Reactivity in the Confinement of Self-Assembled Monolayers: Chain

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Reactivity in the Confinement of Self-Assembled Monolayers: Chain Length Effects on the Hydrolysis of N-Hydroxysuccinimide Ester Disulfides on Gold Barbara Dordi, Holger Scho¨nherr, and G. Julius Vancso* University of Twente, MESA+ Research Institute and Faculty of Science and Technology, Department of Materials Science and Technology of Polymers, P.O. Box 217, 7500 AE Enschede, The Netherlands Received February 21, 2003. In Final Form: April 22, 2003 Two N-hydroxysuccinimide (NHS) ester disulfides, 16,16′-dithiobis(N-hydroxysuccinimidylhexadecanoate) (NHS-C15) and 11,11′-dithiobis(N-hydroxysuccinimidylundecanoate) (NHS-C10), were synthesized and adsorbed as self-assembled monolayers (SAMs) on gold surfaces. These SAMs, together with SAMs of 3,3′-dithiobis(N-hydroxysuccinimidylpropionate) (NHS-C2), were used as model systems for an examination of the factors that affect the kinetics of interfacial reactions. The SAMs and the rate of the base-catalyzed hydrolysis of the incorporated NHS ester groups were characterized by grazing incidence reflection Fourier transform infrared (GIR-FTIR) spectroscopy and contact angle measurements. GIR-FTIR spectroscopy shows that SAMs of NHS-C2 and NHS-C10 undergo a pseudo-first-order hydrolysis with second-order rate constants of (61 ( 11) × 10-2 M-1 s-1 and (4.5 ( 0.4) × 10-2 M-1 s-1, respectively. SAMs of NHS-C15 show a sigmoid behavior with a half reaction time of 1700 ( 20 s in 10 mM aqueous NaOH. The rate constants determined based on the contact angle data and application of the Cassie equation are in excellent agreement with the GIR-FTIR spectroscopy results. The increase in conformational order with increasing chain length and the concomitant improvement of packing of the NHS ester end groups, as seen by GIRFTIR spectroscopy, account for the observed differences in reactivity. Our results imply that surface reactions in SAMs can be controlled via careful design of the adsorbate structure.

Introduction Chemical reactions at organic or polymeric surfaces are of crucial importance for a wide variety of applications, including array technologies for genomics and proteomics,1,2 (bio)sensors,3,4 and surface modifications for biomedical purposes.5 These reactions may be utilized as immobilization steps for coupling active species to sensor or measurement platforms or as surface modification for passive applications in the form of, e.g., corrosion-resistant or protein-adherence-resistant coatings. In particular, many (bio)sensors rely on the interactions or (ir)reversible chemical reactions of analytes with the sensors active centers. Prominent detection techniques, such as surface plasmon resonance (SPR) measurements,3,6 require ultrathin layers, e.g., self-assembled * To whom correspondence may be addressed. E-mail: g.j.vancso@ utwente.nl. Tel: ++31 53 489 2974. Fax: ++31 53 489 3823. (1) (a) Ramsay, G. Nat. Biotechnol. 1998, 16, 40-44. (b) Lipshutz, R. J.; Fodor, S. P. A.; Gingeras, T. R.; Lockhart, D. J. Nat. Genet. 1999, 21, 20-24. (c) Lockhart, D. J.; Winzeler, E. A. Nature 2000, 405, 827836. (2) (a) Walter, G.; Bussow, K.; Cahill, D.; Lueking, A.; Lehrach, H. Curr. Opin. Microbiol. 2000, 3, 298-302. (b) Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol. 2001, 5, 40-45. (3) (a) Biosensor Design and Application; Mathewson, P. R., Finley, J. W., Eds.; ACS Symposium Series 511; American Chemical Society: Washington, DC, 1992. (b) Biosensor and Chemical Sensor Technology: Process Monitoring and Control; Rogers, K. R., Mulchandani, A., Zhou, W., Eds.; ACS Symposium Series 613; American Chemical Society: Washington, DC, 1995. (4) For a recent review see: Rich, R. L.; Myszka, D. G. Curr. Opin. Biotechnol. 2000, 11, 54-61. (5) (a) Bergstrom, K.; Holmberg, K.; Safranj, A.; Hoffman, A. S.; Edgell, A. S.; Kozlowski, A.; Hovanes, B. A.; Harris, J. M. J. Biomed. Mater. Res. 1992, 26, 779-790. (b) Otsuka, H.; Nagasaki, Y.; Kataoka, K. Curr. Opin. Colloid Interface Sci. 2001, 6, 3-10. (c) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1167. (d) Mrksich, M.; Whitesides, G. M. ACS Symp. Ser. 1997, No. 680, 361-373. (6) Knoll, W. Annu. Rev. Phys. Chem. 1998, 49, 569-638.

