Langmuir 1993,9, 3009-3014
3009
Electrophilic Siloxane-Based Self -Assembled Monolayers for Thiol-Mediated Anchoring of Peptides and Proteins Yong Woo Lee,t Joseph Reed-Mundell,? Chaim N. Sukenik,*pt and James E. Zull'J Departments of Chemistry and Biology, Case Western Reserve University, Cleveland, Ohio 44106 Received May 3,1993. In Final Form: August 2 , 1 9 9 P
The synthesis and characterization of long-chain alkyltrichlorosilanes of alkyl halides, benzyl halides, and a-haloacetylsdesigned to form siloxane-anchored self-assembled monolayers (SAMs)for the selective attachment of peptides (via cysteine thiols) is described. Thin film formation by the trichlorosilanes was demonstrated by spectroscopicmeans and by surface wetting properties. Halide exchange could be utilized to produce the more reactive (iodide)surfaces in situ, following their deposition in a more stable (chloride or bromide) form. In solution,these functionalgroups were found to have a range of reactivitywith model thiols which extended from half-lives of minutes to days (essentiallyno reactivity). The order of reactivity is I > Br > C1 within each class of compounds, and a-haloacetyl > benzyl >> alkyl. The reactivity of the S A M s with thiols showed the same order of reactivity. The very reactive a-iodoacetylwas also reactive with amines, but competition experiments demonstrated preference for the thiol under our reaction conditions. S A M reactivity with cysteine-containing peptides was demonstrated with a tripeptide (glutathione)and a nonapeptide (lamininfragment). Both peptides show maximum attachment after 2-3 h of exposure to millimolar concentrations. The attachment was completely blocked by prior treatment of these peptides with dinitrophenylmaleimide or by air oxidation of the thiol. Given that these peptides contain all the nucleophilic side chains found in proteins (thiol,alcohol, phenol, carboxyl, and amine),the selective blocking experiments indicate that these SAMswill be useful for the directed attachment through cysteine side chains in proteins and peptides.
Introduction
carboxyl (Asp, Glu) groups. Indeed, some proteins (e.g. the FAB' fragment of IgGs) contain only a single free thiol. Attachment of proteins and peptides to inorganic Stable and uniform, chemically modifiable surfaces can surfaces is important in the biosensor and biocompatibility be obtained with self-assembled monolayer (SAM)techfields. However, although there are extensive studies on niques. SAMs formed by long-chain alkanethiola on gold protein adsorption a t surfaces,l controlled methods for have been widely studied.6 Mixed thiol f i s have been chemical attachment have been poorly developed. For formed and appear to be quite stable, although detachment attachment to glass, silicon, or metal oxides for biosensor of some of the thiol appears to occur under some applications, short-chain amino silanes have been used, circumstances.6 Such films have been used to develop with subsequent linkage of the protein to the amino-rich specificprotein binding surfaces, utilizing the noncovalent surface by cross-linking through pendant amino2 or biotin-avidin interaction.' However, the thiol-gold linkcarboxyl3 side chains. However, the short chain silane age is too labile to allow extensive manipulation of the linkage is susceptible to hydrolysis and instability of chemical functionality on the SAM surface. protein layers attached by this method has been r e p ~ r t e d . ~ SAMs can also be produced on silicon and glass by Furthermore, the attachment of proteins through amino alkyltrichlorosilanes.8These siloxane-anchored filmsform or carboxyl groups can lead to random linkage through very stable and uniform surfaces which can often be many different attachment points, since proteins are rich modified in situ without apparent damage to the monoin such functionality. Moreover, since the cross-linkers layer.B Thus, there is the potential for developing SAMs cannot distinguish a surface amino group from one on reactive with protein side chains which may provide another protein molecule, cross-linking and oligomerizaopportunities for controlling protein attachment, distrition of the protein molecules may also occur. bution, and orientation on silicon, glass, or metal-oxide surfaces. The creation of more stable surfaces which react with We have previously reportedDthe successful manipuother, less abundant, side chains in peptides and proteins lation of the composition of the surface of covalently-bound is thus of interest. An apparent candidate for such a less siloxane-based monolayer films through the reaction of abundant side chain is the nucleophilic thiol group of the an alkyl bromide functional group with various anionic amino acid cysteine (Cys). This residue is highly reactive nucleophiles. While this approach allowed the incorpoand thus offers the possibility for chemical attachment to ration of a variety of nitrogen- and sulfur-containing electrophilic surfaces, and it also occurs significantly less functional groups, the alkyl bromide is insufficiently often than the residues which contain amino (Lys) and + Department of Chemistry.
