wild-type streptavidin/biotin off-rate is too slow for competitive dissociation to be measured over the time scale of SPR, so the desorption kinetics of engineered streptavidin binding mutants with decreased affinity for biotin were studied instead. To initiate desorption (competitive dissociation) of these mutants after their adsorption on a SAM, a PBS solution containing an excess of biotin (1.0 mM) was injected into the flow cell, while monitoring the resulting decrease in SPR wavelength (protein coverage). Expression of Recombinant Streptavidin. Recombinant wild-type core streptavidin and the W120A and Y43A mutants were expressed and purified as previously described.37

Results XPS Analysis of Mixed SAMs. Film Composition. The solution mole fraction of BAT, χBAT,sol, is defined for the mixture of thiols used for SAM assembly, and similarly χBAT,sur is defined as the surface mole fraction of BAT in a mixed SAM. The surface elemental compositions of pure and mixed BAT/MHD and BAT/OEG SAMs were measured by XPS at θ ) 55° as a function of χBAT,sol (Tables 1 and 2). XPS survey scans detected only gold, carbon, nitrogen, oxygen, and sulfur in all films analyzed. The elemental composition of the pure SAMs agrees very well with that of the bulk thiols, except that the S composition is lower in all cases. This is due to the attenuation of the S photoelectrons emitted from the S-Au interface which must escape through the SAM before reaching the detector.

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Figure 2. Surface nitrogen percentage (right axis) of BAT/ MHD (O) and BAT/OEG (+) SAMs as a function of the solution mole fraction of BAT in the thiol mixture used for assembly (not counting the solvent). The nitrogen percentage in the SAMs was used to estimate the surface mole fraction of BAT (left axis) by normalizing the nitrogen percentage in mixed SAMs to the nitrogen signal in pure BAT SAMs (see text). The BAT/ OEG data were fit to a derived function based on assumptions of first-order Langmuir adsorption (dotted curve) (R ) 4.37, χ2) 0.026, see text for derivation). The BAT/MHD data were also fit to this function (dashed curve) (R ) 0.79, χ2 ) 0.024).

Because the addition of BAT was necessary to produce a detectable nitrogen signal in the mixed SAMs and because the percentage of nitrogen in the pure BAT SAMs was close to the expected value, the percentage of nitrogen in the SAM can be used to estimate χBAT,sur in the mixed films. This gives values for χBAT,sur plotted versus χBAT,sol in Figure 2. Assuming that the adsorption of the two thiols, BAT and a diluent thiol, are both irreversible and follow first-order Langmuir kinetics, then at saturation the ratio of their surface coverages should be proportional to the ratios of their adsorption rate constants, R ) kBAT/kdiluent, times the ratio of their concentrations in the bulk solution:

χBAT,sur χdiluent,sur

)R

(

χBAT,sol

)

χdiluent,sol

(1)

Because χBAT + χdiluent ) 1 in both phases, this simplifies to

χBAT,sur )

RχBAT,sol 1 + (R - 1)χBAT,sol

(2)

The surface compositions estimated from the XPS nitrogen data collected from the BAT/OEG SAMs were well fit to this function (Figure 2, dotted curve). This fit yields R ) 4.22. The fit to the nitrogen data for mixed BAT/MHD SAMs is also shown in Figure 2 (dashed curve), yielding R ) 0.80. Film Thickness. The thickness (d) of BAT/OEG films was determined by relating the Au4f intensity measured while the film is present (I) to the Au4f intensity after sputter cleaning the surface (I0).44

I ) I0e-d/λcosθ

(3)

where λ is the mean free path of Au photoelectrons (42.5 Å with 1486.6 eV photons).45 The thicknesses are shown versus χBAT,sur for the BAT/OEG films in Figure 3. To fit (44) Ertl, G.; Kuppers, J. Low Energy Electrons And Surface Chemistry; VCH: Weinheim, Germany, 1985. (45) Tanuma, S.; Powell, C. J.; Penn, D. R. Surf. Interface Anal. 1993, 21, 165-176.

