Langmuir 1992,8, 2361-2364
2361
Polydiacetylene Monolayers Functionalized with Amino Acids Troy E. Wilson and Mark D. Bednarski' Department of Chemistry, University of California at Berkeley, and the Center for Advanced Materials, Lawrence Berkeley Laboratory, Berkeley, California 94720 Received September 23, 1991. I n Final Form: November 11, 1991 Monomolecular polydiacetylene f i i have been construded from a seriesof lipid monomers functionalized with amino acids. A wide range of surfaces including plastics, glass, and metals can be conveniently coated with peptides using these films. The techniques of X-ray photoelectron spectroscopy, fluorescence microscopy, and contact angle measurements were used to characterize the films. These studies indicate that the amino acid head groups are localized at the film-ambient interface and suggest that the films are structurally ordered both laterally and vertically. Thus, polydiacetylene fiims may provide promising model systems for investigating the fundamental properties of interfaces.
Introduction The control of interfacial properties using organic thinfilm technology has been the subject of considerable attention.' We are currently exploring the relationship between the molecular architecture of thin films and the macroscopic property of biological adhesion. Progress in this area has demonstrated the control of protein adsorption and the construction of spatially defined arrays of oligopeptides.2 Our investigations required methodology to modify a variety of materials with thin films incorporating complex functional groups and a means to assess rapidly the film quality and reproducibility. In our hands, the established methods of molecular self-assembly and Langmuir-Blodgett techniques did not fulfill these req~irements.~-~ This paper reports an alternative approach to surface modification using monomolecular, peptide-functionalized, polydiacetylene films that are formed by the method of two-dimensionalcrystallization. These films allow for a range of surfaces including plastics, glass, and metals to be readily coated with a highly fluorescentorganic monolayers. Characterization of films transferred to glass surfaces by X-ray photoelectron spectroscopy (XPS), contact angle measurements, and fluorescence microscopy confirms that the peptide head groups are localized at the film-ambient interface. These results indicate that polydiacetylene films provide a (1)For reviews, see: (a) Bain, C. D.; Whitesides, G. M. Adu. Mater. 1989,4,110.(b) Swalen, J. 9.; Allara, D. L.; Andrade, J. D.; Chandross, E. A,; Garoff, S.; Israelachvili,J.; McCarthy, T. J.; Murray, R.; Pease, R.
F.;Rabolt,J.F.;Wynne,K.J.;Yu,H.Langmuir1987,3,932. (c)Ringadorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988,27,113. (2)(a) Prime, K.L.; Whitesides, G. M. Science 1991,252,1164. (b) Fodor, S.P. A.; Read, J. L.; Pirrung, M. C.; Stryer,L.; Lu, A. T.; Solas, D. Science 1991,251,767. (3) References concerning the self-assemblyof thiols on gold surfaces include (a) Evans, S. D.; Urankar, E.; Ulman, A,; Ferris, N. J.Am. Chem. SOC. 1991,113,4121.(b) Widrid, C. A.; Alves, C. A.; Porter, M. D. J. Am. Chem. SOC. 1991,113,2805.(c)Hickman,J. J.;Ofer,D.;Zou,C.;Wrighton, M. S.; Laibinis, P. E.; Whitesides, G. M. J. Am. Chem. SOC.1991,113, 1128. (d) Nuzzo, R. G.; Korenic, E. M.; Dubois, L. H. J. Chem. Phys. 1990,93,767. (e) Chidsey, C. E. D.; Bertozzi, C. R.; Putvinski, T. M.; Mujsce, A. M. J. Am. Chem. SOC.1990,112,4301. (4)Referencea for the modificationof silicon and glassviaself-assembly include (a) Tidswell, I. M.; Ocko, B. M.; Pershan, P. S.; Wasserman, S. R.; Whitesides, G. M.; Axe, J. D. Phye. Reu. B 1990,41, 1111. (b) Waseerman, S.R.; Tao, Y.-T.; Whitesides,G . M. Langmuir 1989,5,1074. (c) Ariga,K.;Okahata,Y.J.Am. Chem.Soc. 1989,111,5618.(d) Tillman, N.;Ulman, A.; Schildkraut, J. S.; Penner, T. L. J. Am. Chem. SOC.1988, 110, 6136. (e) Andersson, L. I.; Mandenius, C. F.; Mosbach, K.
Tetrahedron Lett. 1988,29,5437. (5)References for surface modification using Langmuir-Blodgett methodologyinclude(a)MBhwald,H.Angew. Chem.,Int.Ed.Engl. 1988, 27,728. (b)Gainea,G.L.InsolubleMonolayers at Liguid-C~Interfaces; Intarscience: New York, 1966. (c) Ringsdorf, H.; Schlarb,B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988,27,113.
promising alternative for surface modification methods incorporating complex head groups.