of Biology. Abstract published in Advance ACS Abstracts, October 1,1993. (1) (a) Andrade, J. D.; Hlady, V. Adu. Polym. Sci. 1986,79. (b) Brash, J. L.; Horbett, T. A. Proteins at Interfaces; ACS Symposium Series 343; American Chemical Society: Washington, DC, 1987. (2) Bataillard,P.; Gardies, F.;Jaffrezic-Renault, N.; Martelet, C. Anal. Chem. 1988,60, 2374. (3) Gebbert, A.; Alverez-Icaza, M.; Stochlein, W.; Schmid, R. D. Anal. Chem. 1992,64, 997. (4) Gardies, F.; Martelet, C. Sensors Actuators 1989, 17, 461. 1 Department
(5) (a) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whiteaiden, G. M.; Nuzzo, R. G. J. Am. Chem. SOC.1989,111,321. (b) Evans, S. D.; Ulman,A. Chem. Phys. Lett. 1990, 170,462. (c) Chidsay, C. E. D.; Loiacono, D. N. Langmuir 1990,6,682. (6) Chidsey, C. E. D.; Bertozzi, C. R.; Putvinski, T. M.; Mujsce, A. M. J. Am. Chem. SOC.1990,112,4301. (7) Haussling, L.; Michel, B.; Ringsdorf, H.; Rohrer, H. Angew. Chem., Znt. Ed. Engl. 1991,30, 569. (8) Netzer, L.; Sagiv, J. J. Am. Chem. SOC.1983, 105,674. (9) Balachander, N.; Sukenik, C. N. Langmuir 1990,6,1621. (10)Bright, F. V.; Betta, T. A.; Litwiler, K. S. Anal. Chem. lSsO,62, 1065.
0743-7463/93/2409-3009$04.00/00 1993 American Chemical Society
Lee et al.
3010 Langmuir, Vol. 9, No. 11,1993
lb X s I
2b X = l
3b X e l
4b X - I
Figure 1. Long chain alkenes and trichlorosilanes. dicyclohexylsolvent (5mL) in a glovebox under inert atmosphere and transferred to the bench top. SAM surfaces were prepared by immersion of the substrate (using Teflon-coated tweezers) into thissolution in a 10-mLbeaker with a magnetic stir bar. The substrate is quickly withdrawn from the solution after 30 min, washed with CHCl3 and water, and finally cleaned in hot CHC4 in a Soxhlet extractor for 10 min. c. Contact Angle Measurements. Contact angles were measured by using a h e - H a r t Model 100 contact angle goniometer equipped with a controlled environment chamber. Advancing contact angles were determined by depositing a drop of HzO from a syringe, advancing the periphery of the drop by adding more liquid, withdrawing the syringe, and measuring the advancing contact angle within 30 s of application of the drop. Receding contact angles were measured by withdrawing part of the liquid from the drop and measuring the angle. The temperature of the measurements waa 22 f 2 OC. Reported values are averagesof four to six measurements taken at different points on the surface and have error ranges of f 2 deg. Experimental Section d. X-ray Photoelectron Spectroscopy. XPS measurements A. General Procedures. NMR spectra are reported in 6 were carried out on a Perkin-Elmer ESCA 5400. Analyses were units and were recorded on a Varian Gemini 300 spectrometer done by using Mg Ka lines at a pressure of 10-8 Torr with a in CDC4 solvent. 1H-NMR spectra (300 MHz) are referenced takeoff angle of 4 5 O . The Multiplex acquisitions were performed to CHCl3 at 7.24 ppm and 13C-NMR spectra (75 MHz) are in the utility mode (pass energies 35-70 eV, maximum power 400 referenced to the center of the CDCl, solvent triplet at 77.0 ppm. W [Mg anode]), with acqusition times of 30 min. Peak positions Solution infrared spectra were recorded on a Perkin-Elmer 1600 were assigned by referencing the polymethylene C 1s peak at Series FTIR and high-resolution mass spectra were recorded on 284.7 eV and have error ranges of f0.2 eV. a Kratos MS 25RFA spectrometer. UV measurements were done e. In Situ Transformationsof Monolayer Functionality. on a Gilford 240 spectrometer. HPLC used a Waters 590 