Langmuir 1991, 7, 2317-2322
2317
Functionalized Thin Films: Synthesis, Characterization, and Potential Use Shlomo Margel,* Orit Sivan, and Yehudit Dolitzky Department of Chemistry, Bar-Iian University, Ramat- Gan 52900, Israel Received May 24, 1991 Thin film coatings of trichloroalkylsilanecompounds, such as SiC13(CHdnX,wherein n = 3 and 16 and X = CH3 and CN, onto silicon and glass surfaces have been prepared and characterized. The coatings prepared from the longer chain alkylsilane compounds (n = 16) were superior to coatings prepared from the short ones (n = 3). The reduction of the w-nitrile groups of the previously described coating resulted in the formation of films containing w primary amine groups. Studies demonstratingthe effect of solvents, such as water and chloroform, on coatings containing w primary amine groups (hydrophilic surfaces) and w-methyl groups (hydrophobic surfaces)have been accomplished. The potential use of coatingscontaining w primary amine groups for cell immobilization have been demonstrated.
Introduction Maoz and Sagivl have demonstrated that self-assembled monolayer coatings prepared by the interaction of appropriate trichloroalkylsilane compounds with hydroxylbearing surfaces are similar in organization and packing to Langmuir-Blodgett monolayer films. These studies opened the door to the creation and studies of new types of monolayer coatings. Monolayer films containing at the w position various functionalgroups, e.g., olefins,l esters,2J thioesters,' hydroxyls,2*6amides,6 ethers,6 nitrile^,^ t h i ~ e t h e r sbromides: ,~ and sulfoxides7 have been previously described. These functionalized monolayer surfaces have been used for purposes such as organized multilayer formati0n,2*~1~ biomolecule immobilization,"" and chemical reactions, such as hydrolysis of esters,12J3reduction of nitrile groups,7 and transformation of olefins to alcohols" or acids.12 In this article the synthesis and characterization of thin coatings containing functionalized groups such as CN and primary amine groups are described. Self-assembled thin films containing w-nitrile groups were prepared by the interaction of trichloroalkylsilane compounds, such as SiCl3(CH2),CN, n = 3 and 16, with hydroxyl-bearing surfaces, such as glass. The formed w-nitrile films continuously grow during coating time due to self-polymerization of the w-nitrile trichloroalkylsilane compounds. The reduction of the w-nitrile groups of the previous films resulted in coatings containing w primary amine groups. In these studies it was also demonstrated that the coatings derivatized with the long alkyl chain are superior to the (1) Moaz, R.; Sagiv, J. J. Colloid Interface Sci. 1984, 100, 465. (2) Pomerantz, M.;Semuller, A,; Nezer, L.;Sa&, J. Thin Solid Films 1986, 232,153. (3) Tillman, N.; Ulman, A.; Penner, T. L. Langmuir 1989,5, 101. (4) Waeserman, S. R.; Biebuyck, H.; Whitesides, G. M. J. Mater. Res. 1989.4.888. -, (5) Bain, C. D.; Whitesides, G.M. Angew. Chem., Int. Ed. Engl. 1989, 28,508. (6) Tillman, N.; Ulman, A.; Schildkraut, J. S.; Penner, T. L. J. Am. Chem. SOC.1988,110,6136. (7) Balachander, N.; Sukenik, C. H. Langmuir 1990,6, 1621. (8) Nezer, L.; Iscovici, R.; Sagiv, J. Thin Solid Films 1983, 99, 235. (9) Ebersole, R. C.; Miller, J. A.; Moran, J. R.; Ward, M. D. J. Am. Chem. SOC. 1990, 112, 3239. (IO) Subramaniam, S.; Seul, M.; McConnell, H. M. Roc. Natl. Acad. Sci. U.S.A. 1986,83, 1169. (11) Margel, S.;Vogler, E. A.; Sogah, D. V. Submitted toJ. Am. Chem.
----.
SOC.
(12) Wasserman, S. R.; Tao, Y. T.;Whitesides, G.M. Langmuir 1989,
5, 1074.