Experimental Section Film Formation Procedure. An aqueous layer (subphase) was applied to a 5 cm X10 cm Teflon trough or a glass slide and heated to 75 f 2 OC. The Teflon and glass surfaces were washed with 1 0 1 H2SO4/H202 followed by exhaustive rinsing with millipore water (>18MQcm) and dryingunder Nagas. Caution! A solution of H202 in H2SOl (*piranha")is an extremely strong oxidant which reacts violently with organic solvents and materials. The temperature of the aqueous subphase was determined using chromel-alumel thermocouples both in the water subphase and on the Teflon or glass surfaces. Solutions of 7-10 in chloroform or 1:l dichloromethane/hexane were prepared, and approximately 20 rL of a 2 mM lipid solution was administered to the water surface. Solution concentrations of lipids 7-10 were determined using lH NMR spectroscopy with benzene as an internal standard. After lipid application, the aqueous subphase was allowed to thermally equilibrate for 5 min and then slowly cool to 10f 2 OC to initiate crystallization.Cooling was effected using gradient heating elements beneath the Teflon or glass slide surfaces.6 Exposure to UV irradiation at 254 nm for 10 min cross-linked the films which took on the red-blue tint indicative of polydiacetylene conjugation. Irradiation was accomplished using a hand-held UV lamp (Model UVGL-25 Multiband UV 254/366 nm, UVP Inc., San Gabriel, CA 91778). The polymerizedmonolayera were then transferred from the water surface to hydrophobic supports by touching the two surfaces together. X-ray Photoelectron Spectra. XPS spectra were obtained using a Phi 5300 spectrometer (Perkin-Elmer Instruments) with Mg Ka radiation of 1253.6 eV, quartz monochromator, concentric hemispherical analyzer operating in fixed analyzer transmission mode, and multichannel detector. The pressure in the chamber was approximately 5 X 10-8Torr. Survey spectra were recorded with a 45O takeoff angle, 180-eV pass energy, 20 mm2 spot, and 400-W electron beam power with an aquisition time of 10 min. High-resolution spectra were recorded with an 18-eVpass energy, l-mm spot, and 400-W power. Signals were referenced to the Ag 3d5p peak at 367.9 eV. Contact Angle Measurements. Receding contact angles were determined by the sessile drop technique using a RameHart Model 100 goniometer at room temperature and under ambient humidity.' Fluorescence Microscopy. Micrographswere recorded using a Zeiss Axioskop fluorescence microscopewith Epiplan Neofluar X10/0.85 and X50/0.85 objectives and BP 450-490 (blue) exciter filters. ( 6 ) A complete description of the methodology and apparatw used to construct the diacetylene films is available in Wilson, T. E.; Bednareki, M. D. Self-Assembled Molecular Films. U.S.Patent 07 617 988, 1990. (7)Dettre, R. H.; Johnson, R. E. J. Phys. Chem. 1966,63,1507.
0743-746319212408-2361$03.00/0 0 1992 American Chemical Society
Letters
2362 Langmuir, Vol. 8, No. 10, 1992
Scheme I A
carbcnYl Shoulder (289.3 ev)
300.0
284.0
282.0 288.0 Blndlng Enorgy (eV)
298.0
280.0
0
Results and Discussion Monomer Synthesis and Film Construction. Lipids 7-10 were readily synthesized from commercially available 10,12-pentacosadiynoic acid (PDA) (1) by the sequence depicted in Scheme I.8 PDA was first treated with N-hydroxysuccinimide (NHS)and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide(EDC) to generatethe activated NHS eater 2 in 94 7% yield. Reaction of compound 2 with ethanolamine (3), alaninamide (4), serinamide (5), and phenylalaninamide (6) afforded the lipids 7-10. The yields for the alaninamide, serinamide, and phenylalaninamide products were approximately60 %5 while the yield for the ethanolaminederivativewas90 9%. We chose amino acids because they allowed access to a diverse set of functional head groups. Diacetylenic lipids were used because they could be polymerized via a noninvasive photochemical initiation to give a strongly fluorescent organicm~nolayer.~ The fluorescenceproperties provided a convenient assay for film quality and coverage. Monolayers of lipids 7-9 were constructed using methodology described elsewheree and polymerized with ultraviolet irradiation a t 254 nm.Io The polymerized films appeared slightly red in color which is indicative of the conjugated polydiacetylene backb0ne.I' After polymerization, the monolayers were readily transferred to hydrophobic supports by touching the supports to the water surface. We have successfully transferred polydiacetylene films to a range of materials, including graphite, polyethylene, cellulose acetate, polystyrene, and silylated (hydrophobic) glass slides and silicon wafers.12 X-ray Photoelectron Spectroscopy. The