pump, Transformations were carried out by dipping the SAM-coated a Rheodyne 7125 injector, Dynamax-GOA (Si 83-12143 semisubstrate (glass slides or ATR crystals) in the reagent solution prep column, Dynamax-GOA (C18 83-2014) analytical column under the indicated reaction conditions and then rapidly (unless otherwise indicated), and a Waters 401 differential withdrawing it with Teflon-coated tweezers. Unless stated refractometer. TLC was done on aluminum-backed 0.2-mm otherwise, all monolayer-coated substrates were cleaned after 60F254 plates from EM Science and used phosphomolybdic acid they were withdrawn from the reaction medium by washing with for visualization. Column chromatography (flash) was done with acetone and chloroform before a final cleaning with hot CHCb silica gel (Aldrich, 230-400 mesh). Dry THF and ether were in a Soxhlet extractor. Characterization of the modified f i e distilled from Na. Hexane for flash chromatography was distilled involved contact angle measurements, IR, and XPS. before use. HPLC grade hexane was used as received. HSiC4 In situ exchange of Br (Cl) for I was achieved as follows. In (Petrarch) was distilled from quinoline. Dicyclohexyl (Aldrich, a dry 100-mL Erlenmeyer flask containing a small magnetic vacuum distilled) was passed through B r o c k ” Activity I stirring bar was placed a solution (1M) of NaI (3g) in dry acetone alumina (3% water by weight, 80-200 mesh, Fisher). DMF (20 mL). The S A M coated substrates were placed in the flask (Fisher) was purified by MgSOd drying and vacuum distillation. and the solution was stirred at room temperature for 4 h. The Acetone (Fisher) was purified by K2C03 drying and distillation. substrate was removed from the flask and cleaned with dry Water was doubly distilled. CHCl3 (Fisher, HPLC), p-niacetone and CHCl,. trothiophenol (Aldrich), a,a’-dibromo-p-xylene (Aldrich), l-deIn situ reactions of SAMs with neutral nucleophileswere done canethiol (Aldrich), glutathione (Sigma, 9&100% ), and the Laminin peptide fragment (Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser- as follows: Solutions of decanethiol (DT), n-decylamine, p-nitrothiophenol (PNTP), glutathione, and the Laminin fragment Arg; purity 97%; peptide content 81%;Sigma) were used as peptide were made under dry nitrogen in a glovebag, using freshly received. distilled, deaerated, DMFas solvent (0.01M). Freshly derivatized B. Syntheses. The syntheses and characterizations of the glass slides were placed in individual oven-dried polyethylehe long chain olefins and alkyltrichlorosilanes (1-6) are available as 3-dL vials. Maintaining the inert atmosphere, a 1-mL portion Supplementary Material. of one of the above solutions was transferred to each vial. After C. Surface Preparation a n d Characterization. a. Prepthe specified reaction time, the slides were rinsed 5 times on each aration of Solid Substrates for Monolayer Coating. Glass side with 1-mL portions of DMF followedby similar rinsing with slides were obtained from Dynalab Corp. (10 X 10) and were chloroform. Some samples were also rinsed in refluxing chlocleaned by washing with doubly-distilled water followed by roform with a Soxhlet extractor. They were stored in a desiccator cleaning with hot CHCl3 in a Soxhlet extractor for 30 min. All prior to XPS analysis. substrates were plasma-cleaned for about 30 min in radioThe specific procedure for amine/thiol competition experifrequency argon plasma (Harrick PDC-3xG Plasma Cleaner), ments was as follows: the glass slides, freshly derivatized with stored in fluorocarbon containers (Fluoroware, Inc.), and used an iodoacetyl SAM, were treated overnight with either decylwithin 12 h. amine, DT, or an equimolar mixture of decylamine/DT. The b. Preparation and Use of Coating Solutions. All alkylslides were rinsed with DMF and CHZC12 and stored for trichlorosilanes used in this work were prepared as 5 X 103 M characterization. solutions in dicyclohexyl. The silane (100 pL) was added to the
