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Functional Monolayers for Improved Resistance to Protein Adsorption: Oligo(ethylene glycol)-Modified Silicon and Diamond Surfaces Tami Lasseter Clare,† Brian H. Clare,‡ Beth M. Nichols,† Nicholas L. Abbott,‡ and Robert J. Hamers*,† Department of Chemistry, University of WisconsinsMadison, 1101 University Avenue, Madison, Wisconsin 53706, and Department of Chemical and Biological Engineering, University of WisconsinsMadison, 1415 Engineering Drive, Madison, Wisconsin 53706 Received February 8, 2005. In Final Form: April 21, 2005 The interaction of proteins with semiconductors such as silicon and diamond is of great interest for applications such as electronic biosensing. We have investigated the use of covalently bound oligo(ethylene glycol), EG, monolayers on diamond and silicon to minimize nonspecific protein adsorption. Protein adsorption was monitored by fluorescence scanning as a function of the length of the ethylene glycol chain (EG3 through EG6) and the terminal functional group (methyl- versus hydroxyl-terminated EG3 monolayer). More quantitative measurements were made by eluting adsorbed avidin from the surface and measuring the intensity of fluorescence in the solution. The attachment chemistry of the tri(ethylene glycol) molecules and monolayer orientation was studied by X-ray photoelectron spectroscopy. Improvement in the selectivity of surfaces modified with EG functionality was demonstrated in two model biosensing assays. We find that high-quality EG monolayers are formed on silicon and diamond and that these EG3 monolayers are as effective as EG3 self-assembled monolayers on gold at resisting nonspecific avidin adsorption. These results show promise for use of silicon and diamond materials in many potential applications such as biosensing and medical implants.

Adsorption of proteins at surfaces is of great importance in a number of applications, including surface-based bioassays,1,2 biosensors,3-5 and biomedical devices such as implants.6,7 For biosensing applications, the chemical properties of the surface must be tailored such that the surface presents functional groups that will specifically bind to proteins of interest while rejecting all other proteins. Previous studies have examined how different types of functional groups can affect the amount of adsorbed protein at surfaces.8-18 Monolayers containing oligo(ethylene glycol) units (EG) have been found to be * Corresponding author. E-mail: [email protected]. † Department of Chemistry. ‡ Department of Chemical and Biological Engineering. (1) Langer, R.; Tirrell, D. A. Nature 2004, 428, 487. (2) Wisniewski, N.; Moussy, F.; Reichert, W. M. Fresenius’ J. Anal. Chem. 2000, 366, 611. (3) Lasseter, T. L.; Cai, W.; Hamers, R. J. Analyst 2004, 129, 3. (4) Yang, W. S.; Hamers, R. J. Appl. Phys. Lett. 2004, 85, 3626. (5) Ha¨rtl, A.; Schmich, E.; Garrido, J. A.; Hernando, J.; Catharino, S. C. R.; Walter, S.; Feulner, P.; Kromka, A.; Steinmuller, D.; Stutzmann, M. Nat. Mater. 2004, 3, 736. (6) DiTizio, V.; Ferguson, G. W.; Mittelman, M. W.; Khoury, A. E.; Bruce, A. W.; DiCosmo, F. Biomaterials 1998, 19, 1877. (7) Castner, D. G.; Ratner, B. D. Surf. Sci. 2002, 500, 28. (8) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164. (9) Luk, Y.-Y.; Kato, M.; Mrksich, M. Langmuir 2000, 16, 9604. (10) Chapman, R. G.; Ostuni, E.; Yan, L.; Whitesides, G. M. Langmuir 2000, 16, 6927. (11) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426. (12) Mrksich, M.; Sigal, G. B.; Whitesides, G. M. Langmuir 1995, 11, 4383. (13) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 5605. (14) Ostuni, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17, 6336. (15) Ostuni, E.; Grzybowski, B. A.; Mrksich, M.; Roberts, C. S.; Whitesides, G. M. Langmuir 2003, 19, 1861. (16) Ostuni, E.; Yan, L.; Whitesides, G. M. Colloids Surf., B: Biointerfaces 1999, 15, 3. (17) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12.

among the most effective types of functional groups at reducing nonspecific adsorption of a variety of proteins.8,19 Most previous studies of protein adsorption have been performed on self-assembled monolayers (SAMs) on gold. However, recently there has been great interest in use of semiconductor materials, especially silicon and diamond, for direct electronic sensing of biomolecular binding at surfaces3-5,20-23 and for their higher chemical and thermal stability.24,25 In those systems, the ability to detect biological binding is directly associated with the semiconducting properties of silicon and diamond because the charge associated with the biomolecules at the surface induces an electric field that penetrates into the semiconductor. These electric field-induced changes are similar to those in a field-effect transistor, leading to the development of devices that are biologically activated field-effect transistors, or bio-FETs.4,20,26-28 Because in electrolyte solutions charges are screened over distances on the order of 1 nm, operation of these devices relies on having the (18) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (19) Li, L.; Chen, S.; Zheng, J.; Ratner, B. D.; Jiang, S. J. Phys. Chem. B 2005, 109, 2934. (20) Scho¨ning, M. J.; Poghossian, A. Analyst 2002, 127, 1137. (21) Fritz, J.; Cooper, E. B.; Gaudet, S.; Sorger, P. K.; Manalis, S. R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 14142. (22) Cai, W.; Peck, J. R.; van der Weide, D. W.; Hamers, R. J. Biosens. Bioelectron. 2004, 19, 1013. (23) Yang, W. S.; Butler, J. E., Russell, J. N., Jr.; Hamers, R. J. Langmuir 2004, 20, 6778. (24) Yang, W. S.; Auciello, O.; Butler, J. E.; Cai, W.; Carlisle, J. A.; Gerbi, J.; Gruen, D. M.; Knickerbocker, T.; Lasseter, T. L.; Russell, J. N., Jr.; Smith, L. M.; Hamers, R. J. Nat. Mater. 2002, 1, 253. (25) Lu, M.; Knickerbocker, T.; Cai, W.; Yang, W.; Hamers, R. J.; Smith, L. M. Biopolymers 2004, 73, 606. (26) Scho¨ning, M. J.; Luth, H. Phys. Status Solidi A: Appl. Res. 2001, 185, 65. (27) Berggren, C.; Bjarnason, B.; Johansson, G. Electroanalysis 2001, 13, 173. (28) Berggren, C.; Stalhandske, P.; Brundell, J.; Johansson, G. Electroanalysis 1999, 11, 156.

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EG-Modified Silicon and Diamond Surfaces