(13) Gun,J.; Sa&, J. J. Colloid Interface Sei. 1986, 112, 457. (14) Netzer, L.;Sagiv, J. J . Am. Chem. SOC.1983, 105, 674.
0743-7463/91/2407-2317$02.50/0
short alkyl chain derivatized coatings. Studies demonstrating the effect of solvents, such as water and chloroform, on films containing w primary amine groups (hydrophilic surfaces) and w-methyl groups (hydrophobic surfaces) have also been carried out. The potential use of thew primary amine coatings for cell immobilization have been demonstrated.
Experimental Section Materials. The following commercial analytical-grade materials were used magnesium turnings, sodium, benzophenone, copper(1) iodide, methyllithium (1.4 M in ether), hexachloroplatinate, trichlorosilane,sodium phosphate monobasicand dibasic, chloroform (HPLC), methylene chloride (HPLC), ethanol (HPLC),acetone (HPLC),tetrahydrofuran(THF),bicyclohexyl (BCH), diborane (1 M in THF), ethyl ether, glutaraldehyde, sodium tetraborate, and 2,4,6trinitrobenzenesulfonicacid VNBS) from Aldrich; n-butyltrichlorosilane [SiC&(CH&CHs],n-octadecyltrichlorosilane [OTS; SiC&(CHz)&H3], and (3-cyanopropy1)trichlorosilane[SiC&(CH&CN]from ABCR, Karlsruhe,West Germany; 1-iodopropanonitrileand 11-undecylenebromidefrom Pfaltz & Bauer, Waterbury, CT. THF was dried over sodium in the presence of benzophenoneand then distilled before use. BCH was passed through basicalumina(Woelm-Pharma,W200super). Pure water was obtained by passing deionized water through Elgastat Spectrum reverse osmosissystems (Elga Ltd, High Wycombe Bucks,England),which combinesreverseosmosis,organic adsorption, and deionization cartridges. Phosphate-buffered saline solution (PBS; 0.1 M, pH 7.2) was prepared by adding sodium phosphatedibasic solution (0.1 M)to an aqueoussolution of sodium phosphate monobasic (0.1 M) until a pH of 7.2 was reached. Sodium chloride was then added to obtain a concentration of 0.15 M. Rabbit human red blood cells and human cancer cells (line K-562)16were obtained courtesy of Z. Malik from the Dept. of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel. A part of these cells were fixed by shaking them for 1 h at room temperature in PBS solution containing 2% glutaraldehyde. The cells were then washed in PBS by four repeated centrifugations. Synthesis of SiCls(CH2)&N. The synthesis of this compound was accomplished by a procedure similar to that described in the literature'J8 according to the following scheme: MI. ctyi. CUI CH,=CH(CH,),CH,Br
-
-
I(CHp)sCN
CH,=CH(CH,),CH,Cu-CH,MgBr+
-
HsiCb
CH2=CH(CH2),,CN SiCl,(CH,),,CN The yield of the formed w-nitrile trichloroalkylsilanecompound (15) Malik, 2.;Chitayat, S. D.; Langzam, Y. Cancer Lett. 1988,41,203. (16) Bergbreiter, D. E.; Whitesides, G.M. J. Org. Chem. 1976,40,779.
0 1991 American Chemical Society
Margel et al.