electrophilic to react with the nucleophiles found in the side chains of proteins (-OH, -NH2, -SH) under the mild conditions generallyrequired to prevent their denaturation or breakdown. We have now synthesized and studied the chemistry of a series of electrophilic long-chain alkenes bearing alkyl halide (l),haloacetyl(2),or benzyl halide (3)groups (Figure 1). These materials have been used to prepare the corresponding alkyltrichlorosilanes (4-6, Figure 11,which form SAMs on both silicon and glass. The relative reactivities of the various electrophilic moieties in these compounds have been determined, both in solution and on surfaces. Finally, the attachment of a tripeptide and a nonapeptide to the surfaces has been demonstrated and shown to occur through the thiol side chain of the Cys residue in the peptides.
Selective Attachment of Monolayers to SAMs The surface reaction kinetics with glutathione were done as follows. Eight iodoacetyl-bearing slides were reacted with glutathione in DMF. The sampleswere removed at the following times (h): 0.5, 1, 1.5, 2, 3, 4, and 5. D. Solution Kinetics. Stock solutions (0.1 M) of lb, 2b, 3b, and PNTP were prepared in dry DMF. After 100 pL of electrophile solution was placed in a small vial with a magnetic stirring bar, 130pL of PNTP solution was added to the reaction vessel at room temperature and reactions were monitored by injecting 100pL of reaction mixture from the vessel directly into the HPLC column (eluent acetonitrile; reverse phase analysis column). Time points between 1 and 30 min were taken. Appearance and disappearance of starting materials and their products (CI~H~I-OCOCH~S-P~-NOZ, ClZHB-Ph-CHrS-PhNOz, and C16H31-S-Ph-NOz) were monitored. At a flow rate of 0.5 mL/min, retention times (min) were as follows: 2b, 23.6; 3b, 27; CisHsi-OCOCHrS-Ph-NOz, 18.4; CizHz3-Ph-CHz-S-PhNOz, 23. A t a flow rate of 1mL/min, retention times (min)were as follows: lb, 27.2; C16H3l-S-Ph-N0~,19.4. The three products were prepared and characterized separately for use as HPLC standards. Reactions between PNTP and lb, 2b, and 3b were each done by stirring solutions (in DMF) of the reactants, under Nz,at room temperature in the presence of triethylamine. The products were separated by flash chromatography followed by HPLC (acetonitrile, reverse phase, semiprep column, 20 mL/ min). Spectroscopic characterization of these products is described in the Supplementary Material. E. Thiol Blocking. Glutathione and the Laminin fragment peptide were treated with (dinitropheny1)maleimide(1000-fold excess)in DMF at room temperature, overnight. The glutathione thiol was also blocked by oxidation by allowing the glutathione solution to remain exposed to the air for 1week. In both cases, thiol blockage was established by testing the solutions for free thiol with Ellman's reagent." A solution (200 pL, 0.1 mM) of 5,5-dithiobis(2-nitrobenzoicacid) in 0.1 M Tris buffer, pH 8, was mixed with an equal volume of thiol/DMF solution. Absorption at 430 nm measuresthe amount of free thiol in solution. Peptides with blocked thiols were then used for surfacereactions as above.