molecules of interest bind as closely as possible to the semiconductor surface. Extension of bio-FETs to protein systems requires developing and characterizing the degree to which comparatively short molecules are able to resist nonspecific adsorption. Although a great deal of research has been conducted on oligo(ethylene glycol) self-assembled layers on gold, the use of these molecules to modify nonmetallic surfaces such as silicon and diamond has not been systematically investigated. In previous work, we showed that well-defined, functionalized monolayers can be prepared on silicon and diamond surfaces using molecules containing a terminal vinyl (CdC) group. Photochemical excitation at 254 nm binds molecules to the surfaces through the vinyl group, leading to well-defined monolayers.29-33 A variety of different functional groups can be exposed at the surface, such as amino groups,4,29-31 which are useful attachment points for linking biomolecules such as proteins to surfaces, and tri(ethylene glycol) groups,34 which provide a way to reduce nonspecific adsorption. Recently, we showed that covalent functionalization of single-crystal silicon and nanocrystalline diamond with tri(ethylene glycol) (EG3) groups greatly reduces the nonspecific adsorption of fluorescently labeled proteins [avidin, bovine serum albumin (BSA), casein, and fibrinogen] and that ratio of specific binding to nonspecific adsorption can be optimized by using mixed monolayers with controlled composition.34 However, surface-based fluorescence measurements cannot be used to quantitatively compare the extent of protein adsorption on functionalized surfaces of different bulk materials (e.g., gold, silicon, diamond) because of fluorescence quenching.35-38 For sensing applications it is important to understand to what extent protein adsorption is affected by the length of the EG molecule, because the sensitivity of field-effect devices is highest when the molecules are as close as possible to the interface. More generally, it is necessary to identify to what extent the functionalization strategies used on gold can be applied to other materials systems that have different chemical and physical properties. Here, we focus on silicon and diamond because both are semiconductors of interest for biosensing and because diamond has been shown to provide superior chemical and thermal stability. In the present work, we report further investigations into the use of EG monolayers and mixed monolayers to minimize the nonspecific adsorption of proteins at surfaces of silicon and diamond. We have monitored protein adsorption as a function of the length of the ethylene glycol chain and the hydrophilic or hydrophobic nature of the terminal functional groups. On-chip fluorescence measurements are complemented by solution-based elution measurements that provide quantitative comparison of avidin adsorbed on diamond, silicon, and gold with a detection limit of only 2.2 fmol/cm2 (150 pg/cm2), or approximately 0.037% of a monolayer. We have charac(29) Strother, T.; Cai, W.; Zhao, X.; Hamers, R. J.; Smith, L. M. J. Am. Chem. Soc. 2000, 122, 1205. (30) Strother, T.; Hamers, R. J.; Smith, L. M. Nucleic Acids Res. 2000, 28, 3535. (31) Strother, T.; Knickerbocker, T.; Russell, J. N, Jr.; Butler, J. E.; Smith, L. M.; Hamers, R. J. Langmuir 2002, 18, 968. (32) Cicero, R. L.; Linford, M. R.; Chidsey, C. E. D. Langmuir 2000, 16, 5688. (33) Stewart, M. P.; Buriak, J. M. J. Am. Chem. Soc. 2001, 123, 7821. (34) Lasseter, T. L.; Clare, B. H.; Abbott, N. L.; Hamers, R. J. J. Am. Chem. Soc. 2004, 126, 10220. (35) Chance, R. R.; Prock, A.; Silbey, R. Adv. Chem. Phys. 1978, 37, 1. (36) Lakowicz, J. R. Anal. Biochem. 2001, 298, 1. (37) Enderlein, J. Biophys. J. 2000, 78, 2151. (38) Lakowicz, J. R.; Malicka, J.; D’Auria, S.; Gryczynski, I. Anal. Biochem. 2003, 320, 13.

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Figure 1. (a) Covalent modification of silicon and diamond. (b) Example monolayers of 100% EG3-, amino-, EG6-, and Me-EG3-terminated silicon or diamond.

terized these surfaces by quantitatively determining the absolute amount of avidin adsorbed and studied the surface attachment chemistry and monolayer orientation using X-ray photoelectron spectroscopy, XPS. Our results show that high-quality EG monolayers are formed on silicon and diamond and that EG3 monolayers on silicon and diamond are as effective as EG3 SAMs on gold at resisting nonspecific avidin adsorption. The work presented here provides information on the factors that control protein adsorption and additional characterization and mechanistic studies of the monolayers, and it explores the utility of EG-modified surfaces in biosensing assays. These results are particularly important for understanding the potential application of functionalized semiconductor surfaces for bioelectronic devices. Materials and Methods Preparation of Mixed Monolayers on Silicon and Diamond Surfaces. In previous studies we showed that molecules bearing terminal vinyl groups will covalently bind to hydrogenterminated surfaces of silicon29,30 and diamond24,31 upon illumination with 254 nm light. Figure 1a illustrates this process. Hydrogen-terminated silicon (111) surfaces were prepared by cleaning in acidic and basic solutions, followed by etching in nitrogen-sparged 40% NH4F for 30 min.29 Hydrogen-terminated diamond surfaces were prepared by acid cleaning followed by hydrogen plasma treatment, as reported previously.31 Covalent monolayers were then formed on these surfaces by exposing the hydrogen-terminated surface to a parent liquid of the desired molecule under UV light for 3 h in the case of silicon or 12 h in the case of diamond.24 To link amino groups to the surface, t-BOC10-aminodec-1-ene (Boc-N-ene) and TFA-10-aminodec-1-ene (TFA-N-ene) were synthesized, covalently attached to silicon or diamond surfaces, respectively, and deprotected after attachment (and before characterization by XPS) as reported previously.24,30,31 Resistance to nonspecific adsorption was conferred by binding vinyl-terminated oligo(ethylene glycol) monolayers to the surface. Tri(ethylene glycol) (EG3-ene), tetra(ethylene glycol) (EG4-ene), penta(ethylene glycol) (EG5-ene), hexa(ethylene glycol) (EG6-

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ene), and monomethyl tri(ethylene glycol) (Me-EG3-ene) modified undec-1-enes were synthesized and fully characterized for these studies according to previously published procedures.17 Illustrations of monolayers formed from these molecules are presented in Figure 1b. Monomethyl tri(ethylene glycol) (EG3-Me) and dimethyl tri(ethylene glycol) (Me-EG3-Me) were purchased from Aldrich. The various mixed monolayers were formed by making parent solutions of different compositions. Preparation of Mixed Monolayers on Gold Surfaces. Au films (100 nm) sputtered onto glass surfaces (GenTel) were cleaned for 15 min by use of a low-pressure mercury vapor quartz grid lamp, which removes adsorbed organic material from gold surfaces. XPS measurements of these gold films (not shown) revealed a clean, carbon-free surface with only a trace of oxygen. The surfaces were then rinsed with H2O followed by ethanol. The clean gold surfaces were immersed for at least 12 h in 2 mM thiol solutions of dodecanethiol (Dojindo); 11-aminoundecanethiol, MUAM (Dojindo); or tri(ethylene glycol) undecanethiol, EG3SH (Prochimia). Protein Adsorption. Fluorescein-labeled casein (Sigma), fluorescein-labeled avidin (Vector Labs), fluorescein-labeled bovine serum albumin or BSA (Biømeda), and fibrinogen Alexa Fluor 546 conjugate (Molecular Probes) were diluted or dissolved in 0.1 M NaHCO3, pH 8.3, to a working concentration of 0.2 mg/mL. To test for nonspecific adsorption, the proteins were spotted onto silicon or diamond surfaces on which a mixed or one-component monolayer had been formed, allowed to adsorb at room temperature for 1 h (the samples were kept in a humidified chamber during that time), briefly rinsed, and then soaked for 15 min in a wash-off buffer consisting of 0.3 M NaCl, 20 mM Na2PO4, 2 mM EDTA, pH 7.4 (2× SSPE buffer from Promega), and 1% Triton-X 100, commonly referred to as 2× SSPE buffer. These adsorption reactions were characterized by on-chip fluorescence imaging (where the intensity of the adsorbed proteins on the surfaces was measured) or solution-based measurements (where adsorbed protein was eluted from the surfaces and the intensity of fluorescence from the eluent was measured with a fluorometer). For the latter method, the samples were soaked in 1.00 mL of an elution buffer consisting of the wash-off buffer with added 1% mercaptoethanol for at least 12 h. Mercaptoethanol is a reducing agent that acts to cleave disulfide bonds in proteins, which aids their elution from the substrates into the elution buffer. The effectiveness of removal was checked by ensuring that little or no fluorescence remained on the surfaces after elution; the fluorescence intensity of the eluent containing the protein was then measured. Fluorescence Measurements. For the on-chip fluorescence measurements (Figures 2, 3, 6a, and 7), the fluorescence intensity of the fluorescein-labeled proteins was measured on a Genomic Systems UC 4 × 4 fluorimager using a 488 nm excitation source and a 512 nm band-pass filter, and the intensity of the Alexa Fluor 546 conjugated fibrinogen was measured by use of a 532 nm excitation source and a 550 nm long-pass filter. In the solutionbased method, fluorescence measurements of proteins collected in the elution buffer (Figures 5 and 6b) were performed on an ISS photon counting spectrofluorometer. Measurements of fluorescein-avidin were made by use of excitation at 480 ( 8 nm, measuring fluorescence at 518 ( 8 nm. Specific Binding. The silicon surfaces were biotinylated by spotting a biotin linker, sulfosuccinimidyl-6′-(biotinamido)-6hexamidohexanoate (Pierce Endogen) onto amino-terminated silicon surfaces as reported previously.3 Avidin diluted (in the bicarbonate buffer as above) to a working concentration of 0.2 mg/mL was spotted onto biotinylated silicon surfaces, allowed to bind for 10 min at 4 °C, briefly rinsed, and then soaked for 15 min in wash-off buffer (Figures 4 and 7). Controls for specific binding, where biotin-saturated avidin in solution was exposed to biotinylated surfaces, showed no fluorescence intensity. Fluorescence intensities were immediately measured as described above. Competitive binding studies were performed with chicken serum purchased from Sigma. XPS Characterization. Molecular layers on silicon were characterized by X-ray photoelectron spectroscopy, on a system equipped with a monochromatized Al KR source and a multichannel array detector. Measurements on diamond surfaces are not reported here except for stability studies because we have

