pubs.acs.org/Langmuir © 2009 American Chemical Society
Esterification of Self-Assembled Carboxylic-Acid-Terminated Thiol Monolayers in Acid Environment: A Time-Dependent Study Annica Myrskog,†,‡ Henrik Anderson,‡,§ Teodor Aastrup,‡ Bj€orn Ingemarsson,‡ and Bo Liedberg*,† †
Division of Sensor Science and Molecular Physics, Department of Physics, Chemistry and Biology, Link€ oping University, SE-581 83 Link€ oping, Sweden, ‡Attana AB, Bj€ ornn€ asv€ agen 21, SE-114 19 Stockholm, Sweden, and § Uppsala University, A˚ngstr€ om Laboratory, Solid State Electronics, P.O. Box 534, SE-751 21 Uppsala, Sweden Received June 23, 2009. Revised Manuscript Received August 20, 2009 This contribution reports on the influence of acids on the quality of carboxylic-acid-terminated self-assembled monolayers (SAMs) on gold prepared from ethanolic solution of HS-(CH2)15-COOH and HS-(CH2)11CONH(EG)6CH2-COOH. Null ellipsometry, contact angle goniometry, and infrared reflection-absorption spectroscopy are used to monitor the physical and chemical changes occurring within the SAMs upon acid post treatment; after incubation with acids present in the solution; and after incubation in aged acid containing solutions. The presence of acid has a positive effect on the crystallinity, packing, and orientation of the supporting alkyl and ethylene glycol subunits of the SAM. Our studies also confirm previous findings stating that the carboxylic groups are rapidly converted into ethyl ester groups in the presence of hydrochloric acid in the incubation solution. It is also evident that the conversion occurs in the presence of the weaker acid, acetic acid, although at a much slower rate than that for hydrochloric acid. This is a new observation that has not been reported on before. The physical and chemical characterization is also complemented with a functional bioaffinity study. The functional evaluation revealed that the present model system was surprisingly insensitive to the degree of esterification of the carboxylic acid groups, but that 4 weeks of storage of the two investigated thiols in hydrochloric acid containing ethanol resulted in SAMs that were completely inactive with respect to immobilization and subsequent binding of the antigen. It was encouraging to note that the nonspecific binding of both antigen and antibody was extremely low on the two SAMs, regardless of the relative amount of ethyl esters on the surface.
Introduction Self-assembled monolayers (SAMs) of ω-substituted alkyl thiols on gold have attracted considerable interest during recent years because of their ease of preparation and the spontaneous formation of well-ordered and chemically robust two-dimensional (2D) architectures. By varying the constituting building blocks, terminal groups, and by mixing of different thiols in the incubation solution, the properties of the outermost layer can be fine-tuned with respect to wettability, lubrication, chemistry, nanotopography, and many more.1,2 They have been frequently used for studying fundamental adsorption phenomena and for controlling/blocking interactions occurring between contacting fluids and metal surfaces. The SAMs also have found numerous applications as model coatings for the development of new biomaterials and biosensors.3,4 Of particular interest is carboxylic-acid-terminated SAMs because the carboxylic group easily can be modified and reacted, for example, with primary amines, to form a strong and stable covalent bond. Thus, numerous strategies based on carboxylic acids have been developed for immobilizing biomolecules on solid supports. For SAMs to be of use in bioanalytical applications, they also need to form densely packed, defect-free, and chemically robust layers to enable regeneration in chemically harsh environ*Corresponding author. E-mail:
[email protected]. Telephone: +46 (0)13 28 18 77. Fax: +46 (0)13 13 75 68 (1) Ulman, A. Chem. Rev. 1996, 96, 1533–1554. (2) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. Rev. 2005, 105, 1103–1169. (3) Ostuni, E.; Yan, L.; Whitesides, G. M. Colloids Surf., B 1999, 15, 3–30. (4) Mrksich, M.; Whitesides, G. M. Annu. Rev. Biophys. Biomol. Struct. 1996, 25, 55–78. (5) Schoenfisch, M. H.; Pemberton, J. E. J. Am. Chem. Soc. 1998, 120, 4502– 5413.