2318 Langmuir, Vol. 7,No. 10, 1991 was approximately 50% by weight. After vacuum distillation (0.02 mmHg, 170"C),this alkylsilane compoundgavethe expected IR, lH NMR, and mass spectra. Preparation of Solid Surfaces. All the work with the solid surfaces was accomplishedthrough the use of Teflon forceps and glass containers. The glass slides (8 X 8 X 1 mm) used in part of these studies were cut from manufacturing microscope glass slides. The glass slides used for cell binding studies were round and 8 mm in diameter. The quarz slides (37.5 X 12 X 1mm) used for this work were purchased from Westdeutsche Quartzschmeltze, D-2054 Geesthacht, Germany. Both the quarz slides and the glass slides were treated by soaking in 10% NaOH aqueous solution at room temperature for 24 h. The slides were then washed a few times in 1% HCl aqueous solution, pure water, and ethanol and then Soxhlet extracted with chloroform overnight. Before these surfaces were treated with the alkylsilane compounds, they were plasma treated for -30 min in an rf argon plasma (Harrick PDC-3xG plasma cleaner, Harrick, Ossining, NY). The silicon ATR plates (45O, 25 X 10 X 2 mm from Harrick) used in these studies were cleaned ultrasonically for 30 mininanethanol/chloroformmixture (l/l,v/v) and thensoxhlet extracted in chloroform for approximately 2 h. Before use, the silicon plates were plasma treated in the same way as the glass plates.8 Coating of t h e Solid S u r f a c e s w i t h t h e Alkylsilane Compounds. The solid substrates, after plasma treatment, were exposed to air for approximately 20 s and then immersed at room temperature for 3 h in an air-free BCH solution containing 0.1 % (v/v) of the studied alkylsilane compound. The substrates were then immersed twice in chloroform and Soxhlet extracted in chloroform overnight. For kinetic studies, after each desired period of time, the solid surface (a Si crystal) was removed from the BCH solution, immersed twice in chloroform, and then Soxhlet extracted in chloroform for 3 h. Then, contact angle and Fourier transform infrared/attenuated total reflection (FTIR/ ATR) measurements were accomplished. Thereafter, the crystal was immersed in chloroform, dried extensively by a stream of argon, and reimmersed in the air-free BCH solution. Most FTIR/ATR trials, e.g., the kinetic studies, have been carried out on a single Si crystal, since it was demonstrated by us as well as by otherd3 that the absorption peaks of the methylene groups of equal OTS films on different Si crystals have significantly different values. The removal of the alkylsilane films from the substrates was accomplished by argon plasma treatment of the coated surfaces until the methylene absorption peaks have disappeared. Reduction of the w-Nitrile Alkylsilane Coatings. The reduction of the w-nitrile groups of the surfaces derivatized with SiCl&H&,,CN (n = 3 or 16) to primary amine groups was accomplished by the immersion of these substrates at 50 O C for 4 h in a THF solution containing 1 M diborane. The reduced surfaces were then washed in 0.1 % HCl aqueous solution, pure water, and acetoneand Soxhlet extracted in chloroform overnight. In the latest experiments, the formed w primary amine coated surfaces were washed in 0.1 % HC1 aqueous solution and pure water and then were kept in pure water. The reduction of the surface nitrile groups to primary amine groups was confirmed through the derivatization of the amine groups with TNBS (a common reagent for determination of amine groups"). The diborane-reducedsampleswere immersed at room temperature for 5 h in an aqueous 0.01 M TNBS solution at pH 9.3 (0.1 M aqueous sodium tetraborate solution). The derivatized substrates were then washed with pure water and acetone and dried by argon stream. The absorption spectrum of the complex formed between TNBS and thew primary amine groups of the modified surfaces was then recorded. Binding of Cells onto the w Primary Amine Alkylsilane Coated Glass Slides. The coated glass slides were immersed at room temperature for 1h in PBS solution, pH 7.2, containing the desired concentration of cells (fixed or living). The slides were then removed from the cell solution and washed free of unbound cells by three successive immersions in PBS. The cellbound slides were then prepared for scanningelectron microscopy (SEM) examination by immersing the slides in increasing
-
(17) Snyder, S. L.; Sobocineki, P. Z. Anal. Biochem. 1976,64, 284.