Results The syntheses of compounds 1-3 were all straightforward. Compound la9is the precursor to lb (NaI/acetone) and to 2a (conversion of the Br to OH via the acetate, followed by esterification with chloroacetyl chloride) and 2b is derived from 2a NaUacetone. Compound 3a is made by the Grignard coupling of undecenyl bromide with a,a'dibromo-p-xylene, and 3b is obtained by halogen exchange. In principle, hydrosilylation of each of these olefins gives rise to the respective alkyltrichlorosilanes (4-6). Due to the relative instability of the iodide-bearing versions of 5 and 6, insufficient amounts for proper characterization and subsequent use were obtained. Compounds 4-6 were deposited from dilute dicyclohexyl solution as covalentlybound, siloxane-anchored monolayer films. Iodide bearing surfaces were created by direct deposition (4b)or by exchanging bromide or chloride for iodide on an already deposited film. The wetting properties of each S A M were determined. All directly deposited films showed a contact angle hysteresis of 3-4O, and the films created by in situ halide exchange (iodoacetyl and benzyl iodide) showed hysteresis of 5 and 6O, respectively. The advancing contact angles for each SAM were as follows: alkyl bromide, 84; alkyl iodide, 85;chloroacetyl, 69; iodoacetyl, 71; benzyl bromide, 86; benzyl iodide, 88. XPS analysis of each surface showed the expected carbon polymethylene binding energy along with a carbon signal at 293.8-293.9 eV for the halogen-bearing carbon and at 289.0-289.1 for the carbonyl of the acetyl surfaces. Halogen XPS signals for Cl(199 eV), Br (70-70.5 eV), and I (618.9-620.6) were easily detected. (11) Ellman, G.L.Arch. Biochem. Biophys. 1959,82, 70.
Langmuir, Vol. 9,No. 11, 1993 3011 The benzyl iodide and a-iodoacetyl surfactants were found to be quite labile. To retain iodide, precautions with regard to trace water in solvents, atmospheric moisture, and light were required, and generation of the a-hydroxyl form of the ester surfactants (presumably by trace moisture) was apparent by NMR in some experiments. Thus, direct SAM formation with the iodide form of these compounds was not undertaken. Rather, the more stable bromo and chloro compounds were first deposited and iodide was then exchanged onto the surface. XPS experiments indicated that exchange is complete under the conditions described, with bromine and chlorine signals completely replaced with iodine. These results suggested that the desired iodoacetyl functionality is present on the surface (asconfirmed by the selectivereactivity with thiols, shown below) but do not establish that a uniform layer of iodide is present. Indeed, given the lability of the iodoacetyl functionality, such a uniform layer seems unlikely. Nonetheless, the in situ exchange approach was preferable to direct formation of the SAMs with the iodine derivatized surfactants. The relative electrophilicityof our variousfunctionalities as determined by their reactivity with organic thiols was studied both in solution and as SAM films. The classical leaving group reactivity trend of I > Br > C1was observed, and having the iodide version of all three systems (lb,2b, 3b) allowed us to make direct comparisons among structural types. By using two thiols of different nucleophilicity @-nitrothiophenol (PNTP) and decanethiol (DT)), relativereactivities over a wide range could be assessed. Given the goal of protein attachment under mild conditions, all reactions were done in dry, distilled DMF solvent at room temperature. With the focus on relative reactivity, no attempt was made to precisely determine rate constants. In solution, only minimal reaction of compounds la and lb could be detected after days of reaction with either thiol. Relative reactivities were examined by competition experiments and product formation was detected by HPLC. To the extent that it could be assessed by using the more reactive PNTP, reaction with la was slower than with lb,as expected. Similar observations were made for 2a vs 2b and for 3a vs 3b using both PNTP and DT. In comparison of lb vs 2b vs 3b, it was found that the haloacetyl substrate was more reactive than the benzyl which was much more reactive than the alkyl. Finally, meaningful comparisons could be made between 2a,2b, 3a,and 3b. Using both PNTP and DT, a reactivity order of 2b > 2a > 3b > 3a was confirmed. The possibility that amines react with the electrophilic functional groups in solution was also examined. Indeed, in the absence of competing thiol, some reactivity with amines was observed. However, the