Figure 3. Mixed BAT/OEG SAM film thicknesses. XPS sputtering data are indicated by (]), and SPR data are indicated by ([). The XPS data were fit to eq 6 as derived in the text (solid line).

these data, we derive here a relationship between the composition of a mixed SAM composed of thiols with different lengths and its average thickness, d, which is

d ) fBtB + fDtD

(4)

where fi is the fraction of the surface area occupied by thiolate i, ti is the thickness of a pure SAM composed of thiolate i, B is BAT, and D is the diluent thiolate (OEG in this case). Note that fi ) AiNi where Ni is the total number of thiolates i per unit area and Ai is the area occupied per thiolate i, which we will assume here to be equal to Ai in a pure monolayer of I (i.e., we assume that each type of molecule on the surface occupies the same area, independent of its neighbors). If Ni is expressed as χi,surNtot, where Ntot is the total number of thiolates (B + D) per unit area, then

d ) (ABtBχB,sur + ADtDχD,sur)Ntot

(5)

Aiti is the molecular volume, Vi, and Ntot is just NtotBχB + NtotDχD, where Ntoti is the packing density of a pure i SAM, so

d ) (VBχB,sur + VDχD,sur)(NtotBχB,sur + NtotDχD,sur) (6) Equation 6 was used to fit the thickness data from the BAT/OEG SAMs versus surface percentage of BAT as shown by the curve in Figure 3. The fit results in values of 0.0238 molecules/Å2, 0.0423 molecules/Å2, 557 Å3/ molecule, and 985 Å3/molecule for the parameters NtotD, NtotB, VD, and VB, respectively. These values of VD and VB correspond to bulk densities of 1.12 and 1.21 gm/cm3, respectively. This value for OEG compares well to the measured bulk density of 1.13 gm/cm3. Note that the packing densities estimated from the XPS thickness of the pure OEG and pure BAT SAMs (using these bulk densities) are 2.7 × 1014 and 4.0 × 1014 molecules/cm2, respectively. The inverses of the parameters NtotD and NtotB give the mean molecular areas (Ai) for OEG and BAT, ∼42 and ∼24 Å2/molecule, respectively. Also shown in Figure 3 are thicknesses of pure and mixed SAMs, measured in situ by following the shift in the SPR wavelength upon adsorption from ethanol solution. (These shifts were converted to absolute thicknesses following ref 43, using a refractive index for OEG of 1.48

Surface Optimization for Immobilizing Streptavidin

Figure 4. Angle-resolved XPS measurements of the N1s/C1s integrated intensity ratio for four different surface mole fractions of BAT in mixed BAT/OEG SAMs. Data from each series of angles are normalized to ratios from θ ) 0° so that different compositions can be compared on the same graph, because nitrogen percentage changes as a function of composition. Data shown are χBAT,sur ) 0.14 (4), χBAT,sur ) 0.28 (0), χBAT,sur ) 0.95 (O), and χBAT,sur ) 1.0 (3). Lines shown are included as guides for the eye only. The dotted line is for χBAT,sur ) 1.0. (Inset) High-resolution XPS spectra taken from 100% BAT SAM at θ ) 0° and θ ) 80° as indicated. Note the distinctive peak in the θ ) 0° spectrum centered around 162.5 eV corresponding to chemisorbed S. Its absence in the θ ) 80° spectrum indicates that unbound sulfur occurs mainly near the SAM surface, and the bound sulfur is present only at the Au/thiolate interface.

measured in refs 46 and 47. We assumed here a refractive index for the BAT of 1.49 to give the same thickness for the pure BAT film as measured by XPS. We also assumed that the BAT and OEG had the same densities (1.1346,47). These SPR thicknesses are similar to those from XPS. For comparison, a thickness of 2.5 nm was reported based on XPS for a thiol identical to this OEG but with two more -OCH2CH2- units,48 and an ellipsometric thickness of 3.5 nm was reported for a pure monolayer of a molecule identical to this BAT but shorter by one -CH2CH2Ounit.7 Composition Depth Profiling. The composition depth profile of the pure and mixed SAMs was examined using angle-resolved XPS. Carbon, nitrogen, oxygen, sulfur, and gold photoelectron intensities were measured at five different takeoff angles (0, 39, 55, 68, and 80°). The N1s/ C1s ratios measured at each angle for a series of BAT/ OEG SAMs are plotted in Figure 4. The N/C ratio was chosen as an indicator of the position of the biotin moiety of the BAT molecule relative to the gold surface of the film because of the asymmetric distribution of nitrogen and carbon in the biotinylated thiol (Figure 1). For instance, if the biotin headgroups of the BAT thiols in the SAMs are located near the surface, a high N/C ratio would be expected when measuring at shallow takeoff angles (θ ) 80°). As shown in Figure 4, the N/C ratio of the 100% BAT films does not vary with takeoff angle. This suggests that in these films the thiolate chains do not adopt a preferential orientation relative to the surface normal. However, as increasing amounts of the OEG diluent are mixed into the SAM, the angular dependence increases (Figure 4). The same result is obtained with BAT/MHD mixtures (46) Jung, L. S.; Campbell, C. T. Phys. Rev. Lett. 2000, 84, 51645167. (47) Jung, L. S.; Campbell, C. T. J. Phys. Chem. B 2000, 104, 1116811178. (48) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426-436.