concentrations of ethanol/water, up to 100% ethanol, and then immersing them in increasing concentrations of Freon 113/ethanol, up to 100% Freon 113. The conjugated slides were then dried in air. The percent coverage of the derivatized surfaces with the cells was determined with Image analysis computer system, Quantimet 900, Cambridge Instruments, Cambridge, England. Fourier Transform Infrared/Attenuated Total Reflection Spectra. The FTIR/ATR spectra (unpolarized) were collected on a Nicolet 60 SX spectrometer with an MCT detector. Typically, 1000spectra were averaged for each ATR experiment. All spectra manipulations were done with standard Nicolet software. Electron Spectroscopy for Chemical Analysis (ESCA). ESCA (also called XPS, X-ray photoelectron spectroscopy)data were obtained with a PHI-Unicam Perkin-Elmer instrument using Mg Ka lines, at 1X lO+'Torr, with a takeoff angle of 45O. Survey spectra were recorded on a 1-mm spot with 150-eV pass energy, 200-W electron beam power, and an acquisition time of 7 min. High-resolution multiplex spectra of individual elements were done on a 1-mm spot, with 50-eV pass energy, and an acquisition time of 30 min. Peak areas were measured and maxima were calibrated against the C 1s peak defined at 285.0-eV binding energy. Contact Angle Measurements. Contact angles were measured with a Rame-Hart Model 100 contact angle goniometer equipped with an environmental chamber, as described previously.18 The humidity in the environmental chamber was 100%. The temperature was not controlled and varied between 20 and 25 O C . The volume of the water drops used was always 10 pL. All reported values are the average of at least seven measurements taken at different locations on the derivatized surface and have a maximum error of f3O. Scanning Electron Microscopy. SEM photomicrographs were obtained on a JEOL,JSM-840 instrument. Dried, coated glass slides were mounted with double-face adhesive tape on a stud and then coated under vacuum with a thin layer of gold to a depth of -50 A.
Results and Discussion Figure 1 demonstrates FTIR/ATR spectra of films prepared from OTS (A) and from SiCl@HZ)l&N (B)onto a Si crystal. Figure 1C describes the spectrum of coatings formed by the reduction of the w-nitrile groups of the coatings described in Figure 1B. The coatings of OTS and of the w-nitrile alkylsilane compound were prepared at room temperature for 3 h in BCH solution containing 0.1 % of the alkylsilane compound. The films derivatized with w primary amine groups (Figure IC)were prepared by reduction of the w-nitrile groups of the nitrile-alkylsilane coating with 1M diborane in THF. The absorption peak at ca. 2245 cm-l corresponds to the nitrile group stretching band. The absorption peaks at ca. 2850 cm-l and a t ca. 2920 cm-l correspond to -CHZ-stretching bands. The absorption peak at ca. 2960 cm-' corresponds to the -CH3 stretching band. Previous studies have demonstrated that the asymmetric -CHz- stretching band at ca. 2920 cm-l can be selected as best suited for quantitative correlation of similar coatings prepared on solid surfaces.1,8J+21 This band is dominant in all measured FTIR/ATR spectra of alkylsilane compounds and its shape does not change. For example, it was demonstrated in some of the cited references that the absorption peak at ca. 2920 cm-l for two or three monolayers of coatings prepared from OTS and/or arachidic acid are 2 or 3 times higher, respectively, than the absorption peak obtained for a single monolayer coating of the same quality. The (18)Holmes-Farley, S. R.; Reamey, R. H.; McCarthy, T. J.; Deutch, J.; Whitesides, G. M.Langmuir 1985, 1, 725. (19) Levine, 0.; Zisman, W. A. J. Chem. Phys. 1957, 66, 1068. (20) Shafrin, E. G.; Zisman, W. A. J. Phys. Chem. 1962,66, 740. (21) Shafrin, E. G.; Zisman, W. A. Adu. Chem. Ser. 1969, No. 87, 20.
Functionalized Thin Films
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Langmuir, Vol. 7, No. 10, 1991 2319 I
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Figure 2. Absorption spectrum for a coating prepared from the nitrile-reduced SiCl3(CH2)sCN+ TNBS onto a quarz slide. A quarz slide derivatized with the nitrile-reduced SiC13(CH2)&N was immersed for 5 h at room temperature in an aqueous 0.01 M TNBS solution at pH 9.3. The derivatized slide was then washed according to the experimental procedure.