Clare et al. characterized their functionalization previously24,31 and because the high carbon content of both substrate and molecular layers limits the quantitative information. Spectra reported here were recorded with an analyzer resolution of 0.18 eV. The percent EG moiety on the surface was calculated by fitting the carbon spectrum to two peaks and the nitrogen spectrum to one peak (Figure 5).39

Results In our studies, we used a variety of experimental measurements. On-chip fluorescence measurements were used to investigate qualitative trends in the reduction of nonspecific adsorption as a function of monolayer composition. Unfortunately, on-chip fluorescence intensities cannot be quantitatively compared between substrate types (i.e., silicon versus diamond) due to substratedependent fluorescence quenching.37 More quantitative measurements for comparison of adsorption on different substrates were made by eluting adsorbed avidin and measuring the fluorescence of the eluent as described above. I. Effect of EG Chain Length on Protein Adsorption. On the basis of our earlier work showing that EG3 monolayers on silicon and diamond resist the adsorption of proteins,34 we investigated how increasing the length of the EG chain affected nonspecific protein adsorption. In these studies, fluorescently labeled proteins were allowed to adsorb to functionalized silicon or NC diamond, and the protein remaining was measured by on-chip fluorescence imaging. Figure 1a shows the reaction scheme for the chemical modification of silicon and diamond, and Figure 1b depicts the covalently bound monolayers that resulted when the hydrogen-terminated surfaces were exposed to Boc-N-ene (silicon) or to TFA-N-ene (diamond) and then deprotected, to EG3-ene, to EG6-ene, or to Me-EG3-ene. Characterization of the monolayers by XPS was performed and is presented in part IV. Measurements of the fluorescence intensity after the fluorescently labeled proteins (avidin, BSA, casein, and fibrinogen) were adsorbed to separate areas of the functionalized surfaces and rinsed (as described above) are shown in Figure 2, panels a (diamond) and b (silicon). The data presented in Figure 2 were normalized to the amino-terminated surfaces in order to highlight the dramatic reduction of nonspecifically adsorbed protein that occurs when EG units were incorporated into the monolayer. The left panels show the fluorescence intensity due to nonspecific adsorption of proteins onto mixed monolayers of Boc-N-ene and EG3-ene on silicon and diamond, while the right panels show the effect of increasing EG chain length for pure EG monolayers. The trend from the data in the left panels of Figure 2 show that the fluorescence intensity arising from each of the four proteins investigated decreases as more EG3 functionality is incorporated into the monolayers.34 The 100% EG3 monolayer yields a reduction in fluorescence intensity by as much as 60% (silicon) and 70% (diamond) compared with the amino-terminated surfaces; if the fluorescence intensity is assumed to be proportional to surface concentration, then this corresponds to a 60-70% reduction in nonspecific adsorption. Repeated experiments showed a variation in fluorescence intensity of approximately 25% for each data point in Figure 2; thus, the (39) The percent EG moiety was calculated from XPS data by use of the following equation: X ) % EG moiety, 100 - X ) % Boc-N-ene (100 - X)/(X) ) [(low BE carbon area)/(high BE carbon area - nitrogen area)] × (no. of C having high BE)/(no. of C having low BE). The nitrogen area was corrected for the sensitivity factor difference between nitrogen and carbon.


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Figure 3. Fluorescence intensity from proteins adsorbed to mixed EG3-ene and Me-EG3-ene monolayers on silicon. The intensities are normalized to that of the 100% amino-terminated surface.

Figure 2. Normalized intensity of fluorescence from fluoresceinlabeled proteins onto EGX-modified (where X ) 3, 4, 5, or 6) surfaces of (a) diamond and (b) silicon. The left panels show the intensity of fluorescence due to nonspecific adsorption as a function of amino percentage within mixed EG3-amino monolayers; the right panels show the effect of EG chain length on the fluorescence intensity from nonspecific protein adsorption onto pure EG monolayers. The data shown here represent one complete data set. Repeated experiments show approximately 25% variation between data points.

slight difference between diamond and silicon is not significant. More importantly, these results show that EG3 monolayers effectively reduce nonspecific adsorption on both silicon and diamond surfaces by similar amounts. The data in the right panels of Figure 2 show how the fluorescence intensity from adsorbed proteins varies as the EG chain increased from three to six EG units. These data illustrate that although EG3 is effective at reducing nonspecific adsorption, the amount of adsorbed protein can be further reduced by increasing the number of EG units. In the longest oligomer study, the EG6 molecule yields an additional reduction of 50-90% on silicon and 30-80% on diamond compared with EG3, varying somewhat between different proteins. Thus, we conclude that EG6 is significantly more effective than EG3 at resisting nonspecific protein adsorption. II. Effect of Methyl-Terminated EG Monolayers on Protein Adsorption. Because the hydroxyl group of EG3 can, in principle, be oxidized to a (charged) carboxylate group, the hydroxyl-terminated EG3-ene could lead to increased nonspecific adsorption of positively charged proteins.13 Replacing the terminal hydroxyl with a methoxy group to form Me-EG3-ene could therefore provide additional resistance to protein adsorption, especially under oxidizing conditions. However, the terminal methyl group is also expected to be more hydrophobic. Previous studies of SAMs on gold have reported that methylterminated EG3 (Me-EG3) and hydroxyl-terminated EG3 are comparably effective at reducing the amount of nonspecifically adsorbed protein.10,13

Represented in Figure 3 are the on-chip fluorescence intensity data of avidin, BSA, casein, and fibrinogen adsorbed to monolayers of varying composition of EG3 and Me-EG3-ene on silicon. The fluorescence intensity from BSA, casein, and avidin adsorbed to the hydroxylterminated EG3 monolayers is only 20-40% of that observed on the methyl-terminated Me-EG3 monolayers, indicating that the methyl group leads to a substantial increase in the amount of nonspecific adsorption. However, the additional methyl group did not affect the amount of fibrinogen that adsorbed to the surfaces, consistent with earlier reports of Me-EGx SAMs on gold.10,11,13,18,40,41 On the basis of these observations, we conclude that the hydroxyl-terminated EG3-ene molecule is generally more effective than the methyl-terminated Me-EG3-ene molecule at resisting nonspecific adsorption on silicon, but the difference in effectiveness is protein-dependent. III. Comparative Elution Measurements on Different Surfaces. While the above studies provide good qualitative insights into how the monolayers affect nonspecific adsorption, on-chip fluorescence measurements cannot be easily used for absolute, quantitative analysis or even comparisons between different substrates (i.e., gold, Si, and diamond) because of fluorescence quenching. Fluorescence quenching arises from interactions of fluorophores in close proximity to other fluorophores and/or to a conductive substrate.35 The effects of quenching can be eliminated by eluting the nonspecifically adsorbed proteins from the surface and measuring the fluorescence intensity in a dilute solution, rather than measuring the intensity while adsorbed to the surface.37 We chose stringent elution conditions under which the fluorescence intensity of the substrate was reduced by approximately 99% or more, indicating that nearly all the adsorbed protein was eluted into solution. The concentration of avidin in the eluted solution was calculated by comparing the fluorescence intensity of the eluted protein solution to a calibration curve (made from standards of known avidin concentration). The avidin calibration curves showed a linear dependence of fluorescence emission on concentration, with an r2 value of greater than 0.999 over more than an order of magnitude in concentration; the (40) Benesch, J.; Svedhem, S.; Svensson, S. C. T.; Valiokas, R.; Liedberg, B.; Tengvall, P. J. Biomater. Sci., Polym. Ed. 2001, 12, 581. (41) Herrwerth, S.; Eck, W.; Reinhardt, S.; Grunze, M. J. Am. Chem. Soc. 2003, 125, 9359.