monolayers (SAMs),7,8 for the immobilization of receptors due to the limited sampling depth of the evanescent field of the surface plasmons. Other approaches rely on monolayer coupling chemistry, such as several approaches in the mentioned array technologies.1,2 Interestingly, it has been realized that chemical reactivity in ordered ultrathin organic films, such as Langmuir monolayers at the air-water interface8,9 or SAMs on solid supports,7,8 can be very different compared to reactions carried out in solution. Since the functional groups or molecules involved in these reactions are immobilized at interfaces or on surfaces, these differences can be attributed to “confinement effects”.10,11 In some cases, an increase in reactivity has been reported for reactions in constrained environments, while in most cases reactions are retarded and the corresponding rate constants are significantly lower than those obtained in solution.12-14 For example, To¨llner et al. reported a significantly enhanced catalysis of acetone hydrogenation because of an enforced favorable orientation of a rhodium complex incorporated in corresponding Langmuir(7) (a) Dubois, L. H.; Nuzzo, R. G. Annu. Rev. Phys. Chem. 1992, 43, 437-463. (b) Ulman, A. Chem. Rev. 1996, 96, 1533-1554. (8) Ulman, A. An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self-Assembly; Academic Press: New York, 1991. (9) See, e.g.: LB Films; Roberts, G., Ed.; Plenum Press: New York, 1990. (10) Ahmad, J.; Astin, K. B. Langmuir 1990, 6, 1797-1799 and references therein. (11) For a recent review on reactions and reactivity in SAMs, see: Chechik, V.; Crooks, R. M.; Stirling, C. J. M. Adv. Mater. 2000, 12, 1161-1171. (12) To¨llner, K.; Popovitz-Biro, R.; Lahav, M.; Milstein, D. Science 1997, 278, 2100-2102. (13) Neogi, P.; Neogi, S.; Stirling, C. J. M. J. Chem. Soc., Chem. Commun. 1993, 1134-1136. (14) van Ryswyk, H.; Turtle, E. D.; Watson-Clark, R.; Tanzer, T. A.; Herman, T. K.; Chong, P. Y.; Waller, P. J.; Taurog, A. L.; Wagner, C. E. Langmuir 1996, 12, 6143-6150.

10.1021/la0343066 CCC: $25.00 © 2003 American Chemical Society Published on Web 06/04/2003

Reactivity in the Confinement of SAMs

Blodgett films.12 By contrast, monolayers of aliphatic esters with the carboxyl group buried ∼1 nm below the layer surface and well-ordered monolayers of isonicotinate esters show a significantly reduced reactivity in transesterification13 and hydrolysis reactions,14 respectively. Among the factors that affect reactivity in confinement or constrained environments are the choice of solvent, steric, and anchimeric effects of the reactants.11 These factors may alter the reactivity through, for instance, shifts in surface pKa values,15 prevented or hindered access of reactive species from solution to the reaction centers,13,14 or interactions of neighboring functional groups with the reaction center. Additional effects include interactions with the substrate resulting in altered nucleophilicity16 and restricted reorientations of functional groups at the monolayer surface.17 We have previously reported on the alkaline hydrolysis of terminal ester groups in SAMs on gold.18 Monolayerstructure-related reactivity differences, observed by inverted chemical force microscopy on the nanometer scale, agreed well with average macroscopic behavior observed by Fourier transform infrared (FTIR) spectroscopy. Our results indicated that the reaction spreads from defect sites for SAMs with closely packed, well-ordered ester groups, while loosely packed, disordered ester groups show the expected pseudo-first-order kinetics.18,19 Activated esters, such as N-hydroxysuccinimide (NHS) esters, are very attractive functional groups utilized for covalent coupling of biologically relevant molecules to surfaces. Activation of surface-bound carboxylic acid groups,20 as well as direct derivatization of, e.g., gold and silicon surfaces with NHS esters have been reported.21,22 Immobilization of NHS esters in SAMs on gold leads to a marked reduction of their reactivity, for instance, in simple base-catalyzed hydrolysis reactions. For the hydrolysis of SAMs of 11,11′-dithio-bis(N-hydroxysuccinimidylundecanoate) (NHS-C10) on gold in aqueous KOH, Wang et al. reported a reduction in rate constant by as much as 3 orders of magnitude compared to solution.23-25 This observation can be attributed to restricted access of the hydroxide ions to the reactive carbonyl carbon centers. Strikingly, SAMs of NHS-C10 are close to the wellestablished transition of the conformational order of alkane chains from disordered to ordered in SAMs on gold.8,26 Together with the qualitative relation between order and reactivity (vide supra),18,19 this observation may (15) These can be assessed by AFM approaches, see, e.g.: (a) Scho¨nherr, H.; Hruska, Z.; Vancso, G. J. Macromolecules 2000, 33, 45324537. (b) Scho¨nherr, H.; van Os, M. T.; Fo¨rch, R.; Timmons, R. B.; Knoll, W.; Vancso, G. J. Chem. Mater. 2000, 12, 3689-3694. (16) Chechik, V.; Stirling, C. J. M. Langmuir 1997, 13, 6354-6356. (17) Vaidya, B.; Chen, J.; Porter, M. D.; Angelici, R. J. Langmuir 2001, 17, 6569-6576. (18) Scho¨nherr, H.; Chechik, V.; Stirling, C. J. M.; Vancso, G. J. J. Am. Chem. Soc. 2000, 122, 3679-3687. (19) Scho¨nherr, H.; Chechik, V.; Stirling, C. J. M.; Vancso, G. J. ACS Symp. Ser. 2001, No. 781, 36-57. (20) (a) Frey, B. L.; Corn, R. M. Anal. Chem. 1996, 68, 3187-3193. (b) Lahiri, J.; Isaacs, L.; Tien, J.; Whitesides, G. M. Anal. Chem. 1999, 71, 777-790 and references therein. (21) (a) Wagner, P.; Kernen, P.; Hegner, M.; Ungewickell, E.; Semenza, G. FEBS Lett. 1994, 356, 267-271. (b) Wagner, P.; Hegner, M.; Kernen, P.; Zaugg, F.; Semenza, G. Biophys. J. 1996, 70, 20522066. (22) Wojtyk, J. T. C.; Morin, K. A.; Boukherroub, R.; Wayner, D. D. M. Langmuir 2002, 18, 6081-6087. (23) Wang, J.; Kenseth, J. R.; Jones, V. W.; Green, J.-B. D.; McDermott, M. T.; Porter, M. D. J. Am. Chem. Soc. 1997, 119, 1279612799. (24) Cline, G. W.; Hanna, S. B. J. Org. Chem. 1988, 53, 3583-3586. (25) The rate of reaction could be enhanced locally by scanning with an atomic force microscopy (AFM) tip (ref 23). (26) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559-3568.