thiol was highly preferred. In addition, in these experimentsthe possibility for a competing reaction in which amines displace the acetyl functionality to form amides was also examined. This reaction was not observed, as determined by the absence of free alcohol in the reaction mixtures. Having established the relative electrophilicities of the various functional groups in solution, we turned to the chemistry of their SAM analogs. The reactivity of these SAMs with PNTP and DT was examined by XPS. Both reacted readily, but since PNTP was more reactive and since its attachment to the surfaces could be followed by examination of both sulfur and nitrogen (from ita nitro group) signals, the reactions with PNTP are presented here. As illustrated in Figure 2, following 3 h of treatment, both the benzyl iodide and the iodoacetyl surfaces showed clear nitrogen and sulfur signals, with no remaining iodide
Lee et al.
3012 Langmuir, Vol. 9, No. 11,1993 6000
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Figure 2. XPS monitoring of p-nitrothiophenol reaction with three different kinds of SAMs: (top) nitrogen signal for alkyl iodide (l), benzyl iodide (21, and iodoacetyl (3) SAMs treated with the thiol as described in the Experimental Section; (middle) iodide Signal retained on SAMs formed with alkyl iodide (3), iodoacetyl(2),and benzyl iodide (1)compounds; (bottom)sulfur signalwen with iodoacetyl (l), benzyl iodide (2),and alkyl iodide (3) SAMs following treatment with thiol. In all XPS data the
Y axis is relative signal intensity in counts per electrovolta and the X axis is binding energy in electronvolta.
signal. The sulfur and nitrogen signals were stable to extensive washing with organic solvents and water and to refluxing overnight in CHCl3. Thus, the thiol is stably bound to the surface, and the data strongly support the notion that nucleophilic displacement of iodide by thiol has occurred. Consistent with this, under the same reaction conditions, the alkyl iodide surface did not show either nitrogen or sulfur signals and did retain the iodide signal. In general, the relative rates of PNTP attachment to each of our six fundionalized surfaces, as evidenced by rate of growth of both the nitrogen and sulfur XPS signals
Figure 3. Time course for attachment of p-nitrothiophenol to
six S A M s bearing iodoacetyl (01, iodobenzyl (a),bromobenzyl (A), chloroacetyl (A),iodoalkyl ( O ) , and bromoalkyl (m) functionality. Top panel shows sulfur signal and the bottom panel the nitrogen signal. Data are the mean of two experimenta. (Figure 3),mirrors the above described order of reactivity in solution. In further exploring the reactivity of our SAM systems, it was found that the a-iodoacetyl surfaces also react with alkyl amines. However, when competition experiments with decylamine and DT were conducted, the thiol was preferentially bound to the surface. This issue is again addressed in the peptide experiments described below. Initial studies of peptide attachment were conducted with the tripeptide glutathione (a-glutamyl-cysteinylglycine), which contains two carboxyl groups, an amino group, and a thiol. This peptide was found to react with SAM-modified surfaces in a way that was comparable to that of the model thiols described above. Attachment was demonstrated by the parallel appearance of XPS signals for both sulfur and nitrogen and the disappearance of iodide. Figure 4 shows the time course for the reaction of glutathione with an iodoacetyl surface. These results parallel those obtained for reaction with PNTP. Moreover, whereas PNTP attachment to various SAMs left the surface wetting properties minimally changed (advancing water contact angles of 75-78'; receding contact angles of 7&75'), attachment of glutathione, with its polar functionality, created hydrophilic surfaces with advancing water contact angles below 30'. Since the glutathione peptide contains nucleophilic amino and carboxyl residues in addition to the thiol side chain of the Cys, further examination of the role of the Cys residue in peptide attachment was undertaken. First, the peptide was modified with a maleimide commonly used to block thiols in proteins ((dinitrophenyl)maleimide, DNPM) and the disappearance of free thiol was established by using Ellman's reagent. As indicated in Figure 5,
Langmuir, Vol. 9, No. 11, 1993 3013
Selective Attachment of Monolayers to SAMs l4O0
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Figure. 5. XPS nitrogen signalfor iodoacetyl SAM treated with glutathione (1)and with maleimide-blocked glutathione (2).