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linearity of the calibration curves confirms that intermolecular fluorescence quenching is negligible in solution under our conditions. The calibration curves also establish a detection limit of approximately 1.4 pg/mL or 2.2 fmol/ mL avidin. To establish a baseline corresponding to a full “monolayer” of avidin, we first applied this method to surfaces that were modified with biotin, which is bound strongly by avidin and is expected to produce a densely packed layer of avidin molecules. As shown in Figure 4a, silicon and diamond surfaces were first amino-terminated, then biotinylated with a linker containing a disulfide bond, and finally exposed to fluorescein-labeled avidin. Avidin that bound to the surfaces was then eluted off by cleaving the disulfide bond in the biotin linker by use of mercaptoethanol in the elution buffer. Figure 4b,c shows the amount of avidin bound to the surfaces. Biotinylated surfaces of gold, silicon, and diamond adsorbed 6.9, 4.9, and 7.7 pmol/cm2 avidin, respectively. As a point of comparison, the percent monolayer equivalent (% ML equiv) of a close-packed layer can be estimated from the molecular dimensions of avidin42 (40 Å × 50 Å × 56 Å).43 Assuming that avidin adsorbs via its smaller (40 Å × 50 Å face), biotinylated gold binds 83% of a close-packed monolayer of avidin, silicon binds 60% of a monolayer, and diamond binds 93% of a monolayer. We emphasize that while the concentration in picomoles per square centimeter is an absolute measurement directly traceable to known standards, the representation in terms of monolayers is dependent on knowing the effective size of an avidin molecule on the surface; however, the almost cubic shape of avidin makes this a useful way of representing the data. The data show that (1) all three surfaces bind less than what would be expected for a close-packed layer and (2) the three starting surfaces bind different amounts of avidin. While a close-packed monolayer would correspond to 8.3 pmol/cm2, steric hindrance between avidin molecules and random binding (not close-packing) would likely prevent a 100% monolayer from forming on any surface. The diamond surface may have bound slightly more avidin than one would expect because the surface of NC diamond is rough due to the increased surface area from the nanocrystalline (50-200 nm) diamond film.44 Comparing these results to other data in the literature, it has been reported that 125I-labeled avidin immobilized on a biotinylated Teflon surface bound approximately 5.4 pmol/ cm2 or approximately 66% of a monolayer,45 which falls within the range of our data (4.9-7.7 pmol/cm2). The results from our measurements and good correspondence with previous results from radioactive methods provides confidence that the use of elution combined with solutionbased fluorescence measurements is a highly sensitive, accurate method for quantitatively analyzing avidin adsorption and, by avoiding the problems associated with quenching, provides a good way to quantitatively compare different surfaces. After ensuring that the elution buffer and fluorometer measurements yielded accurate results on biotinylated (42) Pugliese, L.; Coda, A.; Malcovati, M.; Bolognesi, M. J. Mol. Biol. 1993, 231, 698. (43) The amount of avidin bound to the surface was based on a molecular mass of 62 400 Da. Percent monolayer equivalent was calculated from the size of avidin (5.6 nm × 5.0 nm × 4.0 nm). A complete monolayer of avidin is between 3.6 × 1011 and 5.0 × 1011 molecules/cm2 (or between 6.0 and 8.3 pmol/cm2). (44) Clare, T. L.; Hamers, R. J. Manuscript in preparation. (45) McFarland, C. D.; Jenkins, M.; Griesser, H. J.; Chatelier, R. C.; Steele, J. G.; Underwood, P. A. J. Biomater. Sci., Polym. Ed. 1998, 9, 1207.

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Figure 4. (a) Schematic depicting the formation of a biotinylated monolayer containing a cleavable linker that allowed bound avidin to be eluted from the surface and collected for analysis by fluorometry. (b) Amount of avidin bound or adsorbed by a variety of monolayers on diamond, silicon, or gold surfaces, measured by fluorometry of the avidin eluted into solution. Values are listed in picomoles per square centimeter and as percent monolayer equivalent. (c) Adsorption of avidin versus substrate material and monolayer type.

silicon, NC diamond, and gold, the effect of different surface terminations on nonspecific protein adsorption was studied. Figure 4b tabulates the absolute amount of avidin removed (in picomoles per square centimeter) and the equivalent surface coverage (in percent monolayer equivalents, assuming each avidin molecule occupies a 50 Å × 40 Å area) from elution experiments. In these experiments, avidin was allowed to adsorb to surfaces


with different terminations. The avidin on the surface was then eluted overnight under conditions that removes >99% of the nonspecifically adsorbed avidin, and the fluorescence from the eluent was measured in solution and compared against known standards. To measure specific binding of avidin, the surface was biotinylated, whereas nonspecific adsorption of avidin was measured on amino-, EG3-, or EG6-terminated monolayers, and the results are graphed in Figure 4c. The data show that for silicon and diamond, functionalization with the amino group reduces the amount of nonspecific adsorption by approximately a factor of 10 compared with the biotinylated surfaces (i.e., a full monolayer), while aminotermination of gold reduced the nonspecific adsorption by a factor of 2. For all three surfaces, modification with EG3 further reduced the amount of avidin adsorbed to them. Gold and NC diamond adsorbed approximately 0.24 pmol/ cm2 (3% ML equiv) avidin, while silicon adsorbed less, 0.074 pmol/cm2 (1% ML equiv). Silicon and diamond were also functionalized with EG6 (EG6-termination on gold was not studied) and the data show that this yields a further reduction in the amount of adsorbed avidin, to 0.16 pmol/cm2 or ∼2% ML equiv (diamond) and 0.056 pmol/ cm2 or ∼0.7% ML equiv (silicon). These experiments demonstrate several important points. First, our data show that modification with EG3 monolayers very effectively reduces nonspecific avidin adsorption on silicon, diamond, and gold surfaces. A comparison of the surfaces shows that EG3-modified diamond surfaces resist nonspecific adsorption as effectively as EG3 SAMs on gold and that EG3-modified silicon samples are the most effective of all. Finally, our data show that although EG3 is effective at reducing nonspecific adsorption of avidin, further reduction can be obtained by using longer EG chains. These experiments underscore the sensitivity of fluorescence methods as shown by the extremely low detection limit of the fluorometer, 2.2 fmol/mL. Assuming a washoff volume of 1 mL, a 1 cm2 sample, and avidin molecular weight of 62 408 g/mol, this would correspond to a detection limit of only 2.2 fmol/cm2 (140 pg/cm2) or ∼0.027% ML equiv. This limit is somewhat lower than the typical detection limits of ∼(500 pg-1.2 ng)/cm2 reported for SPR46,47 and considerably lower than typical values of 1-4% ML for ellipsometry.41 These experiments show that elution followed by fluorescence measurements can detect much less than 1% of a monolayer of adsorbed avidin with approximately 25% variation between nominally identical samples (Figure 4b). These solution-based measurements are particularly useful because they can be applied even to textured surfaces, they avoid the quenching problems commonly known to plague surface-based fluorescence measurements, and they can be used to compare a wide range of materials (e.g., metallic, nonmetallic, nonreflective, polymeric) having different optical properties. IV. Characterization of Monolayers. Having shown that a greater percentage of EGX within a monolayer made silicon and diamond more effective at reducing nonspecific protein adsorption (Figure 2), we wished to address a few basic surface chemistry questions, specifically the following: (1) How accurately does the composition of the surface-bound molecules reflect the composition of the parent solution? (2) Do any side reactions between functional groups other than the terminal vinyl and the surface occur? (3) Is a homogeneously oriented EG (46) Silin, V.; Weetall, H.; Vanderah, D. J. J. Colloid Interface Sci. 1997, 185, 94. (47) Shumaker-Parry, J. S.; Campbell, C. T. Anal. Chem. 2004, 76, 907.