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imply that the reactivity is sensitive to variations in adsorbate chain length. As reported here, we investigated the reactivity of NHS esters in SAMs of the corresponding disulfides on gold systematically as a function of chain length in order to elucidate the relation of conformational order on one hand and chemical reactivity on the other hand for a prime model system of interfacial reactions in thin organic films. Experimental Section General Methods. The 1H NMR spectra were recorded on a Bruker AC 250 spectrometer (250 MHz). 1H NMR chemical shifts are stated relative to residual CHCl3 (7.25 ppm). Fast atom bombardment mass spectrometry (FAB-MS) was carried out on a Finnigan MAT 90 spectrometer using m-nitrobenzyl alcohol (NBA) or o-nitrophenyl octyl ether (NPOE) as a matrix. Reagents. 3,3′-Dithio-bis(N-hydroxysuccinimidylpropionate) (NHS-C2) was obtained from Fluka. All the organic solvents, except for ethanol (p.a.; Merck), were purchased from Biosolve and used as received. Synthesis. (a) 11,11′-Dithio-bis(N-hydroxysuccinimidylundecanoate) (NHS-C10). Sodium thiosulfate (Aldrich) (7 mmol, 1.737 g) was added to a solution of 11-bromoundecanoic acid (Aldrich) (7 mmol, 1.8564 g) in 50% aqueous 1,4-dioxane (20 mL). The mixture was heated at reflux (90 °C) for 2 h until the reaction to the intermediate salt was completed (clear solution). The oxidation to the corresponding disulfide was carried out in situ by adding iodine in portions until the solution retained a yellow to brown color after heating at 60 °C for 3 h. The excess of solid iodine was neutralized with 15% of sodium pyrosulfite (Aldrich) in water. After removal of the solvent by rotary evaporation, the creamy suspension was filtered to yield the product. Recrystallization from ethyl acetate/tetrahydrofuran provided 11,11′-dithio-bis(undecanoic acid) as a white solid, which was dried in a vacuum. N-Hydroxysuccinimide (Aldrich) (2.53 mmol) was suspended in 10 mL of tetrahydrofuran, and 1.15 mmol of 11,11′-dithiobis(undecanoic acid) was added. The mixture was cooled to 0 °C, then 2.53 mmol of EDC HCl (Aldrich) was added, and the resulting solution was stirred for 3 h. Afterwards the reaction mixture was allowed to warm to room temperature and stirred for 24 h. The solvent was removed under vacuum by rotatory evaporation, and the creamy suspension was dissolved in 50 mL of ethyl acetate. The solution was washed with 10% aqueous KHSO4, H2O, 5% aqueous NaHCO3, and H2O, respectively, and dried over MgSO4. The solvent was removed under vacuum to yield the product as a white solid. Isolated yield 0.27 g, 0.43 mmol, 37%. 1H NMR (250 MHz, CDCl ): δ (ppm) 2.83 (s, 4H), 2.68 (t, 2H, 3 J ) 7.3 Hz), 2.60 (t, 2H J ) 7.3 Hz), 2.68 1.79-1.62 (m, 4H), 1.43-1.26 (m, 12H). FAB-MS m/z 627.3 [M - H], calcd for C30H48N2O8S2 628.8. (b) 16,16′-Dithio-bis(N-hydroxysuccinimidylhexadecanoate) (NHS-C15). 16-Mercaptohexadecanoic acid (Aldrich) (4 mmol, 1.15 g) was suspended in 100 mL of chloroform. The oxidation to the corresponding disulfide was carried out in situ by adding KI3 (aqueous) (under vigorous stirring) until the solution retained a yellow to brown color. The excess of iodine was neutralized with 15% of sodium pyrosulfite in water. After removal of the solvent by rotary evaporation, the suspension was filtered to yield 16,16′-dithio-bis(hexadecanoic acid) as a white solid. The solid was eventually dried in a vacuum. N-Hydroxysuccinimide (1.54 mmol) was suspended in 10 mL of tetrahydrofuran, and 0.7 mmol of 16,16′-dithio-bis(hexadecanoic acid) was added. The mixture was cooled to 0 °C, then 2.1 mmol of EDC HCl was added, and the resulting solution was stirred for 3 h. Afterwards the reaction mixture was allowed to warm to room temperature and was stirred for 24 h. The solvent was removed under vacuum by rotatory evaporation, and the creamy suspension was dissolved in 50 mL of ethyl acetate. The solution was washed with 10% aqueous KHSO4, H2O, 5% aqueous NaHCO3, and H2O, respectively, and dried over MgSO4. The solvent was removed under vacuum; recrystallization of the product from acetone-diethyl ether gave the product as white powder.