treatment of the a-iodoacetyl surface with the DNPMblocked peptide, did not generate a nitrogen signal on the surface, suggesting that a free thiol is required for attachment. Although DNPM modification is specific for thiols in aqueous solution under conditions where amino groups are protonated, it was possible that, in DMF, DNPM might also have blocked peptide amino groups. Therefore, the reactivity of air-oxidized peptide was also examined. As shown in Figure 6, using air-oxidized peptide which contained no free thiol, no peptide attachment to the a-iodoacetyl SAM surfaces could be detected. Since air oxidation of amines is very slow under these conditions, this result further implicates the thiol residue as the site of peptide attachment. Additional studies were conducted with a nonapeptide fragment of the protein Laminin (CysAspProGlfl?yrIleGlySerArgNHa). With the exception of the e-amino group of Lys, this peptide contains all the nucleophilic functionality of typical proteins including a-amino, carboxyl, thiol, phenol, and alcohol groups. As indicated in Figure 7,this peptide also reacted readily with the surface. The key diagnostic of peptide attachment is the nitrogen XPS signal. That reaction is through the thiol side chain and is again indicated by the lack of reaction with the DNPMblocked peptide. As with glutathione, the Laminin fragment strongly attached to the surface and the iodide was displaced. With DNPM-blocked peptide, no iodide displacement occurs and no nitrogen XPS signal was detected.
Binding Energy
Figure 6. Sulfur (top) and nitrogen (bottom) XPS signals of iodoacetyl SAMs treated with glutathione and air-oxidized glutathione. Oxidation of the peptide thiol was confirmed by test for free thiol with Ellman's reagent.
Further confirmation of peptide attachment was obtained by monitoring surface wetting properties. Since the Laminin fragment is, overall, a less polar molecule than glutathione, it was expected that surfaces bearing this peptide would not be as wettable as those bearing glutathione. This was confirmed by observing water contact angles of 45O (adv)-40° (rec) for surfaces bearing the Laminin fragment peptide. Interestingly, with surface-boundLaminin we observed no sulfur X P S signal, even with an angle-resolved X P S experiment. This result is presumably a combination of the inherently small sulfur signal being even further attenuated by the mass of the attached peptide. This is consistent with the sulfur being the attachment site for the peptide.
Discussion In many of the applications requiring biologically active proteins attached to inorganic surfaces (e.g. biosensors), it is important to have surfaces which are structurally well defined and still versatile enough to attach different proteins or peptides for different applications. For example, with immunosensors, one can envision surfacebound antigen peptides attached to a sensor capable of detecting a specific antibody. Each such peptide will be of different composition and may require a surface with different chemical and/or physical properties. Although the amino and carboxyl side chains are the most likely candidates for chemical attachment sites and the most often used, these side chains are also likely to be involved in the biological activity of the peptide or protein, and
3014 Langmuir, Vol. 9, NO.11,1993 4000
Lee et al.