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intensity of the absorption peak at ca. 2920 cm-' of the w-nitrile alkylsilane coating is slightly higher than that obtained from OTS (0.027and 0.02 cm-', respectively). Previous publications have demcinstrated that films formed from OTS are composed from highly organized closepacked monolayer structure.'p2I8 Therefore, it may be possible to assume that under the indicated experimental conditions the coverage obtained from the w-nitrile alkylsilane compound films is slightly higher than a monolayer coverage. Figure 1Cdemonstrates that the reduction of the w-nitrile groups of the nitrile surfaces was complete, as indicated by the disappearance of the nitrile band at ca. 2245 cm-l. This FTIR/ATR behavior was obtained for both the long- and the short-chain w-nitrile alkylsilane films. The presence of primary amine groups formed by the reduction of the surface nitrile groups has been demonstrated by the reaction of the formed w-amine-modified surfaces with TNBS.17 Figure 2 presents an adsorption spectrum for coatings prepared from the nitrile-reduced SiC13(CH&CN onto a quarz slide, indicating the expected absorption peaks of the TNBS complex at 420 and 350 11m.l' Further evidence for the presence of primary amine groups was also obtained by ESCA studies. Despite the relatively broad signals, the various kinds of nitrogencontaining functional groups could be distinguished: the nitrile signal was a t 399.9 eV and the amine signal was at 400.6-401 eV. The reaction of surfaces containing hydroxyl groups, particularly silica and glass surfaces, with trichloroalkylsilane compounds has been intensively studied.12 6 7 12,22,23 The structure of the coatings prepared by this reaction is highly complicated and not yet defined. The absence of 9
9
9
9
(22) Yang, S.S.; Gilpin, R. K. J. Chromatogr. 1988,449, 115. (23) Nawrocki, J.; Buszewski, B. J. Chromatogr. 1988, 449, 1.
chlorine atoms in the formed coatings, as obtained by our ESCA studies as well as by others,12 indicates that each silicon atom of the obtained coatings must have formed three silicon-oxygen bonds. Generally, only one chlorine of the trichloroalkylsilane compound will react with the hydroxyl surface; the remaining chlorine groups can either be cross-linked or be hydrolyzed and then may undergo a further reaction with excess reagent.1J2*22*23 Table I demonstrates the characterization of the various coatings prepared from the trichloroalkylsilane compounds onto glass and silicon crystals obtained by FTIR/ATR, ESCA, and contact angle measurements. This table demonstrates that the films formed by the long-chain alkylsilane compounds are superior to those formed by the short ones; e.g., advanced contact angles of llOo and 100° with hysteresis of 5 O and 20° were obtained for coatings prepared from OTS and from SiC13(CH2)3CH3, respectively. Similar behavior was obtained for coatings prepared from the long-chain w-nitrile alkylsilane compound and the short one; e.g., contact angles of 70° and 48O with hysteresis of 6 O and 10' were obtained for coatings prepared from SiCl3(CH2)&N and from SiCldCH2)3CN, respectively. The superiority of coatings prepared from long-chain trichloroalkylsilane compounds to coatings prepared from the short ones has also been demonstrated in previous p ~ b 1 i c a t i o n s . l The ~~~~ long-chain trichloroalkylsilane compounds may form highly organized closepacked monolayer films which are considered to be equivalent to the monolayer structures obtained by the Langmuir-Blodgett (LB) trough technique25in the condensed phase, while the structure of films formed by the short-chain trichloroalkylsilane compounds are less ordered and packed and may be considered to be equivalent to the structures obtained by the LB method in the liquid pha~e.'~?~~ Table I also demonstrates that the advanced contact angles of the coatings prepared from both the long- and short-chain alkylsilane compounds decreased as a consequence of the reduction of thew-nitrile groups to primary amine groups from 70' to 5 8 O and from 48' to 38O, respectively. This decrease in the advanced contact angles can be explained by the increased hydrophilic nature of the reduced coatings. Furthermore, the relative small decrease in the FTIR/ATR absorption peak intensity a t ca. 2920 cm-' of coatings containing w-amine groups compared to coatings containing w-nitrile groups (Figure 1and Table I) and the insignificant difference in the atomic percent concentrations of nitrogen and carbon of these (24) Maoz, R.; Sagiv, J. Langmuir 1987,3, 1034. (25) Gaines, G. L. In Insoluble Monolayers at Liquid-Gas Interfaces; Intersciences Publishers: New York, 1966.