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(data not shown). This suggests that (1) in mixed BAT/ OEG SAMs, particularly those with low χBAT,sur, the majority of the biotin headgroups can be found near the SAM surface and (2) in pure BAT SAMs, the biotin headgroups are uniformly distributed throughout the depth of the film. High-resolution C1s spectra (not shown) of 100% BAT films taken at normal (0°) and shallow (θ ) 80°) takeoff angles were essentially indistinguishable in the intensity of the peak(s) between ∼288 and 288.5 eV (caused by the carbonyl- and ureido-type C atoms, respectively49) relative to the major unresolved doublet at 285-286.5 eV (resulting from alkyl- and ether-type C atoms, respectively49). Consistent with the data from elemental ratios, this lack of angular dependence in the high-resolution C1s spectra shows that the biotin is uniformly spread through the film depth. To determine if this uniformity is caused either by “hairpinning” of the BAT by coordination of both the sulfhydryl and the biotin thioether to the surface or by nonspecific physisorption of BAT, high-resolution sulfur spectra were taken at both normal and glancing incidence from 100% BAT films. Whereas the spectrum taken at a takeoff angle of 80° showed only one peak (actually a doublet) centered around ∼164 eV, the spectrum taken at a takeoff angle of 0° had distinct peaks at 164 and 161.8 eV (see inset, Figure 4). Previous studies have shown that XPS sulfur peaks centered around ∼164 eV correspond to unbound thiol species, and peaks centered around ∼162 eV are indicative of bound thiolate species bound directly to the Au.41 The presence of the bound sulfur species that is detectable only at normal incidence indicates that the sulfur species near the gold/thiolate interface is indeed chemisorbed. The other S species that is not bound to gold (∼164 eV) is clearly near the surface of the SAM because its signal is strongest at 80°. This demonstrates that the BAT thiol is chemisorbed at the gold/SAM interface and that only one end of the molecule is bound to the interface and not hairpinned. This result does not rule out the binding of the biotin thioether to the gold surface, but in our hands the thioether group in biotin binds to gold weakly, if at all, and it is readily displaced by sulfhydrylbased thiolates (unpublished XPS data). NEXAFS Determination of Order within Alkylthiolate Monolayer. The long-range chain alignment of the mixed SAMs was probed using NEXAFS spectroscopy. For these experiments, polarized X-rays illuminated the samples at different angles of incidence and the absorption at different X-ray energies was measured using partial electron yield. Because X-ray absorption by molecular orbitals is strongly dependent on the favorable overlap of bond orbitals with the electric field vector of the incident X-rays, if the thiol chains are well aligned with each other the NEXAFS adsorption spectra will show a strong angular dependence. If there is very little interchain alignment or long-range order in the SAM, the X-ray absorption spectra will not vary with angle of incidence. The observation of polarization dependence in the X-ray spectra is thus indicative of molecular orientation and order in the SAM. NEXAFS spectra for a pure MHD SAM are shown in Figure 5a. There is a strong polarization dependence similar to spectra previously reported for hydrocarbon SAMs 29 and Langmuir-Blodgett films.50 The peak at 287.9 eV has been assigned to the transition from the C1s (49) Beamson, G.; Briggs, D. High-Resolution XPS of Organic Polymers; John Wiley and Sons: New York, 1992. (50) Outka, D. A.; Stohr, J.; Rabe, J. P.; Swalen, J. D. J. Chem. Phys. 1988, 88, 4076-4087.