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Scheme 1. Schematic of the Formation of Self-Assembled Monolayers of NHS-Cn with n ) 2, 10, and 15 on Gold

Isolated yield 0.12 g, 0.16 mmol, 23%. 1H NMR (250 MHz, CDCl ): δ (ppm) 2.83 (s, 4H), 2.68 (t, 2H, 3 J ) 7.3 Hz), 2.60 (t, 2H J ) 7.3 Hz), 1.79-1.62 (m, 8H), 1.431.26 (m, 18H). FAB-MS m/z 768.3 [M - H], calcd for C40H68N2O8S2 769.1. Monolayer Preparation. All glassware used to prepare monolayers was immersed in piranha solution (solution containing 70% concentrated sulfuric acid and 30% hydrogen peroxide) for 15 min and then rinsed with large amounts of high-purity water (Millipore Milli-Q water). Caution: Piranha solution should be handled with extreme caution; it has been reported to detonate unexpectedly. Gold substrates (200 nm of Au on 2 nm of Ti primer on glass) were purchased from SSENS bv (Hengelo, The Netherlands). The substrates were cleaned immediately before use by oxygen plasma (5 min, 30 mA, 60 mTorr) (SPI Supplies, Plasma Prep II) and soaked subsequently in ethanol for the same time. The freshly cleaned gold substrates were immersed with minimal delay into 0.1-1.0 mM adsorbate solutions in ethanol. The substrates were removed from solutions after >12 h assembly time and rinsed extensively with chloroform, ethanol, and water to remove any physisorbed material. Contact Angle Measurements. The advancing and receding contact angles θadv and θrec were measured with Millipore water as a probe liquid by using a contact angle microscope (Data Physics, OCA 15plus). Contact angles were determined at room temperature and ambient humidity. For this purpose a 1 µL drop of water was placed on the monolayers; liquid was then added to (removed from) the drop until the front was seen to advance (recede) across the surface. Once observable motion has ceased, the advancing (receding) contact angle was measured without removing the needle from the drop. A set of at least three different locations was averaged per monolayer sample. X-ray Photoelectron Spectroscopy (XPS). XPS spectra were recorded on a PHI Quantum 2000 scanning ESCA microprobe using a monochromated X-ray beam (Al-anode); 100 µm diameter/25 W X-ray beam scanned over 1000 µm × 500 µm area at a takeoff angle of 30°. Atomic concentrations were determined by numerical integration of the relative peak areas in the detailed element scans using the following sensitivity factors: C 1s [0.314], N 1s [0.499], O 1s [0.733], S 2p [0.717].27 Polarized Grazing Incidence Reflection Fourier Transform Infrared (GIR-FTIR) Spectroscopy. The FTIR spectroscopy data were collected on a BIO-RAD model FTS575C FTIR spectrometer using a GIR accessory (BIO-RAD) and a liquidnitrogen-cooled cryogenic mercury cadmium telluride (MCT) detector. Measurements were performed at room temperature; the spectrometer was continuously purged with nitrogen. The spectra were collected at an angle of incidence of 87° relative to the surface normal. 1024 scans recorded with a resolution of 4 cm-1 were ratioed against the previously obtained background spectrum (SAM of d33-hexadecanethiol on gold). Kinetics Studies. The hydrolysis of NHS ester SAMs was carried out by incubation of the respective monolayers for different reaction times in a 1.00 × 10-2 M solution of NaOH for studies of NHS-C15 and NHS-C10, and a 1.00 × 10-3 M solution of (27) The calculated atomic composition is also sensitive to the energy of the primary X-ray beam, variations in photoionization cross section with chemical structure, the takeoff angle, and the elemental distribution perpendicular to the surface, in addition to the actual composition of the monolayer (Wagner, C. D.; Riggs, W. M.; Davis, L. E.; Moulder, J. F. Handbook of X-ray Photoelectron Spectroscopy; Muilenberg, G. E., Ed.; Perkin-Elmer Corporation: Eden Prairie, MN, 1979.)