2320 Langmuir, Vol. 7, No. 10,1991
Margel et al.
Table I. Characterization by FTIR/ATR, ESCA, and Contact Angle Measurement6 of Coatings Prepared from Trichloroalkylsilane Compounds onto Glass and Si Crystals.
0.02
110
5
100 70 69 58 58 48 48 39 38
20
20.0 0.0035 0.027 26.3
1.7
0.06
0.06
28.9
1.8
0.06
0.06
24.2
7.0
0.28
0.25
28.5
5.8
0.20
0.25
0.026 0.016 0.018
6 10
a Si ATR crystals and glass slides were immersed for 3 h at room temperature in a BCH solution containing 0.1% (w/v) of the trichloroalkylsilane compounds. The derivatized surfaces were then washed according to the description in the ExperimentalSection. The terminal nitrile derivatized surfaces were reduced in a THF solution containing 1 M diborane,according to the description in the ExperimentalSection.
coatings (Table I) demonstrate the relative stability of these coatings toward the reduction conditions. Rate of Formation of Coatings Prepared from the Trichloroalkylsilane Compounds onto a Si Crystal. Figure 1A,B demonstrates, as previously described, that under similar coating conditions, e.g., coating a Si crystal for 3 h at room temperature in BCH solution containing 0.1 r0 of the alkylsilane compounds, the intensity of the absorption peak at ca. 2920 cm-l corresponding to the coating of SiCl3(CH2)&N is slightly higher than that obtained for the coating prepared from OTS (0.027 and 0.02 cm-l, respectively). These results may indicate, as previously discussed, that under these experimental conditions the coverage of the Si crystal surface by the nitrile-alkylsilane compound may be slightly higher than a monolayer coverage. ESCA survey spectra data of coatings onto glass surfaces prepared from various trichloroalkylsilane compounds under the same conditions (coating for 3 h at room temperature in 0.1% BCH solution) are presented in Table I. Monolayer coverage of the surfaces by coatings prepared from SiCl3(CH2)&N and from SiCls(CH2)3CNshould give an atomic concentration ratio of nitrogen of 1/1and an atomic concentration ratio of carbon of 17/4, respectively. However, the nitrogen and carbon atomic concentration ratios for the long-chain compound compared to the short one were found to be 1.7/7.0 and 26.3/24.2, respectively, as shown in Table I. These results may indicate that the mole coverage of the surfaces by the short-chain w-nitrile alkylsilane compound is approximately 4 times higher than the coverage obtained for the long one. The higher coverage of the surfaces obtained with SiC13(CH2)&N compared to OTS and that of the short-chain w-nitrile alkylsilane compound compared to the long one may be explained by self-polymerization of the w-nitrile alkylsilane compounds in the BCH solution. The polymerization process may be happened through the formation of Si-0-Si bonds before or after the binding of the w-nitrile alkylsilane compound to the solid surfaces. Indeed, if the coating of the glass surfaces is continued for a longer time, e.g., 12 h instead of 3 h, the glass surfaces become opaque due to the accumulation on the surface of a high concentration of this polymer. The polymerization process described here requires the presence of an extremely low concentration of water, which can hardly be prevented. Figure 3 describes the kinetic behavior of films prepared from OTS (A) and from SiCl3(CH2)&N (B), as demonstrated by the change in the absorption peak at ca. 2920 cm-1 with coating time. The absorption peak at ca. 2920 cm-1 of the film prepared from OTS becomes almost constant after approximately 3 h while that of SiC13(CH2)16CNcontinuously increased
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Figure 3. Change in the absorption peak at ca. 2920 cm-l with time of coatings prepared from OTS (A) and from SiC&(CH2)16CN (B) onto a Si crystal. A Si crystal was immersed at room temperature in BCH solution containing 0.174 of the studied alkylsilanecompound. At each noted interval of time, the crystal was removed from the BCH solution and washed for the FTIR/ ATR measurements according to the experimental procedure. during the coating process, indicating the continuous formation of this film. A similar behavior was obtained for films prepared from SiCl3(CH2)3CH3 (Figure 4A) and from SiCls(CH2)3CN (Figure 4B). The absorption peak at ca. 2920 cm-l of the film prepared from SiCh(CH2)sCH3 becomes almost constant after approximately 8 h, while that prepared from SiC&(CH2)3CN continuously increased during the coating