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Figure 5. Angle-resolved NEXAFS spectra collected from different BAT/MHD and BAT/OEG mixtures: (a) pure MHD SAM, (b) mixed BAT/MHD SAM, (c) pure BAT SAM, (d) mixed BAT/OEG SAM, and (e) pure OEG SAM. Angles of X-ray incidence with respect to the surface are shown in the figure.

to the C-H* orbital.29,50 This peak is enhanced when the X-ray beam is at normal incidence to the sample surface (φ ) 90°). At normal incidence, the electric field vector (E) of the polarized X-ray source is parallel to the surface. The overlap of this E vector with the C-H bond orbital causes the transition to the C-H σ* orbital and indicates that the C-H σ* bond is nearly parallel to the surface. The peak at 293 eV, which has been assigned to the transition to the C-C σ* orbital,29,50 is enhanced when the X-rays are at glancing incidence. This indicates that the C-C σ* orbital has more of a perpendicular orientation. From the polarization dependence of the MHD NEXAFS spectra, it was calculated that the alkyl chain of MHD was tilted 35° ( 5° from the surface normal. The C K-edge spectra of a mixed BAT/MHD surface show a similar polarization dependence to the 100% MHD surface (Figure 5b). These results indicate that the alkyl chains of this mixed monolayer, like the 100% MHD monolayer, are fairly well-ordered and tilted ∼35° from

the surface normal. The new peak at 289.5 eV is assigned to a Rydberg type transition from the CH2 species between the ether linkages in the BAT molecule.51 The minimal polarization dependence of this peak indicates that very little ordering may exist in the upper oxygen- and nitrogencontaining regions of this film. The C K-edge spectra of the 100% BAT surface (Figure 5c) show relatively little polarization dependence, although enough at 293 eV to indicate some order in the alkyl chains. The peak at 289.5 eV is more pronounced in the 100% BAT spectra than in the mixed monolayer because of the higher concentration of ether linkages in the pure BAT SAM. NEXAFS results showed that as the solution percentage of BAT in the BAT/ MHD mixture increased from 40% to 60% to 80% the 289.5 eV peak became more prominent and the amount of polarization dependence or orientation decreased (data (51) Urquhart, S. G.; Hitchcock, A. P.; Priester, R. D.; Rightor, E. G. J. Polym. Sci. 1995, 33, 1603-1620.

Surface Optimization for Immobilizing Streptavidin

Langmuir, Vol. 17, No. 9, 2001 2813 Table 3. Dissociation T1/2 and Quantities of Specifically and Nonspecifically Adsorbed Protein for Mutants Bound to BAT/MHD SAMs of Various Surface Compositions at 23 °Ca

Figure 6. Pseudo-saturation wild-type streptavidin coverage (in buffer containing 0.05 mg/mL of SA) as a function of the surface percentage of BAT for BAT/MHD (0) and BAT/OEG (1) measured by SPR. The SPR wavelength shift has been converted to absolute coverage (in ng/cm2) according to the formalism presented in ref 43. Also included is this plot is the N/C ratio measured at 80° takeoff angle at the indicated surface composition as a measure of the relative concentration of surface biotins (+).

not shown). This is consistent with the compositional data indicating an increased amount of BAT in these SAMs and suggests that the regions of these films that contain ether linkages are not orientationally ordered. Monolayers made with the OEG thiol, either alone or mixed with BAT, showed very little polarization dependence regardless of the mixture (Figure 5d,e, data taken at 20-100% BAT but not shown). The 100% OEG monolayer showed a strong peak at 289.5 eV resulting from the relatively large percentage of the C being in ether linkages (Figure 5e). This peak is broader than expected, likely because of an unresolved contribution from a small 287.9 eV peak caused by the alkyl chains. The low intensity at 287.9 eV at φ ) 90° suggests that even the alkyl chain parts of the pure OEG film are not orientationally ordered. SPR Measurement of Protein Adsorption to Pure and Mixed SAMs. The saturation amount of wild-type recombinant streptavidin (WTSA) adsorption to both pure and mixed SAMs was measured in situ using SPR. Here, we define “saturation” as the coverage reached after 10 min of adsorption, at which point the rate of adsorption had decreased by several orders of magnitude below its initial value. As shown in Figure 6, striking differences were observed in maximal protein coverage when different monolayer compositions were used for binding. For BAT/ OEG SAMs, the lowest χBAT,sur studied (0.1% solution ) 0.34% surface BAT) resulted in a relatively low streptavidin (SA) coverage. Intermediate BAT coverages (10% solution ) ∼32% surface) bound almost 5-fold more protein. When no BAT was incorporated in the film (100% OEG), very little SA was adsorbed (∼2 ng/cm2). In contrast, very large amounts of SA adsorbed to BAT/MHD SAMs at the lowest BAT concentrations, even when no BAT was present (100% MHD). This is due to the well-known nonspecific adsorption of proteins to alkyl-terminated surfaces (for example, see ref 52). Included in Figure 6 is a plot of the Nls/Cls XPS intensity ratio measured for the BAT/OEG films at θ ) 80°, which is the most surface sensitive angle. This ratio is a measure of the concentration of biotin headgroups nearest to the surface. One can see that the saturation SA coverage on the film correlates well qualitatively with this value. This again verifies the specific nature of the bonding of the SA (52) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1166.