to surfaces. Indeed. wine a different chemical amroach. J Bright et al.lo h a v e ' d e s c h d a fiber optic imm;;nofluoNitrogen
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Figure 7. Attachment of a nonapeptide laminin fragment to iodoacetyl SAMs. (top) XPS signal for nitrogen obtained from SAMs exposed to native peptide and maleimide blocked peptide; (bottom) XPS signal for iodide observed with native and maleimide blocked decapeptide.
they are the least likely to provide a unique site for attachment. Thus, it is important to develop surfaces based on a range of attachment methods to accommodate various applications. The thiol side chains of Cys residues provide the most obvious alternative to amino or carboxyl pointa of attachment. Cys is a relatively low abundance amino acid, and in proteins ita thiol is often involved in disulfide bonds which further reduces the availability of thiol nucleophiles. Also,in synthetic peptides known to represent epitopes of specific antigens, it is less common to find Cys residues than amino or carboxyl groups. Thus, addition of a Cys residue to a synthetic antigen may well render it reactive with surfaces such as those we describe, while not interfering with biological interactions important for ita function. A second example of a unique attachment site for a protein through a Cys side chain is the FAB' fragment of IgGs. These antibody fragmenta have a single reduced Cys a t a site which is distant from the antigen-binding domain of the protein, and which therefore provides the opportunity for uniform attachment of antibodymolecules
rescent sensor system in which a FAB' fragment is attached through this Cys residue. Our studies with the set of electrophilic SAMsdescribed above indicate that they are quite specific for the thiol side chain in peptides. In addition, recent experimentsin our laboratories confirm that the amino groups in these thiol-attached peptides are still available for further modification, which would not be the case if attachment to the SAM were through the amino group. However, this selectivity is not absolute and, in the absence of competing thiol, organic amines do attach to the iodoacetyl surfaces. Furthermore, the a-amino group in the peptides we have used is less nucleophilic than the €-aminogroup in the common amino acid, lysine. Thus, we cannot rule out the possibility that a peptide rich in lysine functionality might react to some extent through this residue. Indeed, the highly electrophilic tresyl surfaces described by Bright et al.l0were found to react with amino groups at pH values above 5, and thus the specificity of that system for thiols was compromised. However, the ability to control the electrophilic character of the surface should allow customization of surface reactivity to adjust for such circumstances. For example, a-bromoacetyl or the benzyl iodide surfaces will be less electrophilic than the a-iodoacetyl surfaces utilized here, and specificity for thiols should be enhanced. Further studies of the specificity of the surfaces with a range of peptides will be informative. A second advantage of the flexibility of electrophilic character inherent in our systems is related to the solvent systems which can be used for peptide or protein attachment. Here we utilized DMF as solvent,to avoid hydrolysis of the very reactive a-iodoacetyl surface. However, some peptides and many proteins may not be soluble in nonaqueous systems or may lose biological activity in such solvents. Thus, the benzyl halide surfaces may prove more useful in application with aqueous systems, since these surfaces may be somewhat more stable to hydrolysis. Further exploration of the scope and limitations of these materials for the attachment of various biomolecules will be needed. In summary, this paper presents the first step in development of a set of chemical tools for controlled attachment of peptides and proteins to silicon, glass, and metal oxide surfaces as stable, uniform monolayer f h s . Further exploration of these approachesmay enhance our ability to construct surfaces which have protein or peptide species displayed in a desired orientation with a density and distribution favorable for a particular biochemical interaction. These tools enhance the possibilities for creation of surfaces useful for new biocompatible inorganic materials and biosensors. Acknowledgment. Financial support of both the Edison Biotechnology Center of the State of Ohio and the NIH (GM 45678) is gratefully acknowledged. Supplementary Material Available: Descriptions of the synthesis and characterization of compounds 1-6 and the productaof the solutionkinetics experiments (7 pages). Ordering information is given on any current masthead page.