Table 1. Contact Angles (H2O) of SAMs of NHS-C2, NHS-C10, and NHS-C15 θadv (deg)

θrec (deg)

60 ( 2 59 ( 2 60 ( 2

39 ( 2 43 ( 2 50 ( 2

n)2 n ) 10 n ) 15

Table 2. Atomic Concentration of NHS-Cn Ester Monolayers on Gold Determined by XPS observed composition (atom %) n)2 n ) 10 n ) 15

expected composition (atom %)

C

N

O

S

C

N

O

S

55.1 84.1 86.0

9.1 2.2 1.3

30.9 12.0 10.2

5.8 3.3 2.3

53.8 71.4 76.9

7.7 4.8 3.8

30.8 19.0 15.5

7.7 4.8 3.8

NaOH for studies of NHS-C2. All experiments were carried out at 30 °C. After the reaction, the samples were rinsed with H2O, 1 M HCl, H2O, and finally with CHCl3.

Results and Discussion The investigation of the reactivity of the NHS esters immobilized in self-assembled monolayers (SAMs) on gold is based on the characterization of the as-prepared SAMs and the subsequently measured changes in composition or wettability as assessed in GIR-FTIR spectroscopy and contact angle measurements, respectively. Characterization of SAMs of NHS-Cn on Gold. All the disulfides investigated in this study form SAMs on gold by spontaneous assembly from solution (Scheme 1). The structure and conformational order of the monolayers, and in particular the SAM formation of the novel compound NHS-C15, were verified by a multitechnique characterization using contact angle (CA) measurements, GIR-FTIR spectroscopy, and XPS. The values for the advancing and receding contact angles, θadv and θrec, using ultrapure water as probe liquid, of SAMs of the investigated disulfides are summarized in Table 1. The influence of the length of the alkyl chain is obvious. While the advancing contact angles are the same for NHS-C2, NHS-C10, and NHS-C15 to within the experimental error, the receding contact angles increase with increasing hydrocarbon chain length. Consequently the hysteresis, as a measure of surface roughness, disorder, or inhomogeneity,8 decreases with increasing number of methylene groups in the chain. The values obtained for NHS-C10 are in fair agreement with literature data.21,23 XPS provided additional confirmation about the formation of SAMs of NHS-Cn on gold. Table 2 shows the atomic compositions derived from the acquired XPS spectra. These spectra confirm the presence of the expected elements in the monolayers and allow one to calculate the atomic composition of the SAMs. The absolute compositions derived from XPS should be interpreted with caution, since photoelectrons from the subsurface atoms are attenuated by the overlying material.27 For all the monolayers studied,

Reactivity in the Confinement of SAMs

Langmuir, Vol. 19, No. 14, 2003 5783 Table 3. Band Assignments and Corresponding Peak Positions for NHS-C2, NHS-C10, and NHS-C15 Adsorbed on Gold νas(CH2) (cm-1) νs(CH2) (cm-1) ν(CdO)a (cm-1) ν(CdO)a (cm-1) ν(CdO)b (cm-1) ν(C-O) (cm-1)

n)2

n ) 10

n ) 15

1816 1789 1748 1216, 1078

2922 2852 1815 1785 1748 1217, 1082

2918 2850 1816 1786 1748 1214, 1076

a Splitting of the band of the ester carbonyl CdO stretching vibration. b Succinimidyl carbonyl group.

Figure 1. High-energy region of GIR-FTIR spectra of SAMs of NHS-Cn with n ) 2, 10, and 15 on gold showing the C-H stretching vibrations.

Figure 2. Low-energy region of GIR-FTIR spectra of SAMs of NHS-Cn with n ) 2, 10, and 15 on gold showing the succinimidyl and ester carbonyl CdO stretching vibrations. The spectra have been normalized to the absorbance of the C-D stretching vibrations of d33-hexadecanethiol on gold used to record the background spectra.

only the expected elements were observed. The observed and calculated compositions agree reasonably well with each other, taking takeoff angle, photoelectron attenuation, and possible variations in coverage into account. The FTIR spectra of SAMs of NHS-C2, NHS-C10, and NHS-C15 are shown in Figures 1 and 2. The most prominent bands are the asymmetric C-H stretching vibration, νas(CH2), at ca. 2920 cm-1, the symmetric C-H stretching vibration, νs(CH2), at ca. 2850 cm-1, the CdO stretching vibrations, ν(CdO), at ca. 1748 cm-1, and the C-O stretching vibrations, ν(C-O), at ca. 1216 and 1078 cm-1 (ν(C-O), data not shown). Only the spectra of SAMs of NHS-C15 and NHS-C10 show the asymmetric and symmetric C-H stretching vibrations of the methylene groups. These modes are apparently absent in the NHSC2 SAMs. For the latter SAMs these modes are probably too broad to be detected. The complete band assignments and peak positions are summarized in detail in Table 3.21,28 For all the SAMs, the (CdO) band at 1748 cm-1, assigned to the succinimidyl carbonyl group (the two peaks at higher wavenumbers, which correspond to the ester carbonyl are seen with very low absorbance), and the two (C-O) bands at 1217 and 1082 cm-1 (data not shown) are diagnostic of the ester groups of the NHS esters (Figure 2). The (28) Duhachek, S. D.; Kenseth, J. R.; Casale, G. P.; Small, G. J.; Porter, M. D.; Jankowiak, R. Anal. Chem. 2000, 72, 3709-3716.