χBAT,sur (BAT/MHD)

T1/2 (s)

specifically bound (ng/cm2)

nonspecifically bound (ng/cm2)

0.03 (Y43A) 0.05 (W120A) 0.55 (W120A) 1.00 (W120A) 0.55 (WTSA)

1.1 × 103 3.7 18.3 40 1.8 × 105

39 111 195 61 229

251 153 24 10 3

a T 1/2 was estimated from single-exponential fits of the SPR response following biotin-buffer-induced protein dissociation. The amount of specifically bound protein was determined by subtracting the amount of protein dissociated following biotin injection from the amount bound just prior to biotin injection. The remainder is the amount that adsorbed nonspecifically.

to these films. The reason for the decrease in SA coverage with χBAT,sur at high BAT concentrations is simply that the concentration of available biotin (i.e., biotin headgroups protruding from the topmost parts of the film) decreases with BAT concentration in this region. This is due to the loss of upright orientation of the BAT in BATrich films and the inaccessibility of biotin headgroups at the film surface (bound biotin is inserted ∼1.4 nm into the SA binding pocket).53,54 The trend in Figure 6 for the amount of adsorbed streptavidin on these BAT/OEG films agrees with that reported for binary SAMs containing a similar biotinylated alkylthiol diluted with 11-mercapto undecanol by both Spinke et al.5 and Pe´rez-Luna et al.7 The competitive desorption kinetics of the surface-bound SA was also measured with SPR to clarify the relative extent of specific binding to surface biotins versus nonspecific binding to other functionalities in the thiolate films. WTSA and several different mutants were selected for this study, because they have dramatically different biotin-SA dissociation rates in homogeneous solution.36,37 Thus, their relative desorption rates should indicate whether their binding to the surface is specific. As with measurements in homogeneous solution, the desorption was induced by exposure to a high concentration of biotin (1 mM), which competes with surface biotin for the SA binding sites and thus induces their dissociation from the surface. Desorption experiments with the BAT/MHD films are summarized in Table 3. As can be seen, at low χBAT,sur (∼0.05) the majority of the protein binds nonspecifically, whereas specific binding dominates at high χBAT,sur (g0.55). For the small fraction of specifically adsorbed W120A, the half-lives for χBAT,sur ) 0.05 vary with mutation in a way quite similar to their half-lives in homogeneous solution (WT ) 1.7 × 105 s, Y43A ) 1.1 × 103 s, and W120A < 60 s at 25 °C38) and also similar to the results at very low χBAT,sur for BAT/OEG films (WT ) 5 × 104 s, W120A ) 3 s 8). Also, as with the BAT/OEG films, the t1/2 values for W120A at χBAT,sur g 0.55 are only slightly longer than at χBAT,sur ) 0.05.8 No experiments were done using BAT/ MHD films with compositions in the range 0.05 e χBAT,sur e 0.55 (except at 1.00). A thorough study of the BAT/OEG films at all compositions from 0.034 e χBAT,sur e 1.00 with WTSA and the mutants W120A, N23E, and S27A is reported elsewhere.8 It proved that the attachment of SA to these BAT/OEG surfaces is dominated by specific binding at all these compositions, and almost all the adsorbed SA could be (53) Weber, P. C.; Ohlendorf, D. H.; Wendoloski, J. J.; Salemme, F. R. Science 1989, 243, 85-88. (54) Hendirckson, W. A.; Pahler, A.; Smith, J. L.; Satow, Y.; Merritt, E. A.; Phizakerley, R. P. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 21902914.