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monolayer is necessary to confer the best resistance to nonspecific protein adsorption? The composition of the molecules that are immobilized on a surface does not necessarily reflect the composition of the parent solution due to factors such as steric hindrance34 or preferential van der Waals interactions.48 Two side reactions, one between the terminal hydroxyl group of the EG3 moiety and the hydrogen-terminated silicon surface49 and one between oxidized ether linkages in the EG chain and the silicon surface, are both possible. The extent to which both of these side reactions occur was carefully studied to determine whether the EG3 molecules are oriented with the EG end exposed at the surface or whether they react directly with the silicon surface, leaving the EG end buried within the monolayer. Since the monolayers containing EG groups were found to be effective at resisting nonspecific protein adsorption,34 it would be expected that a significant fraction of the molecules are oriented such that the EG portion is exposed and not buried within the monolayer. However, if it is determined that the molecules are not all oriented with their EG ends exposed, that implies that a well-organized monolayer is not necessary to confer resistance to protein adsorption. Quantitative measurements of the monolayer composition were performed by mixing the molecules of interest with Boc-N-ene in varying mole fractions in the parent mixture and analyzing the resulting monolayer composition from the intensity of the C(1s) peaks. Figure 5 presents a summary of silicon monolayer data obtained from a number of different parent solutions of varying composition. Figure 5a provides representative XPS spectra; Figure 5b graphically summarizes the composition of the surface monolayers as determined by XPS for various parent compositions, while Figure 5c gives some specific values of surface composition. The labels (A-E) in each part of this figure are consistent, facilitating comparison of the XPS spectra in Figure 5a with specific surface compositions in Figure 5b,c. To identify the molecules bound to the surface, we use the fact that, in the EG molecules, the carbon atoms directly bound to oxygen atoms are shifted to a relatively high binding energy of 287.3 eV,11,17,50 giving rise to the peak at this energy that can be observed in the C(1s) spectra in Figure 5a. The carbon atoms in the hydrocarbon chain appear at a lower binding energy of 285.8 eV.11,17,50 (The t-Boc group of Boc-N-ene was removed under deprotection conditions prior to XPS characterization.) Thus, measuring the areas of these peaks and correcting for the known number of carbon atoms of each type in the parent molecules allows us to determine the surface composition.39 We first address the composition of mixed monolayers of EG3-ene and Boc-N-ene. Figure 5a shows XPS data for five different solution compositions, and the square data points in Figure 5b show the composition of the resulting surface layers obtained by analyzing these data. These data show that when the mole percentage of EG3-ene in the parent solution is greater than 70%, the mole percentage on the surface accurately reflects the parent solution composition (points C-A in Figure 5). However, when the parent solution contained less than 70% EG3ene (and therefore more than 30% Boc-N-ene), the surface (48) Martins, C. L.; Ratner, B. D.; Barbosa, M. A. J. Biomed. Mater. Res. 2003, 67A, 158. (49) Niederhauser, T. L.; Lua, Y.-Y.; Jiang, G.; Matheson, R.; Hess, D. A.; Mowat, I. A.; Linford, M. R. Angew. Chem., Int. Ed. 2003, 41, 2353. (50) Huang, N.-P.; Michel, R.; Voros, J.; Textor, M.; Hofer, R.; Rossi, A.; Elbert, D. L.; Hubbell, J. A.; Spencer, N. D. Langmuir 2001, 17, 489.

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Figure 5. (a) Representative XPS spectra of EG monolayers on silicon and the structures of the EG molecules that were mixed with Boc-N-ene and used in these studies. (b) Graph of percent EG moiety on surface (from XPS measurements) versus percent EG moiety in parent solution. Points A-E are discussed in the text. (c) Numerical data from XPS spectra of points A-E.

showed a higher EG3 concentration than the parent solution did, as demonstrated by the points that lie in the “more than expected” region of Figure 5b. We attribute this deviation to steric hindrance between the bulky Boc groups, which allow the smaller EG3 molecules to more effectively access the surface and thereby increase the amount of EG3 relative to Boc-N-ene on the surface.34 A second question is whether other hydroxyl or ether groups in the EG molecule react with the surface. To identify the predominant binding configuration, we also

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did experiments using two control molecules: one in which the terminal vinyl group was replaced by a methyl group (yielding EG3-Me) and one in which both the terminal vinyl and terminal hydroxyl groups were replaced by methyl groups (Me-EG3-Me). Solutions containing BocN-ene and either monomethyl EG3 (EG3-Me) or dimethyl EG3 (Me-EG3-Me) were mixed and allowed to react with hydrogen-terminated Si surfaces, which were analyzed for percent EG content. For mixtures of EG3-Me and Boc-N-ene (2 in Figure 5b), the XPS data show that the EG3-Me molecules are incorporated much less effectively than the Boc-N-ene molecules are. For example, point D shows that a parent solution consisting of 75% EG3-Me/25% Boc-N-ene yields a monolayer containing only 30% EG3-Me. Thus, the larger Boc-N-ene molecules, which contain a vinyl group, react more efficiently than does the EG3-Me molecule, which contains a hydroxyl group and ether linkages. We further note that comparison of points C and D shows that when mixed monolayers of similar composition are formed (approximately 70% EG-containing moieties and 30% BocN-ene), the EG3-Me molecule is incorporated into the monolayer much less effectively than the EG3-ene (30% EG3-Me vs 69% EG3-ene) in the monolayer. These differences in solution and surface composition show that the reaction between the terminal vinyl groups and the silicon surface is faster than reactions involving the hydroxyl group or ether linkages. Finally, we investigated whether the ether linkages in the EG molecules would react with the silicon surface. This reaction was investigated by using a control molecule that lacked all other possible reactive groups, Me-EG3Me (no terminal hydroxyl or terminal vinyl groups). In Figure 5c, data point E shows that mixed solutions composed of 82% Me-EG3-Me (and 18% Boc-N-ene) yielded only 11% Me-EG3-Me on the silicon surface.51 Comparison with similar data for EG3-Me indicates that Me-EG3-Me is incorporated significantly less effectively than EG3Me; this difference suggests that most of the reactivity of EG3-Me likely arises from the hydroxyl group rather than the ether linkages. These studies allow us to rank the order of reactivity of the functional groups with H-terminated silicon: terminal vinyl > terminal hydroxyl > ether linkages. While in principle reaction rates could be used to predict the surface composition from a known solution composition, XPS spectra cannot identify the fraction of molecules that bind to the surface through each functional group (i.e., via the terminal vinyl, terminal hydroxyl, or ether linkages) when all three groups are present, as in EG3ene. However, these studies do establish the relative reaction rates for each functional group, showing that the reaction through the terminal vinyl group is significantly faster than the reactions of the terminal hydroxyl and ether linkages with the silicon surface. These results indicate that the reaction between the terminal vinyl and the silicon surface is the primary reaction pathway. Consequently, we conclude that EG3-ene molecules bound to silicon are primarily oriented with their EG portion distal to the surface. V. Stability of EG Monolayers. The stability of functionalized surfaces can be very dependent on the experimental conditions. For example, self-assembled monolayers on gold can be quite stable under some (51) When H2O2, a radical initiator in the presence of UV light, was added to the 82% Me-EG3-Me solution, 42% Me-EG3-Me was found on the surface (data not shown), which suggests a radical-mediated attachment mechanism.


conditions but can be rapidly degraded in the presence of certain compounds, including surfactants that are sometimes used to help eliminate nonspecifically adsorbed molecules at surfaces24,52 and under applied electrical potentials,53 both of which may have important ramifications in biosensing. Recent studies have shown that surfaces of silicon and, especially, diamond are more robust under harsh conditions such as elevated temperatures,25 in the presence of surfactants,24 and in the presence of applied electrical potentials.54,55 We performed stability studies on EG-functionalized surfaces over a period of 10 days. EG6-functionalized silicon and diamond and EG3-functionalized gold monolayers were prepared and stored in deionized water for 0, 1, 2, or 3 days. After the prescribed time period, the samples were removed, 0.2 mg/mL of avidin was allowed to adsorb to the surfaces for 1 h, and the fluorescence intensity was measured by on-chip fluorescence. Figure 6a shows the resulting amount of avidin that adsorbed to the EGmodified surfaces. The absence of any significant change in intensity shows that all three surfaces retained their ability to resist nonspecific adsorption for 3 days. A second set of experiments was performed by the solution-based elution method. One set of samples was stored in water for 3 days and then exposed to avidin (0.2 mg/mL, 1 h); a second set of samples was stored in water for 3 days and then in 2× SSPE + 1% Triton-X buffer for 7 more days before being exposed to avidin. The chart in Figure 6b shows the amount of avidin that was removed from these two sets of samples. The EG6-modified silicon and the EG3-modified gold sample both show some increase in the amount of nonspecific avidin adsorption after 10 days, while EG6-modified diamond shows only a very small increase, within the error of the measurement. These results show that EG6-modified diamond is more stable than EG6-modified silicon and EG3-modified gold. To investigate whether these changes in the ability to resist avidin adsorption were correlated with changes in the EG monolayers, XPS measurements were made of EG monolayers on diamond, silicon, and gold at the beginning of the experiment and after 3 and 10 days. Representative spectra of EG-functionalized samples that were exposed to water for 3 days and then to 2× SSPE + 1% Triton-X buffer for an additional 7 days are shown in Figure 6c-e, for diamond, silicon, and gold, respectively. The relative stability of the monolayer on the surface and the relative degradation of the EG molecules during the course of the experiment can be measured. For EG6modified diamond, the peak areas of the C(1s) peaks at 287.3 eV (carbons in ether linkages) and 285.8 eV (carbon atoms in bulk diamond or hydrocarbon-like environments) provide information on the stability of the monolayers. The area ratio, A287.3/A285.8, decreased by only 3% over the course of the 10-day experiment; this is within the error of the experimental measurement, thereby indicating that there is no significant loss of the EG6 monolayer on diamond (Figure 6c). For silicon, that information is obtained from the Si(2p) and C(1s) spectra (Figure 6d). The XPS spectrum of a sample after 3 days in water shows a peak due to silicon oxidation that constitutes 6.6% ( 1.7% of the total Si(2p) area, while at the end of the 10(52) Yang, G.; Amro, N. A.; Starkewolfe, Z. B.; Lui, G. Langmuir 2004, 20, 3995. (53) Boubour, E.; Lennox, R. B. J. Phys. Chem. B 2000, 104, 9004. (54) Granger, M. C.; Witek, M.; Xu, J. S.; Wang, J.; Hupert, M.; Hanks, A.; Koppang, M. D.; Butler, J. E.; Lucazeau, G.; Mermoux, M.; Strojek, J. W.; Swain, G. M. Anal. Chem. 2000, 72, 3793. (55) Kuo, T. C.; McCreery, R. L.; Swain, G. M. Electrochem. Solid State Lett. 1999, 2, 288.