integrated absorbance of the succinimidyl CdO stretching vibrations in the normalized spectra shown in Figure 2 suggests a significantly lower coverage for decreasing chain length of the disulfide, provided that the mean orientation of the transition dipole moments is similar. Furthermore, the peak width at half-maximum increases monotonically for decreasing chain length, which is indicative for a more disordered arrangement of the NHS ester end groups in the short chain disulfides. An examination of the spectra in more detail provides insight into the orientation and conformational order of the three SAMs. In general, polarized grazing incidence reflection FTIR spectroscopy yields information about the direction of the transition dipole moments in the sample.8 The peak positions of νs(CH2) and νas(CH2) for the NHSC15 monolayers are shifted to lower frequencies compared the NHS-C10 monolayers (Table 3, Figure 1). These modes are unrecognizable in SAMs of NHS-C2, probably due to a broadening of the bands. A broadening of the bands attributed to the C-H stretching vibrations is already obvious for SAMs of NHS-C10. The peak width at halfmaximum for the νs(CH2) and νas(CH2) vibrations estimated from normalized spectra increases from 14.7 and 11.2 cm-1 to 15.2 and 13.0 cm-1, respectively, with a decrease in chain length from 15 to 10 methylene groups in NHS-C15 vs NHS-C10. In the bulk, the corresponding peak positions and widths were found to correlate with the physical state of the compounds.26 Compared to hydrocarbon solids, liquids show a shift to higher wavenumbers of about 5 cm-1. Solid CH3(CH2)21SH in KBr shows the νas(CH2) and νs(CH2) stretching modes at 2918 and 2851 cm-1, respectively, while for the liquid CH3(CH2)7SH these transitions are shifted to 2924 and 2855 cm-1, respectively.26 Similar compounds adsorbed in SAMs on gold show the same trend; CH3(CH2)17SH shows the νas(CH2) and νs(CH2) stretching modes at 2917 and 2850 cm-1, compared to 2921 and 2852 cm-1 for CH3(CH2)5SH.26,29 Following this analogy, we conclude from our data that SAMs of NHSC15 show identical peak positions that have been previously attributed to nearly crystalline-like packing in SAMs of n-alkanethiols,8,26 while SAMs of NHS-C10 and NHS-C2 are more disordered, resembling liquidlike SAMs. Base-Catalyzed Hydrolysis. SAMs of NHS-C15, NHS-C10, and NHS-C2 were hydrolyzed in aqueous sodium hydroxide solution to form the corresponding carboxylate-terminated SAMs, as shown in Scheme 2. The progression of the hydrolysis of the ester groups confined in the monolayers was determined by GIR-FTIR spectroscopy. These measurements were performed in an ex situ mode for samples immersed in the appropriate solutions for variable periods of time followed by extensive rinsing with H2O, 1 M HCl, H2O, and finally CHCl3. The kinetics was determined by measuring the decrease of (29) Chidsey, C. E. D.; Loiacono, D. N. Langmuir 1989, 6, 682-691.

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Scheme 2. Schematic Illustration of the Reaction between NHS-Esters and Sodium Hydroxide

Figure 3. GIR-FTIR spectra of NHS-C10 adsorbed on gold after different reaction times with 1.00 × 10-2 M NaOH at 30 °C.

the integrated intensity of the succinimidyl carbonyl band as shown in Figure 3 for a NHS-C10 monolayer hydrolyzed in 1.00 × 10-2 M NaOH. The strong band at 1748 cm-1 decreases in absorbance with the progress of the reaction. The carboxylic acid groups in a hydrolyzed monolayer are expected to exist in the deprotonated carboxylate form when immersed in 1.00 × 10-2 M NaOH. However, the carboxylate groups on the surface appear to partially protonate upon rinsing.30 We observed a very broad band at ∼1466 cm-1 (data not shown), which is consistent with deprotonated carboxylate species; however, due to the sensitivity of the IR data to the orientation of the transition dipole moments of the corresponding vibration, we cannot detect the relevant species in case of a likely in plane orientation.31 Additional XPS experiments on NHC-C2 SAMs confirmed that even the short-chain SAMs stay intact after partial or complete hydrolysis. The changes observed in the FTIR spectra are all consistent with the conversion of the ester groups to carboxylate groups as the hydrolysis progresses. The extent of the reaction x can be expressed as a function of hydrolysis time

x)

A0 - At A0 - A∞

(1)

where A0 is the integrated absorbance of the succinimide ester carbonyl band at time zero, At at time t, and A∞ at infinitive time. The progression of the hydrolysis was also determined from the corresponding changes in contact angles as a function of time. Hydrolysis causes a significant increase (30) Arnold, R.; Azzam, W.; Terfort, A.; Wo¨ll, C. C. Langmuir 2002, 18, 3980-3992. (31) The CA and FTIR characterization data of all reaction products agree well with the data obtained for SAMs of the corresponding neat mercaptoalkanoic acids and disulfides.