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Figure 7. Schematic illustrating key configurational features of alkylthiol components in pure and mixed biotin-containing SAMs. When mixed with BAT, MHD promotes ordering of the aliphatic region of the mixed BAT/MHD SAM, whereas the upper oxyether region of the SAM is relatively less ordered as evidenced by NEXAFS (Figure 5b). Pure BAT SAMs are poorly ordered and do not show appreciable orientation of biotin headgroups as indicated in NEXAFS and ARXPS results. Similarly, mixed BAT/OEG and pure OEG SAMs are highly disordered and show decreasing surface density relative to pure BAT SAMs as the surface mole fraction of OEG increases. Gray ovals ) carbonyl groups, light gray circles ) oxygen, dark gray circles ) nitrogen, and circles at Au interface ) sulfur. The ureido oxygen, nitrogens, and sulfur atom present in the biotin headgroup are not shown for clarity (cf. Figure 1).

induced to desorb upon exposure to excess biotin, at least for the weakly binding mutants. (This could not be proven for WTSA because of its extremely tight binding and long half-life.) The BAT/OEG films showed a very interesting result in this coverage range: t1/2 increased by 1-3 orders of magnitude relative to the values for χBAT,sur e 0.05 and χBAT,sur g 0.55, and the relative off-rates between the mutants were amplified (approximately squared). This is strong evidence for binding of SA to the surfaces with two biotin linkages at ∼30% BAT, whereas it binds mainly with only one biotin linkage for χBAT,sur e 0.05 and χBAT,sur g 0.55.8 The similarity in half-lives seen here for BAT/ MHD films at χBAT,sur ) 0.05, 0.55, and 1.00 to those seen at these same χBAT,sur values with BAT/OEG films strongly suggests that the specifically bound component of the adsorbed SA is also bound via a single biotin to these BAT/MHD films. Discussion An important result obtained from XPS measurements is the correlation between the solution and surface composition of both mixed BAT/MHD and BAT/OEG SAMs. This has allowed us to (1) compare the architecture of the two different binary composition SAMs at similar BAT percentages, (2) study how the different diluents affect the structure of BAT in the SAM, and (3) relate the protein binding behavior of the different binary composition mixed SAMs to the actual surface ligand concentration and to the choice of diluent thiol at the same surface ligand concentration. We observed strikingly different solution/ surface concentration correlations for the two different mixtures, and both of these correlations are different than the correlation reported by Pe´rez-Luna et al. who used a somewhat shorter biotin thiol and a different diluent thiol (11-mercapto undecanol).7 This is most likely a result of the different relative solubilities of the diluent thiols, with ethanol being a more favorable solvent for OEG than for MHD in this case. The composition data fit fairly well to a model based on simple first-order Langmuir adsorption kinetics with relative adsorption rate constants increasing as OEG < MHD < BAT. This ranking for OEG and MHD agrees with direct kinetic measurements55 and is exactly as would be expected if their relative solubilities in ethanol solution determined their adsorption rates, with the higher (55) Jung, L. S.; Nelson, K. E.; Stayton, P. S.; Campbell, C. T. Langmuir 2000, 16, 9421-9432.