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Figure 6. Stability test of silicon and diamond modified with EG6-ene and Au modified with EG3-SH. Stability was tested by immersion in water for the indicated times, followed by exposure to avidin (panels a and b only). Panels c-e show XPS spectra (dark lines) and fits used for area measurements (light lines) of the samples after immersion in water for 3 days. (a) Amount of avidin adsorbed measured by on-chip fluorescence. (b) Amount of avidin adsorbed after 0, 3, and 10 days, measured by elution. (c) C (1s) from EG6-modified NC diamond after 3 days in water. (d) C (1s) and Si (2p) from EG6-modified silicon after 3 days in water. (e) C (1s) and Au (4f) from EG3-modified gold after 3 days in water.

day experiment the oxidized Si(2p) peak increased to 8.4% ( 1.2%. Also, the C(1s)/Si(2p) area ratio decreased from 2.4 (after 0 and 3 days) to 1.8 (after 10 days). These data indicate that approximately 23% of the monolayer was lost between days 3 and 10, possibly from hydrolysis of the Si-C bond, which resulted in an increase in the amount of oxidized silicon peak observed. In the case of EG3 on gold, the XPS results were similar to those for silicon. The C(1s) to Au(4f) ratio after 0 and 3 days was 3.2 (Figure 6e), but after 10 days, the ratio decreased to 2.6, suggesting that approximately 20% of the monolayer was removed from the surface. For both silicon and gold surfaces, the C(1s) area ratio, A287.3/A285.8, remained constant over the course of the experiment even though the total amount

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of carbon on the surfaces decreased. This result shows that changes observed on silicon and gold arise from the entire EG molecule desorbing from the surface and not from degradation of the EG molecule itself. Overall, our experiments show that silicon, diamond, and gold are all stable for 3 days in water at room temperature, but after 10 days silicon and gold have each lost approximately 20% of the EG monolayer, while diamond has lost an insignificant amount (3% based on peak XPS areas, but not different from zero within the error of the area measurement). The EG molecules remaining on the surfaces have not degraded during this time course. The correlation between XPS and elution measurements suggests that the ability to resist nonspecific adsorption is directly tied to the chemical stability of the interface. VI. Optimization for Biosensing. The above results show that EG6 is very effective at reducing nonspecific adsorption. However, for biosensing applications one desires a monolayer that not only resists nonspecific adsorption but also contains embedded probe molecules to specifically recognize target molecules in solution. Two questions in the design of this type of biosensor are as follows: (1) What is the optimum density of probe molecule on the surface that gives the highest ratio of specifically captured target to nonspecifically adsorbed target molecule? (2) Is it possible to detect a given target molecule within a solution that contains many different types of molecules? These questions were addressed by use of mixed monolayers of EG6 and biotin, the model probe molecule, on silicon and exposing the surface to avidin, the model target molecule. Chicken serum was used as a model complex protein solution. The optimum density of probe molecules was explored by forming mixed amino- and EG6-terminated monolayers on silicon. To evaluate specific binding and nonspecific adsorption in a single experiment, we functionalized the entire surface with a mixture of EG6-ene and Boc-N-ene that was subsequently deprotected to produce a mixed monolayer consisting of amino groups separated by EG6 molecules. By use of a microfluidic circuit, the terminal amino groups in some locations were then reacted with a biotin linker, while the monolayer on the rest of the surface was left alone. This process produces a mixed monolayer that is composed of molecules that resist nonspecific adsorption (EG6) with a controlled number of embedded biotin molecules that act as sites for specific binding of avidin, as shown in Figure 7a,b. Surfaces functionalized with varying densities of biotin were then exposed to a 20 µg/mL fluorescein-avidin solution, and the adsorption of avidin was then characterized by onchip fluorescence imaging; the intensity of fluorescence in the biotinylated regions was attributed to specific binding, while that in the nonbiotinylated region was attributed to nonspecific adsorption. The overall quality of the surface can be parametrized by the ratio of specifically bound avidin to nonspecifically adsorbed avidin, which we define as the S/NS ratio. When no EG6 was present in the monolayer, the fluorescence intensity was high on the regions that were biotin-modified, but the S/NS ratio in Figure 7a was low. However, in Figure 7b, the percentage of EG6-ene in the parent solution was increased to 90% (10% Boc-N-ene), which improved the contrast of the fluorescence image dramatically. The graph in Figure 7c shows the substantial increase in the S/NS ratio by incorporation of EG units into monolayers. In the case of the EG6 monolayer, the optimum parent solution composition (90% EG6 and 10% amino) resulted in a factor of 19 improvement over the

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Figure 7. Schematic of avidin specifically binding to biotinylated silicon and nonspecifically adsorbing onto (a) 100% amino-terminated and (b) 90% EG6/10% amino-terminated monolayer on silicon. (c) Optimization of specific (S) and nonspecific (NS) binding of avidin to silicon covalently modified from mixed solutions of EG6-ene/Boc-N-ene ([) and EG3-ene/ Boc-N-ene (9). The data points presented are for 1%, 10% 25%, 50%, 75%, and 100% Boc-N-ene. ‡Data from panel a; *data from panel b. (d) Comparison of mixed biotinylated/EG6 monolayers and 100% biotinylated monolayers on silicon for their ability to detect fluorescein-labeled avidin in undiluted chicken serum.

100% amino monolayer (22.8/1.21). A maximum occurred at 10% amino/90% EG6-ene because the intensity of the specifically bound avidin was almost equal to the intensity on a 100% amino surface (controlled by steric effects from adjacent avidin molecules), and most importantly, the amount of nonspecifically adsorbed avidin was dramatically reduced. These results as well as previously published results on EG3-modified silicon are presented in the graph in Figure 7c. The maximum S/NS ratio when EG3 monolayers were used was 9.10, but use of EG6 instead of EG3 in the monolayer increased the S/NS ratio by a factor of 2. It should be noted that the x-axis in Figure 7c is the percent amino that existed in the parent solution, not the percent amino that actually attached to the surface, and as discussed in part IV, these values can vary significantly. XPS characterization of EG3 mixed monolayers showed that 70% or more EG3-ene in the parent solution resulted in the same percentage of EG3 on the surface. However,