process. Solvent Effect on thecontact Angle of thecoatings. Our preliminary studies indicated that the water advanced contact angles of the w primary amine surfaces were not consistent. For example, coatings prepared by reduction, according to the experimental procedure, of the nitrile groups of surfaces derivatized with the short-chain w-nitrile alkylsilane compound resulted in a contact angle of approximately 39". However, when these derivatized surfaces were Soxhlet extracted in chloroform, the contact angle significantly increased, up to approximately 50". The effect of water and chloroform on the change in the advanced contact angle with time of films derivatized with w primary amine group and films derivatized with CH3 groups (OTS coating) is demonstrated in Figure 5. The contact angle of coatings prepared from OTS was not effected by these solvents. On the other hand, the contact angle of the w primary amine surfaces decreased in hot water, slightly increased in chloroform a t room temperature, and rapidly increased in hot chloroform. This behavior was also found to be reversible. The progression of the contact angle in chloroform may suggest that the
Functionalized Thin Films
Langmuir, Vol. 7, No. 10,1991 2321
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Figure 4. Change in the absorption peak a t ca. 2920 cm-l with time of coatings prepared from SiC13(CH2)3CH3 (A) and from SiCl&H2)3CN (B) onto a Si crystal. A Si crystal was immersed at room temperature in BCH solution containing 0.1% of the studied alkylsilane compound. A t each noted interval of time, the crystal was removed from the BCH solution for the FTIR/ ATR measurements according to the experimental procedure. H,O-75"C
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TIME (h) Figure 5. Change in the advance contact angle with time of coatings immersed successively in H2O at 75 OC for 26 h, then in chloroform at room temperature for 51 h, and then in chloroform at 55 "C for 31 h. (A) demonstrates the behavior of glass surfaces coated with the w primary amine short-chain alkylsilane compound; (B) demonstrates the behavior of glass surfaces coated with the w primary amine long-chain alkylsilane compound; (C) demonstrates the behavior of glass surfaces coated with OTS.
polar primary amine groups disappear from the interface by diffusion. The o primary amine groups restructure in chloroform and minimize the free energy of the system by burying the amine groups as much as possible and exposing more CH2 groups to the surface. This reconstruction process is slow at room temperature and becomes faster
Figure 6. SEM photomicrographs demonstrating the binding of rabbit human red blood cells (107 cells/mL) onto glass slides coated with SiCls(CH2)&!N (A) and with its nitrile-reduced form (B).
at higher temperatures. Such "surface reconstruction" has been postulated for other hydrophilic surfaces, e.g., oxidized polyethylene, which consists largely of exposed carboxylic acids, with smaller amounts of ketones and aldehyde groups,26and gold surfaces coated with @-hydroxyl alkylthiol (e.g., OH(CH2)21SH) monolayer^.^^ Cell Binding onto the o Primary Amine Coated Glass Surfaces. It is common to bind cells onto glass slides for microscopypurposes by coating the glass surfaces with p ~ l y l y s i n e The . ~ ~binding ~~ of cells to the polylysinecoated glasssurfacesis based on electrostatic forcesexisting between the positively charged amino groups of polylysine (pK, of the free amine is basic, -10) and the negatively charged cell membrane. Usually, the coating of glass surfaces with polylysine is accomplished by soaking the glass surfaces in 0.1 % aqueous polylysine solution. The glass surfaces are then removed from the polylysine solution and ai~dried.~*~O The polylysine-coating procedure is based on weak physical attraction forces between polylysine and the glass surfaces. Therefore, this procedure should be carried out a short time before the cell attachment work. The polylysine coating on the glass is also not stable and can easily be peeled off the glass surfaces. Hereby, preliminary studies demonstrating the (26) Holmes-Farley, S. R.; Reamey, R. H.; NUZZO, R.;McCarthy, T. J.; Whitesides, G. M. Langmuir 1987,3, 799. (27) Evans, S. D.; Sharma, R.; Ulman, A. Langmuir 1991, 7,156. (28) Hopkins, C. R. J. Cell Biol. 1977, 73, 685. (29) Yavin, E.; Yavin, Z. J. Cell Biol. 1974,62, 540. (30)Liberman, I.; Ove, P. J. Biol. Chem. 1985,233,637.