energy (less soluble) thiol adsorbing faster. This suggests that kinetics are largely responsible for determining the composition of the SAM. Indeed, kinetics must control the composition, because the thiolates are strongly chemisorbed. The thickness of SAMs composed of BAT and OEG could be fit to a model which assumes that the packing density (or area per molecule) of the different thiol species remains constant regardless of the composition of neighboring molecules. Parameters for SAM packing density and molecular volume of the different thiolates could be extracted from this model and in general agreed well with measurable macroscopic quantities. The model parameters for the OEG SAMs indicated an unexpectedly low packing density (large excluded area per molecule) for these thiolates. Taken together with the lack of angular dependence in the NEXAFS data (discussed below), this suggests that OEG SAMs are highly disordered. Previous studies of EG6-OH SAMs on Au based on XPS and IR by Harder et al.48 have reported a helical structure in the oxyether region of these SAMs but note considerable structural variation ranging from crystalline to amorphous to mixtures of the two. Our data suggest that the shorter EG4-OH thiols yield films that are more characteristic of the amorphous, disordered states. In contrast to the OEG SAMs, BAT thiolates packed at a higher density but still showed very little angular dependence in their NEXAFS spectra, although the alkyl chains of pure BAT SAMs did show more orientational ordering than the alkyl chains of pure OEG SAMs. We note that pure BAT SAMs also showed a significant amount of nonspecific protein adsorption (Figure 6 and ref 8, discussed below.) Further insight into the order of the three monolayer systems was provided by NEXAFS characterization. As expected from previously published reports (see refs 11, 15, and 16 and references therein), pure MHD SAMs showed a high degree of angular dependence in the NEXAFS spectra indicative of significant lateral order. Similar results in the aliphatic regions of mixed BAT/ MHD SAMs were also observed, albeit lower in the magnitude of angular dependence. In contrast, neither pure OEG, mixed BAT/OEG, nor pure BAT SAMs showed any significant orientational ordering in either the aliphatic or the oxyether/biotin regions of the films. Thus, it appears that the MHD diluent was responsible for inducing order in the aliphatic region of mixed BAT/MHD

Surface Optimization for Immobilizing Streptavidin

SAMs, whereas the OEG diluent, itself disordered in pure OEG SAMs, was not able to induce measurable order in mixed BAT/OEG SAMs (see Figure 7). This disorder within the OEG SAMs may be connected to previously suggested mechanisms by which OEG may inhibit nonspecific protein adsorption, either by allowing access for water molecules to hydrogen-bond with the EG ether oxygens, thereby creating a layer of tightly bound water which must be displaced for protein adsorption to occur,48,56,57 or through the energetic penalty associated with loss of OEG configurational entropy upon protein adsorption (see, for example, ref 58). In contrast, the mixed BAT/MHD SAMs present a more hydrophobic interface than do mixed BAT/OEG SAMs, and they are also more structurally ordered as evidenced by the NEXAFS results (Figure 5b,d). It is thus reasonable to expect a lower entropic penalty upon nonspecific protein adsorption to mixed BAT/MHD SAMs. Taken together, the more hydrophobic interface and preordered structure of BAT/ MHD SAMs could underlie their propensity to promote nonspecific protein adsorption (see Figure 6). Furthermore, as noted earlier, pure BAT SAMs also showed a significant amount of nonspecific protein adsorption despite the usage of an EG3 linker between the methylene chain and the biotin headgroup. In this case, the stabilization of nonspecific protein binding could be a result of both their higher surface density and the increase in exposure of functional groups other than biotin at the interface that arises from poor packing. This is evidenced by the angleresolved XPS measurements which revealed an absence of angular dependence of the N/C atomic ratio at very high χBAT,sur (0.95-1.00) in BAT/OEG SAMs but a strong dependence at low χBAT,sur (Figure 4). This finding implies that a lower percentage of the biotin headgroups and a higher percentage of the methylene and amide groups are exposed to solution in pure BAT SAMs relative to the mixed SAMs with low χBAT,sur. These amide and methylene groups may be responsible for the observed increase in nonspecific interactions between streptavidin and the nearly pure BAT SAM.8 Increasing the amount of either MHD or OEG in the mixed monolayers tended to promote the exposure of the biotin headgroups at the topmost part of the film (thus exposing them to aqueous solution, see Figure 4). Thus, the increase in specific SA binding with the mixed BAT/OEG monolayers, compared to pure BAT, is not due solely to the nonfouling properties of OEG but is also due to the increased interfacial concentration of biotin and the concomitant reduction in exposure of parts of the BAT molecule (like aliphatic regions) that promote nonspecific interactions. The overall protein adsorption properties of these monolayers must therefore be determined by the energetic interplay between all of the components, including their conformation and packing density, their configurational mobility, the chemical properties of the exposed atoms of these different chains,26,48,52 and the presence of a hydrogen-bonded layer of water at the SAM-liquid interface. The structural studies can be directly connected to functional SPR studies of streptavidin interfacial binding. SPR spectra were collected using a full range of mixed SAM compositions exposed to WTSA. We observed with SPR that maximal streptavidin coverage occurred over a wide range of χBAT,sur (∼0.15-0.55) (see Figure 6). At higher surface BAT densities, the maximal coverage decreased (56) Feldman, K.; Hahner, G.; Spencer, N. D.; Harder, P.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134-10141. (57) Pertsin, A. J.; Grunze, M. Langmuir 2000, 16, 8829-8841. (58) Everett, D. H. Basic Priciples of Colloid Science; Royal Society of Chemistry: London, 1988.