in the case of EG6 monolayers, this rule does not hold. A mixed monolayer made from a parent solution of 90% EG6ene and 10% Boc-N-ene resulted in a surface composition of 69% EG6 and 31% amino by XPS (data not shown), the same optimum surface composition found when EG3 was used. From this we conclude that functionalized surfaces composed of approximately 70% EG(3 or 6) and 30% amino resulted in a maximum S/NS ratio of specifically bound to nonspecifically adsorbed avidin. Since biosensing assays typically involve detection of one component within complex mixtures of many components, the selectivity of functionalized silicon surfaces was tested by exposing both biotinylated monolayers and biotin embedded within EG6 monolayers to chicken serum, a complex mixture of proteins, to which fluorescein-avidin was added. Biotin-modified silicon surfaces were prepared from 100% Boc-N-ene (Figure 7a) and from 90% EG6ene/10% Boc-N-ene (Figure 7b), which were then biotinylated with an amine-reactive biotin linker. Chicken serum was spiked with fluorescein-labeled avidin to make serum solutions having avidin concentrations between 20 and 0.2 µg/mL. The biotin-modified silicon samples were then immersed in the avidin/serum solutions for 1 h. The fluorescence intensity was measured in two places on each sample: on the biotinylated stripe (which specifically bound avidin) and on the surrounding area (to which avidin nonspecifically adsorbed). Because the composition of the monolayer was constant for each data set, the nonspecifically adsorbed fluorescein-avidin (NS) was subtracted from the specifically bound fluorescein-avidin (S) and the data were plotted as shown in Figure 7d. The fluorescence intensity of the biotinylated silicon surfaces that had been functionalized with 90% EG6-ene/10% BocN-ene was almost twice as high as that of the biotinylated 100% Boc-N-ene surfaces. This difference indicates that significantly more avidin was able to bind to biotin molecules immobilized on EG6 regions than on the amino regions. We attribute the difference in the intensities of the two types of functionalized surfaces to the nonspecific adsorption of serum proteins, which block fluoresceinavidin from binding biotin on the biotinylated 100% amino surface more than on the biotinylated 10% amino/90% EG6 surface. The detection limit of this assay was approximately 3 nM avidin, which is likely limited by mass transport phenomena.56,57 These results demonstrate that EG-containing monolayers may be used to improve two parameters in biosensors. First, the S/NS ratio may be increased by reducing nonspecific adsorption, and second, the selectivity of monolayers containing EG6 can be enhanced to bind a specific protein while resisting the nonspecific adsorption of others, although the detection limit is not controlled by nonspecific protein adsorption. Discussion While there are many studies examining how surface chemical modification affects protein adsorption, the influence of the underlying substrate has been less frequently investigated. The elution methods employed here provide important new insights on metallic and semiconducting surfaces and can even be employed on rough or nonreflective surfaces to which many alternative detection methods cannot be applied. Perhaps the most important observation is that our elution data show that, upon modification with the EG functionality, the surfaces (56) Skaife, J. J.; Abbott, N. L. Langmuir 2000, 16, 3529. (57) Haake, H.-M.; Schutz, A.; Gauglitz, G. Fresenius’ J. Anal. Chem. 2000, 366, 576.

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of silicon, diamond, and gold are all comparably effective at resisting nonspecific avidin adsorption. In fact, our results show that EG3-modified silicon is slightly more effective than an EG3-thiol SAM on gold is at resisting avidin adsorption. The observed similarity between these three surfaces is important in the context of previous experimental and theoretical studies. These studies agree that the EG functionality reduces nonspecific adsorption of most proteins; however, the physical processes that may be responsible for inhibiting nonspecific adsorption, especially the possible role of structural order within the monolayers, are still not well understood.8,18,58-62 Nearly all the previous data for surfaces modified with short EGX oligomers (X ) 1-7) have been performed on SAMs on silver and gold, linking EG alkanethiols to the surface by Ag-S or Au-S bonds.8,11,17 These previous studies on gold have reported that the EG chains of shorter oligomers such as EG3 are disordered, while longer oligomers such as EG6 may adopt a helical configuration.11,41,63,64 Some of these studies have proposed that hydration of the EG chains is crucial and have therefore proposed that closely spaced, crystallinelike monolayers may be less resistant to nonspecific adsorption than similar layers with structural or chemical disorder that facilitates hydration of the EG groups.11,16,19,41,64,65 I. Comparison of Silicon, Diamond, and Gold. Adsorption at surfaces is a balance between attractive forces and repulsive forces. The van der Waals force is usually parametrized in terms of the Hamaker constant A, reflecting the energy associated with two materials through an intervening medium.66 Hamaker constants of approximately 250 × 10-21 J for Au,67 90 × 10-21 J for silicon,66,68 and 135 × 10-21 J for diamond surfaces66,68 interacting through water have been reported previously. While Hamaker constants depend on the properties of both materials interacting, these data suggest that the van der Waals forces attracting proteins to the surfaces will be largest on gold, next largest on diamond, and smallest on silicon. However, all three materials have Hamaker constants of similar magnitude, suggesting that other factors may also be equally important. A second factor that can potentially contribute to protein adsorption at surfaces of conductive materials, such as gold, is the image charge that is induced in the metal due to the charge on the protein molecules.69 In the image effect, the charge on a molecule near a surface induces an image charge in the underlying metal, leading to a net attractive interaction. Image charge effects would lead to increased adsorption at metallic surfaces compared with semiconducting or insulating materials. Finally, the local rough(58) Yuana, Y.; Oberholzer, M. R.; Lenhoff, A. M. Colloids Surf., A: Physicochem. Eng. Aspects 2000, 165, 125. (59) Halperin, A.; Leckband, D. E. C. R. Acad. Sci. Ser. IV, Phys. 2000, 1, 1171. (60) Mrksich, M. Chem. Soc. Rev. 2000, 29, 267. (61) Satulovsky, J.; Carignano, M. A.; Szleifer, I. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9037. (62) Szleifer, I. Physica A 1997, 244, 370. (63) Wang, R. L. C.; Kreuzer, H. J.; Grunze, M. J. Phys. Chem. B. 1997, 101, 9767. (64) Zwahlen, M.; Herrwerth, S.; Eck, W.; Grunze, M.; Hahner, G. Langmuir 2003, 19, 9305. (65) Schwendel, D.; Dahint, R.; Herrwerth, S.; Schloerholz, M.; Eck, W.; Grunze, M. Langmuir 2001, 17, 5717. (66) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: London, 1992. (67) Biggs, S.; Mulvaney, P. J. Chem. Phys. 1994, 100, 8501. (68) Fernandez-Varea, J. M.; Garcia-Molina, R. J. Colloid Interface. Sci. 2000, 231, 394. (69) Joanny, J.-F.; Castelnovo, M.; Netz, R. J. Phys.: Condens. Matter 2000, 12, A1.

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ness of the surfaces can contribute to protein adsorption.70 The issue of roughness is complicated by the fact that roughness occurs on many length scales. Overall, however, silicon is the smoothest surface in these studies. While it is not possible to identify the specific causes for the relatively small differences in avidin adsorption between the three materials investigated, it is important that the functionalized diamond and silicon surfaces performed at least as well as gold in their ability to resist nonspecific avidin adsorption. II. Influence of the Monolayer. Our monolayers on silicon and diamond are fundamentally distinct from those on gold and silver because on silicon and diamond the functionalization with EG occurs by formation of covalent bonds from the substrate (Si or C) to the terminal vinyl group of the molecules. This difference is significant because whereas conventional SAMs on gold and silver can optimize alkyl chain packing by lateral diffusion, the covalent bonds of molecules to Si or diamond prevent any lateral movement of the molecules. Although we have only limited information about the structure of our monolayers, the rigid bonding to the underlying silicon or diamond lattice is expected to constrain the layers and prevent optimal packing of the alkyl chains, thereby leading to monolayers that are less ordered than SAMs on gold or silver. Additionally, our XPS characterization shows that while the primary reaction route with the silicon surface is through the terminal vinyl group of EG3-ene, two side reactions occur through the hydroxyl and possibly the ether linkages. These side reactions contribute to additional disorder in the monolayers. Consequently, we expect them to be significantly less ordered than those of alkanethiols on Ag or Au. Experimentally, we find that EG3-functionalized diamond, silicon, and gold all resist protein adsorption with comparable effectiveness. Our results clearly demonstrate that EG monolayers lacking crystalline order are highly effective at resisting nonspecific adsorption. For oligomers that are short compared with the size of the proteins, it is reasonable to expect that the very terminus of the molecule will have an especially large influence on the adsorption to that surface. Indeed, our results (Figure 3) show that hydroxyl-terminated and methyl-terminated surfaces show a rather significant difference in their ability to resist nonspecific adsorption of avidin, BSA, and casein. This result suggests that local chemical interactions between the distal end of the molecules and the proteins are very important in controlling nonspecific adsorption. Yet we also find that lengthening the chain from EG3 to EG6 yields substantial additional improvement in the ability to resist nonspecific adsorption, in agreement with previous studies of gold SAMs.18,71 Taken together, the comparison of EG3, EG6, and EG3-Me layers shows that even for short oligomers that are densely packed (that is, the average molecular spacing is small compared to the dimensions of the proteins involved) and where the proteins might be expected to interact only with the terminus of the molecular layer, the ability to resist protein adsorption is clearly affected by a more extended region of the molecule including the terminal group (-OH or -OCH3) and the EG groups. While further studies of the detailed structures of the monolayers are needed to more fully understand these factors, our results indicate that factors such as hydration of the EG (70) Wisniewski, N.; Reichert, W. M. Colloids Surf., B: Biointerfaces 2000, 18, 197. (71) Zhu, B.; Eurell, T.; Gunawan, R.; Leckband, D. J. Biomed. Mater. Res. 2001, 56, 406.