Figure 4. Plot of ln[1 - x] (x: extent of reaction) as function of reaction time for the hydrolysis in 1.00 × 10-3 M NaOH at 30 °C as determined by CA and GIR-FTIR spectroscopy for NHS-C2. The solid line corresponds to a least-squares fit of the GIR-FTIR spectroscopy data.

in wettability. If the two components (NHS and COOH) in a mixed monolayer act independently and the effect of surface roughness and phase separation can be neglected, the experimentally determined advancing contact angles θexp can be described by the Cassie equation32

cos θexp ) χNHS cos θNHS - χCOOH cos θCOOH

(2)

where χNHS and χCΟΟΗ are the surface coverages of the two components and θNHS ) 60° and θCΟΟΗ ) 0° are the experimentally measured advancing contact angles of the two pure SAMs.32 As observed by both FTIR and CA measurements in the case of SAMs of NHS-C2 and NHS-C10, an exponential decrease of NHS ester coverage with increasing reaction time is observed (Figures 4 and 5). For the shortchain ester there may be a possible acceleration present at long reaction times. The exponential decrease can be rationalized assuming a pseudo-first-order kinetics.18 Considering the concentration of the base, we can conclude that SAMs of NHS-C2 react much faster than SAMs of NHS-C10. This result indicates that increased chain length is related to reduced reactivity in this particular case. The calculation of the rate constants for the hydrolysis of NHS-C10 and NHS-C2 was performed according to pseudo-first-order kinetics.33 Linearization of the FTIR spectroscopy data yielded pseudo-first-order rate constants k′ of (4.5 ( 0.4) × 10-4 s-1 for NHS-C10 and (6.1 ( 1.1) (32) Adamson, A. W.; Gast, A. P. Physical Chemistry of Surfaces, 6th ed.; Wiley-Interscience: New York, 1997; p 355.

Reactivity in the Confinement of SAMs

Figure 5. Plot of ln[1 - x] (x: extent of reaction) as function of reaction time for the hydrolysis in 1.00 × 10-2 M NaOH at 30 °C as determined by CA and GIR-FTIR spectroscopy for NHS-C10. The solid line corresponds to a least-squares fit of the GIR-FTIR spectroscopy data.

Langmuir, Vol. 19, No. 14, 2003 5785

Figure 7. Plot of [1 - x] (x: extent of reaction) as function of reaction time for the hydrolysis in 1.00 × 10-2 M NaOH at 30 °C as determined by GIR-FTIR spectroscopy for NHS-C15 and NHS-C10 at the early stages of hydrolysis.

× 10-4 s-1 for NHS-C2 (mean ( 95% confidence limit). The corresponding second-order rate constants k′′ are (4.5 ( 0.4) × 10-2 M-1 s-1 and (61 ( 11) × 10-2 M-1 s-1, respectively. The linearization of the surface coverages calculated from the contact angle data using the Cassie equation yielded a pseudo-first-order rate constant k′ of (4.5 ( 2.3) × 10-4 s-1 for SAMs of NHS-C10 and (5.6 ( 2.3) × 10-4 s-1 for NHS-C2. The corresponding second-order rate constants k′′ are (4.5 ( 2.3) × 10-2 M-1 s-1 and (56 ( 23) × 10-2 M-1 s-1, respectively. The FTIR spectroscopy and CA data appear to be in excellent agreement for the two systems for the initial course of the reaction. At longer reaction times we observe a systematic deviation of the contact angle data (Figure 5 and Figure 6). For this particular system and conversions exceeding 50%, the application of the Cassie equation leads to an overestimate of the surface coverage of NHS ester compared to estimates

by independent methods.34 Hence we included only the contact angle data for conversions 10-12, FTIR spectra and contact angle measurements suggest highly ordered, “crystallinelike” monolayers with very good conformational order.26,35 On the basis of a simple analogy regarding chain-lengthdependent behavior, one would expect to observe already for NHS-C10 a different reactivity, similar to the simple esters mentioned above.18 However, the observation of an induction period, and thus a change in mechanism, only for NHS-C15 can very well be explained, if the effect of

(33) The rate of the reaction is described by: -(d[NHS]/dt) ) k′′[OH-][NHS] ) k′[NHS], where [NHS] and [OH-] denote the concentration of NHS ester and hydroxide ions, respectively, and k′′ and k′ are the second-order and pseudo-first-order rate constants, respectively ([OH-] ≈ constant; k′ ) k′′[OH-]).