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but was never less than 65% of the maximal coverages (∼230 ( 50 ng/cm2) seen on films with optimal BAT densities. The ARXPS and NEXAFS observations presented earlier show that the biotin headgroups in pure BAT SAMs are uniformly distributed throughout the depth of the film and that the thiolates are not well-ordered. This suggests that some of the biotin headgroups may protrude far enough from the 100% BAT film to specifically bind SA. This would explain why substantial specific binding of SA is still seen at χBAT,sur ) 1.0 in Figure 6, which is consistent with previous results from other groups that used a slightly different biotinylated thiolate5,7 (albeit at somewhat higher levels). As noted previously, the disorder in the pure BAT monolayers is likely to expose more hydrophobic portions of the BAT molecule which nonspecifically bind proteins. Indeed, competitive desorption SPR measurements suggest that ∼15-20% of the SA bound to pure BAT SAMs is nonspecifically adsorbed.8 We also observed that the maximal SA coverage as a function of surface BAT density is quite similar, regardless of the diluent (OEG or MHD), at BAT coverages higher than approximately 10%. The higher amount of adsorbed SA on BAT/MHD SAMs at low χBAT,sur ( 0.7, where some nonspecific bonding also occurs (∼20% of the total SA). The current structural studies elucidate this curious behavior at high χBAT,sur. The nearly random distribution of N atoms in the probe depth of XPS shows that the surface biotins are no longer all protruding from the surface of the film, and thus many of the biotins will be unavailable to SA binding pockets. This lack of availability explains why a SA, once bound to the surface by a (rarely occurring) accessible biotin, rarely finds another accessible biotin near enough to its other surface-directed biotin binding pocket. At the same time, it may interact with other (e.g., more hydrophobic) regions of the BAT molecules which promote nonspecific adsorption. Conclusions The composition, orientation, order, and thickness (coverage) of self-assembled monolayers designed for the specific surface immobilization of streptavidin have been characterized. In addition, the relative amount of streptavidin bound to the various surface compositions was also determined. A nonlinear relationship between the monolayer thiolate composition as a function of solution mole fraction of BAT was determined for mixed BAT/MHD and BAT/OEG monolayers, likely because of the different rate constants for adsorption of the different thiols. Pure BAT does not assemble into ordered monolayers, as demonstrated by the angle-resolved XPS and NEXAFS experiments. Addition of the MHD thiol to solutions of BAT improved the orientation of the biotinylated thiol and increased the order within the hydrocarbon region of the film. The addition of the OEG thiol to solutions of BAT also improved the orientation of the biotinylated thiol, although no increase in the order of the alkyl segments within the film was detected by NEXAFS. The OEG diluent promotes specific adsorption of streptavidin by increasing

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the concentration of exposed biotin groups above the film, in addition to disfavoring nonspecific adsorption through its nonfouling capabilities. The degree of specific binding of streptavidin to these surfaces was directly correlated with the amount of ligand (BAT) and the nature of the diluent thiol (MHD or OEG) and thus with the structure and physical properties of the SAMs. Maximal SA coverage varied with surface BAT coverage, passing through a broad maximum before slowly declining at high BAT coverage. This reduced coverage is correlated with lower availability of surface-exposed biotin groups, resulting from loss in orientational order of the BATs in the film. Acknowledgment. The authors gratefully acknowledge support from the National ESCA Surface Analysis Center for Biomedical Problems (NESAC/BIO NIH Grant

Nelson et al.

#RR01296 from the National Center for Research Resources). Additional support was provided by the National Science Foundation and University of Washington Engineered Biomaterials Grant Number EEC-9529161. P.S.S. wishes to acknowledge NIH Grant Number DK49655. We also thank Mimi Mar for her expert technical assistance with the electron beam evaporation. NEXAFS studies were performed at the NSLS, Brookhaven National Laboratory, which is supported by the U.S. Department of Energy, Division of Materials Science and Division of Chemical Sciences. Supporting Information Available: The methods used to synthesize the BAT and OEG thiols. This material is available free of charge via the Internet at http://pubs.acs.org. LA001111E