Langmuir 2010, 26(2), 821–829
ments for repetitive use.5 They should also permit prolonged storage at elevated/varying temperatures to offer a shelf life that is practically acceptable and justified from a commercial point of view. Several reports have been published on the structure of carboxylic-terminated alkyl thiol SAMs with contradictory results. One of the first reports6 showed that a terminal carboxylic acid did not have a big impact on the structure and ordering of the alkyl chains. Others, however, have stated that unstructured carboxylic acid SAMs will be formed from ethanolic thiol solutions.7,8 The addition of acid to the incubation solution has been used as a mean to reduce electrostatic interaction between carboxylate groups that might be formed because of trace amounts of cations in the incubation solution. Thus, the acid will restore the carboxyl group into the acidic form (COOH), which will facilitate the formation of densely packed and ordered assemblies.9 Others have suggested that it reduces the risk of bilayer formation through dimer formation between COOH groups.10,11 Different acids have been investigated for these purposes including trifluoroacetic acid (TFA), hydrochloric acid (HCl), and acetic acid (HAc). When Arnold et al. investigated SAMs formed from HCl-acidified thiol solution with infrared reflection-absorption spectroscopy, the resulting SAM showed (6) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112, 558– 569. (7) Dannenberger, O.; Weiss, K.; Himmel, H. J.; J€ager, B.; Buck, M.; W€oll, C. Thin Solid Films 1997, 307, 183–191. (8) Himmel, H. J.; Weiss, K.; J€ager, B.; Dannenberger, O.; Grunze, M.; W€oll, C. Langmuir 1997, 13, 4943–4946. (9) Arnold, R.; Azzam, W.; Terfort, A.; W€oll, C. Langmuir 2002, 18, 3980–3992. (10) Willey, T. M.; Vance, A. L.; Van Buuren, T.; Bostedt, C.; Nelson, A. J.; Terminello, L. J.; Fadley, C. S. Langmuir 2004, 20, 2746–2752. (11) Wang, H.; Chen, S.; Li, L.; Jiang, S. Langmuir 2005, 21, 2633–2636.
Published on Web 10/15/2009
DOI: 10.1021/la902255j
821
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Myrskog et al.
peaks corresponding to an ester.9 It was proposed that the carboxylic acid reacts with ethanol in the presence of acid to form an ethyl ester. This was not seen for ethanolic thiol solutions acidified with HAc, and it was therefore recommended to use HAc instead of HCl to obtain well-ordered SAMs. Willey et al.10 confirmed better organization in the SAM with HAc present during assembly. On the other hand, a recent article comparing SAMs assembled from thiol solutions with and without HAc concluded that well-organized carboxyl-terminated SAMs were formed in both cases.12 Furthermore, they observed residues of HAc on the surfaces which were difficult to remove. Thus, given the contradictory results presented above, there is undoubtedly room for more detailed analyses of the influence of acids on the structural and chemical characteristics of carboxylic-acid-terminated SAMs, and their ability to be used as anchoring layers for further biomolecular derivatization. In this work, we report on two different compounds: a linear thiol HS-(CH2)15-COOH (MHDA) and an ethylene glycol (EG) containing carboxylic-acid-terminated thiol HS-(CH2)11CONH-(EG)6CH2-COOH (EG6COOH). Most work is conducted on MHDA, and the focus is on three different types of experiments: (1) influence of a short acid rinse on the structure of a SAM that is prepared in pure EtOH; (2) influence of acids in the EtOH incubation solution during formation of the SAM for different periods of time (1 h to 5 days); and (3) influence of acids on the aging of the incubation solution prior to SAM formation for 1 day (0-4 weeks of aging). Ellipsometry, contact angle goniometry, and infrared reflection-absorption spectroscopy (IRAS) are used to monitor the reactions occurring on and within the SAM architecture as a function of acid treatment, incubation time, and solution aging. The accessibility of the COOH groups upon further activation and derivatization with biomolecules and the influence of acid treatment on specific and nonspecific binding are studied with quartz crystal microbalance (QCM). Our experiments reveal that HCl treatment has huge influence on the rate of conversion of chemically reactive COOH groups into nonreactive CO2CH2CH3 groups, whereas the supporting alkyl part of the SAMs seems to withstand the acidic treatment. Interestingly, the crystallinity of the EG portion of the EG6COOH SAM improves substantially upon acidic treatment, resulting in a highly oriented assembly. Our studies support the recommendation proposed Arnold et al.9 However, it is also evident that one should avoid extensive aging of incubation solutions containing HAc because ethyl ester formation also occurs in that case, although at a much slower rate than that observed for HCl.