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monolayers and differences in conformational flexibility in these structures also likely play an important role. Our data show that casein, BSA, and avidin adsorb to silicon surfaces terminated with hydrophobic (i.e, Me-EG3 groups) more than hydrophilic groups (i.e., EG3 groups), in agreement with a general trend identified in many previous studies. Fibrinogen, however, exhibits anomalous behavior, adsorbing in comparable amounts to both types of surfaces. Previous studies have observed a similar effect and have attributed it to the existence of both hydrophobic and hydrophobic domains within fibrinogen, which allow it to interact with both types of surfaces.65,72,73 The unique elongated structure of fibrinogen74 likely contributes to orientation-dependent changes in fibrinogen packing, as these physical packing forces may dominate the adsorption dynamics, thereby weakening the effect of surface termination.48 It has been proposed that methyl-terminated EG monolayers should be more useful than hydroxylterminated monolayers for many in vivo applications because the methyl group cannot be oxidized.14,75 Our results suggest that although hydroxyl groups may be oxidized, they are more effective than terminal methyl groups at resisting protein adsorption. Recent studies have also shown that monolayers terminated with mannitol are resistant to protein adsorption and cell attachment;9 this suggests that the hydroxyl groups may have a direct, beneficial influence on the ability to resist nonspecific adsorption. III. Impact on Technological Applications. The ability to resist nonspecific adsorption on silicon and diamond surfaces is significant because of their technological applications. The semiconducting properties of silicon and diamond can be used to achieve new types of detection via electrical impedance methods22,23,76 and through the fabrication of biologically sensitive field-effect transistors (bio-FETs).4,20,21 The ability to make proteinresistant surfaces by incorporating very short EG oligomers is important for these devices to achieve high sensitivity because in ionic solutions the changes in electrostatic potential are screened over short distances on the order of ∼1 nm, thereby reducing the sensitivity of the detection to targets within ∼1 nm of the semiconductor interface. A second reason for investigating silicon and, especially, diamond is because early attempts to integrate biological systems with microelectronics yielded unstable interfaces due to hydrolysis and/or oxidation. Recent advances in the ability to functionalize silicon via direct Si-C bond formation (without intervening oxides)32,77 and to functionalize diamond via direct C-C bond formation24,31 may provide ways to overcome the earlier limitations. Our XPS data show that functionalized silicon surfaces are approximately as stable as SAMs on gold, while EG-modified diamond surfaces do not appear to show any measurable degradation over the time period of the investigation, in agreement with previous studies.24 These results suggest that the functionalization strategies outlined here for resisting nonspecific adsorption show promise for the use of these materials in complex media (72) Cook, B. C.; Retzinger, G. S. J. Colloid Interface Sci. 1992, 153, 1. (73) Kim, J.; Somorjai, G. A. J. Am. Chem. Soc. 2003, 125, 3150. (74) Fuss, C.; Palmaz, J. C.; Sprague, E. A. J. Vasc. Intervention Radiol. 2001, 12, 677. (75) Faucheux, N.; Schweiss, R.; Lutzow, K.; Werner, C.; Groth, T. Biomaterials 2004, 25, 2721. (76) Kharitonov, A. B.; Wasserman, J.; Katz, E.; Willner, I. J. Phys. Chem. B 2001, 105, 4205. (77) Barrelet, C. J.; Robinson, D. B.; Cheng, J.; Hunt, T. P.; Quate, C. F.; Chidsey, C. E. D. Langmuir 2001, 17, 3460.


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(such as serum) and in applications such as environmental sensing, where stability becomes a particularly important issue. The very high stability of diamond under a wide range of conditions also suggests that diamond thin films deposited on a variety of media may be attractive candidates for use in biomedical applications, where resistance to protein adsorption could be conferred by short EG oligomers (as investigated here) or by longer poly(ethylene glycol) chains. The ability to quantitatively measure surface concentrations by on-chip fluorescence methods is confounded by quenching between the fluorophore and substrate and by self-quenching due to lateral interactions between nearby fluorophores. Furthermore, there is also strong evidence that proteins can adsorb in different orientations, and/or reorient at different surface coverages, and/or change their conformation while adsorbed, all of which can affect fluorescence. Thus, while on-chip fluorescence measurements are very useful for identifying qualitative trends, their use in quantitative studies is more limited.37,38 The use of elution methods provides more quantitative data that also permits a direct comparison of different substrate materials (e.g., gold, diamond, and silicon). The considerable improvement in reducing nonspecific adsorption of proteins to silicon and diamond surfaces suggests potential applications for these functionalized substrates in the design of biosensors. Our data show that substrates that contain approximately 30% amino groups (which may be utilized to incorporate biomolecules into the monolayer) and 70% EG(3 or 6) results in an optimum specific/nonspecific avidin ratio. Similar to previously published results on mixed monolayers of organothiols supported on gold substrates,78 this result demonstrates that mixed monolayers on silicon and diamond substrates are useful for biosensing applications, as they can both detect a specific protein and resist the adsorption of others. Finally, the use of these modified surfaces permits the specific capture and detection of a protein analyte from a complex protein mixture (chicken serum). These surfaces could conceivably be used in direct screening of biological samples for the presence of bloodborne pathogens for medical diagnostics.

minimize the amount of adsorbed avidin to silicon and diamond surfaces and that longer EG chains are more effective than shorter EG3 molecules. Elution combined with fluorometry provides a highly sensitive, quantitative way to measure the amount of nonspecific adsorption that avoids the problems associated with surface quenching and that facilitates comparison of different surfaces. Our measurements show that EG3 monolayers are comparably effective at reducing nonspecific adsorption on singlecrystal silicon, nanocrystalline diamond, and gold surfaces, with EG6-functionalized single-crystal silicon yielding the lowest amount of nonspecific adsorption of all three surfaces studied. XPS characterization shows that the monolayers on silicon are bound primarily through the vinyl group, but with some chemical and structural disorder. Thus, we conclude that structural perfection of the monolayer is not necessary in order to resist nonspecific adsorption, and indeed, some disorder may even be beneficial. We find that hydroxyl-terminated EG3 monolayers are more effective than methyl-terminated EG3 monolayers (i.e., Me-EG3-ene) on silicon for three of the four proteins investigated, which implies that applications requiring the greatest immunity to nonspecific adsorption will likely benefit most by use of hydroxyl-terminated EG3 layers. The incorporation of amino groups into the EG6 monolayers provides a way to link particular biomolecules of interest to the surface. Mixed monolayers with optimized compositions can maximize the ratio of specific binding to nonspecific adsorption. Our results showing that avidin is able to specifically recognize and bind to surfaceimmobilized biotin, even when in chicken serum, demonstrate that these optimized monolayers greatly enhance the ability to detect one specific protein in a complex protein mixture. Overall, these results show that covalent functionalization of silicon and diamond with EG monolayers leads to dramatic improvements in the ability to control the specific binding and nonspecific adsorption of proteins. This, in turn, indicates that EG-functionalized silicon and diamond may be promising substrates for many applications, including biosensing and medical implants.

Conclusions We have demonstrated that monolayers of oligo(ethylene glycol) molecules such as EG6 can be used to

Acknowledgment. We thank Judith N. Burstyn, James E. Butler, John N. Russell, Jr., and Lloyd M. Smith. This work was supported by the National Science Foundation, CHE-0314618 and DMR-0079983, and by Smiths Detection.

(78) Knoll, W.; Zizlsperger, M.; Liebermann, T.; Arnold, S.; Badia, A.; Liley, M.; Piscevic, D.; Schmitt, F.-J.; Spinke, J. Colloids Surf., A: Physicochem. Eng. Aspects 2000, 161, 115.

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Functional Monolayers for Improved Resistance to Protein Adsorption

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