(34) The application of the Israelachvili equation (ref 32) leads practically to the same results. (Scho¨nherr, H.; Feng, C.; Shovsky, A. Unpublished data.). (35) See, e.g.: Scho¨nherr, H.; Ringsdorf, H. Langmuir 1996, 12, 38913897.

Figure 6. Plot of ln[1 - x] (x: extent of reaction) as function of reaction time for the hydrolysis in 1.00 × 10-2 M NaOH at 30 °C as determined by CA and GIR-FTIR spectroscopy for NHS-C15.

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Dordi et al.

Table 4. Rate Constants and Half-Reaction Times Obtained for the NHS-Cn Esters kFTIR′′ (M-1 s-1) n)2 n ) 10 n ) 15 bulkb a

10-2

kCA′′ (M-1 s-1) 10-2

(61 ( 11) × (4.5 ( 0.4) × 10-2

(56 ( 23) × (4.5 ( 2.3) × 10-2

8700 × 10-2 b

8700 × 10-2 b

τ1/2 (FTIR)a (s)

τ1/2 (CA) (s)a

117 ( 5 1540 ( 10 1700 ( 20 0.8b

124 ( 5 1500 ( 10 1700 ( 20 0.8b

Calculated as τ1/2 ) ln 2/k′ for a base concentration of 1.00 × 10-2 M. b Data obtained/recalculated from ref 24 (T ) 25 °C).

intrachain functional groups on conformational order is taken into account. For SAMs of adsorbates comprising intrachain functional groups, such as esters, the transition from disordered to ordered chains requires a longer n-alkane segment than 10 methylene groups to show the mentioned transition from liquidlike (disordered) to solidlike (well-ordered).35 Consistent with this interpretation is the report by Vaidya et al., who did not observe any induction period for the hydrolysis of p-nitrophenyl ester groups in SAMs on gold.17 The systems investigated by these authors possessed no n-alkane segment; thus there is little possibility for interchain interactions resulting in a tightly packed array of ester groups that would form an effective barrier against attacking nucleophiles. The striking differences in rate law, as well as rate constants for the hydrolysis of the three SAMs with different chain lengths is compared with rate constants measured for the hydrolysis of NHS esters of anisole in Table 4.24 The reaction in solution proceeds 2 orders of magnitude faster compared to the fastest reaction presented in this study. A comparison of half reaction time calculated for a base concentration of 1.00 × 10-2 M drastically shows the differences between solution and confined reaction. These reactivity differences observed here may be attributed to differences in orientation, packing, and conformational order of the molecules confined in the monolayers, which can be expected to vary systematically with chain length (vide supra).14,18 As indicated by our FTIR spectroscopy results, SAMs of NHS-C15 possess an increased surface coverage of NHS ester groups. Hence SAMs of NHS-C15 appear to be more tightly packed, which would hinder the attack of the hydroxide ions on the ester groups and consequently reduce the rate constant of the hydrolysis. This interpretation is further supported by the peak positions of the C-H stretching vibrations (Figure 1, Table 2), the width at half-maximum of these bands, and the small contact angle hysteresis (Table 1). For SAMs with shorter chain length, we observed larger contact angle hysteresis and broad (or no) peaks for the C-H stretching vibrations. These results suggest increasing disorder and more facile reorientations of functional groups at the surface of the SAMs. Increasing disorder, reduced steric hindrance, and reduced crowding

at the SAM surface would release the constraint of the local environment and facilitate the attack of the hydroxide ions and thus increase the rate of reaction. This interpretation implies that surface reactions in SAMs can be controlled via proper design of the adsorbate structure. Loosely packed liquidlike SAMs may consequently increase the rate constants of surface reactions and favor completion of reactions after short reaction times, even though decreased order in the SAMs also results in a slightly reduced number of active sites per unit surface area. Conclusions The rate expressions, as well as the rate constants, of interfacial reactions in SAMs on gold were shown for the first time to depend critically on the length of the alkane segment linking the active ester to the (disulfide) anchor group. The alkaline hydrolysis of NHS ester groups confined in the SAMs investigated here is slowed compared to reactions in solution. For a segment length of n ) 15 methylene groups, an induction period was observed by FTIR spectroscopy and contact angle measurements. This induction period was absent for n ) 10 and n ) 2. The hydrolysis of these latter reactions could be described by pseudo-first-order kinetics; the correspondingly calculated second-order rate constants are three, respectively two orders of magnitude smaller than that reported for similar reactions in solution. Our results can be rationalized based on an increase in conformational order and packing density with increasing alkane chain length, as detected by FTIR spectroscopy, as well as contact angle measurements. This increasing conformational order results in a more tightly packed surface of NHS ester groups and in a steadily growing hindrance for nucleophilic attack of the hydroxide ions, which in turn slows down the rate of the reaction for short and more pronounced for intermediate chains lengths. In highly ordered, tightly packed SAMs the mechanism even changes from a homogeneously occurring reaction to a reaction, which spreads from defect sites. Acknowledgment. This work has been financially supported by the Council for Chemical Sciences of The Netherlands Organization for Scientific Research (CWNWO). LA0343066