Experimental Section Sample Preparation. Gold samples were prepared by electron beam evaporation of Ti and Au on cleaned precut standard silicon (100) samples in a Balzers UMS 500P instrument. Twentyfive A˚ of Ti was evaporated at a rate of 2 A˚/s followed by 2000 A˚ of Au at a rate of 10 A˚/s. The gold surfaces were cleaned in a 5:1:1 solution of Milli-Q water (Milli-Q, Millipore), 30% hydrogen peroxide (Merck), and 25% ammonia (Merck) for 10 min at 85 °C, thoroughly rinsed with Milli-Q, and quickly rinsed with ethanol (99.5%, Kemetyl) before incubation in ethanolic solutions containing 20 μM MHDA (Prochimia, Poland) or 20 μM EG6COOH (synthesized in our laboratory, to be published elsewhere). MHDA SAMs formed from three different solvents were compared: ethanol, ethanol with 1% (v/v) 1 M HCl (10 mM HCl), and ethanol with 10% (v/v) HAc (glacial 100%, Merck) (1.75 M HAc) following the recommended protocol by Arnold et al.9 (12) Mendoza, S. M.; Arfaoui, I.; Zanarini, S.; Paolucci, F.; Rudolf, P. Langmuir 2007, 23, 582–588.
822 DOI: 10.1021/la902255j
Scheme 1. Illustration of the Two Main Incubation Experiments
Depending on the incubation solution, the surfaces were treated differently after the incubation. Surfaces incubated in ethanol were rinsed in ethanol, sonicated for 3 min, rinsed in ethanol, and blown dry in nitrogen gas before being analyzed. These samples were also rinsed for 10 s in a solution containing two drops of 1 M HCl (∼0.03% HCl corresponding to ∼4 mM HCl). In the following we refer to the M-values. Surfaces incubated in ethanol with 10 mM HCl were rinsed in ethanol with 10 mM HCl, sonicated 3 min, rinsed in ethanol, and blown dry in nitrogen gas before being analyzed, and surfaces incubated in ethanol with 1.75 M HAc were rinsed thoroughly in ethanol prior to being blown dry in nitrogen gas and analyzed according to the protocol by Arnold et al.9 Scheme 1 illustrates the two main incubation experiments undertaken. For the time study, samples were incubated in freshly prepared solutions for 1 h to 5 days, while for the solution aging study the gold samples were incubated for 1 day in a freshly prepared solution and in solutions that have been aged for 2 and 4 weeks. In one experiment, we also extended the aging to 8 weeks. Ellipsometry. For the ellipsometric measurements, a Rudolph Research AutoEl ellipsometer with a He-Ne laser source was used with λ = 632.8 nm and an angle of incidence of 70°. The refractive index was set to 1.5, and the thickness was calculated as an average of five spots on every sample. The optical constants for the clean gold surface had been determined prior to the incubation experiments and were used as input for the calculation of SAM thickness using the software provided by Rudolph Research. Contact Angle Goniometry. A CAM 200 goniometer (KSV Instruments Ltd.) was used to measure the static contact angle in air. Fresh Milli-Q was used, and the surfaces were measured directly after being blown dry with N2. The contact angles were measured as an average of two spots on every surface. IRAS. All infrared reflection-absorption (RA) spectra were collected using a Bruker IFS66 instrument with a grazing angle of 85° and a nitrogen cooled MCT detector. The measurement chamber was continuously purged with N2. Every measurement was a mean value of 3000 scans (data collection time of