2322 Langmuir, Vol. 7, No. 10, 1991 Table 11. Numbers of Rabbit Red Blood Cells Bound to Various Coatings onto Glass Slides. coatings cells/mL bound cellslareab 10 glass 108 14 glass/Si(CHz)&N 108 14 alass/Si(CHhCN 106 106 235 &ss)Si(CH&NH2 108 317 glass/Si(CHz)rNHz glass/Si(CHz)rNHn 107 345 106 105 glass/Si(CH~)l.~NHz 108 132 glass/Si(CHz)l.~NHz 107 259 glass/Si(CHz)l7NHz a The coated glass slides were immersed for 1 h at room temperature in PBS solutioncontaining various concentrations of the rabbit red blood cells. The slides were then removedfrom the physiological aqueous solution and were treated according to the experimental procedure. b Bound cells were counted in an area of 47 453 Mm*.
use of thew primary amine alkylsilane coated glass surfaces for cell immobilization are described. Figure 6 demonstrates the binding of living rabbit human red blood cells onto glass slides coated with SiC13(CH2)3CN(A) and onto its nitrile-reduced form (B). The w-nitrile-coated surfaces (control) contain a negligible number of attached cells. On the other hand, the w primary amine coated surfaces are covered with a significant number of cells. Table I1 summarizes the binding of different concentrations of rabbit red blood cells to various coatings on glass slides. Similar results were obtained when the rabbit red blood cells were replaced by human cancer cells (line K-562), and when fixed cells were used instead of living cells. The results for each coating represent the average of cells bound to three coated glass slides. The control surfaces, e.g., noncoated glass and w-nitrile alkylsilane coated glass, are covered with a negligible number of cells, 10-14 cells for On the other hand, the w primary each area of 47 453 "2.
Margel et al.
amine alkylsilane coated glass surfaces are covered with a significant amount of cells. The amount of cells attached to the surfaces was higher as the cell concentration increased; e.g., for cell concentrations of lo5, loe and 107, 235,317, and 345 cells per area of 47 453 pm2,respectively, were attached to glass surfaces coated with the short w-amine alkylsilane compound. Increasing the cell concentration to lo8 cells/mL resulted in approximately the same cell coverage as obtained for a cell concentration of lo7cells/mL. Table I1 also demonstrates that the percent coverage of the w primary amine coated surfaces by the cells was higher for coatings derivatized with the short alkylsilane chain than for coatings derivatized with the long one. For example, in the presence of lo5, lo6,and lo7 cells/mL, 235,317, and 345 cells, respectively, were bound to the glass surfaces coated with the short alkylsilane chain and 105, 139, and 259 cells, respectively, were bound to the glass surfaces coated with the long alkylsilane chain. This difference in the percent coverage of the surfaces with the cells may be explained by the higher concentration of primary amine groups of coatings prepared under similar conditions and derivatized with the short alkylsilane chain rather then with the long one (see Table I, ESCA measurements). An additional explanation may be the higher mobility of the short alkylsilane chain films compared to the long. Further detailed studies attempting to improve our understanding of various aspects of this project, e.g., the organization and packing of the w-nitrile alkylsilane films and its reduced form, the reconstruction of w-nitrile and w-amine alkylsilane films as a function of pressure, solvents, temperature, etc., and the relationship between the cell coverageand the advanced contact angle of the w primary amine surfaces, are ongoing in our laboratories.