interfacial composition and the orientation of the amino acid head groups were studied using angle-resolved X-ray photoelectron spectroscopy (XPS).13 Spectra in the regions of (8) The identity of all compounds was established by characterization including IHNMR, 1% NMR, MS, and either HRMS or elemental analysis. (9) Day, D.; Lando, J. B. Macromolecules 1980, 13, 1478. (10) The phenylaleninamide derivative 10 did not form reproducible films. We suspect that the hydrophobic phenylalaninamide moiety is expelled from the aqueous phase which disrupta the monolayer order. For a more detailed discussion, see: MacRitchie, F. Chemistry at Interfaces; Academic Prese: San Diego, 1990; p 21. (11) For referencea concerningthe useof diacetylene lipids toconstruct films,see: (a) Kuo, T.; OBrien, D. F. Langmuir 1991,7,584. (b) GBbel, H. D.; Kjaer, K.; Ah-Nielaen, J.; Mbhwald, H. Thin Solid F i l m 1989, 179,41. (c) Dorn, K.; Ringsdorf, H. Contemp. Top. Polym. Sci. 1984.5, 73. (d) OBrien, D. F.;Whiteoidea,T. H.; Klingbiel, R. T. J . Polym. Sci., Polym. Lett. Ed. 1981, 19, 95. (e) Hub, H.; Hupfer, B.; Ringsdorf, H. Springer Ser. Chem. Phys. 1980,11,253. (0Chance, R. R.; Patel, G. N.; Witt, J. D. J. Chem. Phys. 1979, 71, 206. (12) Hydrophobic glass and silicon supports were prepared using a procedure adapted from one presented in Maoz, R.; Sagiv, J. Thin Solid F i l m 1986,132, 135. (13) Practical Surface Analysis by Auger and X-ray Photoelectron Spectroscopy, 2nd ed.;Briggs, D., Seah, M. P., Eds.; Wiley and Sons: Chicheater, U.K., 1990; Vol. 1, Chapter 4.
I
300.0
I
I
I
206.0
r
I
I
I
I
202.0 288.0 Blndlng Energy (EV)
I
284.0
I 280.0
Figure 1. (A) C 1s region of an XPS spectrum obtained for a PDA-alaninamide film supported on a silylated glass slide. A computer-assistedpeak separation for the C 1s signal shows two overlappingpeaks. The higher binding energy shoulder at 289.3 eV correspondsto the heterosubstituted carbons,and the parent peak at 286.5 eV corresponds to the substituted carbons of the lipid portion. (B)C l e signal intensity for the PDA-alaninamidef i i as a function of takeoff angle. Decreasingtakeoff anglea (75', 60°, 4 5 O , 30°, and 15') are displayed from back to front, and lower values for the takeoff angle~correspondto shallower XPS sampling. Note that the signal intensity for the parent peak at 286.5 eV declines steadily with decreasing takeoff angle while the shoulder at 289.6 eV remains independent of takeoff angle.
*
.......
-c-
C l s (satd.)
Cls (shoulder) Si25
I
04 0.2
0.4 0.6 0.8 Sine (Take Off Angle)
I
1.0
Figure 2. A composite presentation of the C 1s (saturated),C 1s (shoulder),N Is, Si 28, and Si 2p peak areas as a function of takeoff angle. The data are plotted as intensityversus the sine of the takeoff angle. Larger values for the sine reflect XPS sampling from atoms deeper in the bulk. Note that the Si 2s, Si 2p, and saturated C 1s peaks increase steadily, showing the increasing contribution of the silicon substrate and the atoms of the saturated carbon chains. In contrast, the N 1s signals due to the amidenitrogens and the high binding energy C 1s shoulder are nearly independent of takeoff angle. interest were obtained as a function of takeoff angle. (Recall that the takeoff angle is defined as the angle between the surface plane and the detector axis.) Rotation of the specimen holder allowed the takeoff angle to be varied from 75' to 15'. The angle between the incident X-rays and the electrons collected by the energy analyzer was fixed at 45' so that the angular dependence of the
Langmuir, Vol. 8, No.10, 1992 2363
Letters
1 .
,
C
Figure 3. (A)A fluorescence micrograph of a PDA-ethanolaminefilm supportedon a silylated glass slideobtainedat X 100 magnification using rhodamine filters. A grain boundary between two microcrystallinedomains is just visible near the center of the image (scale 20 pm). (B)A fluorescence micrograph of a PDA-alaninamide film at XlOO magnification (scale 20 pm). Visible in this image are several tears in the monolayer due to the transfer process. (C) A magnified view (X500) of the PDA-ethanolamine film depicted in (A) (scale5 pm). The dark spots on the image are pores formed from contraction of the monolayer during the polymerizationreaction. (D) A magnified view of the PDA-alaninamide film shown in (B)(scale 5 pm). The morphology of the pores is not identical to the PDA-ethanolamine film which may reflect unequal cooling rates or different steric demands on the head groups.
photoelectric emission cross section did not affect the angle-resolved XPS spectra. The presence of heterosubstituted (carbonyl) carbons on the lipid monomers introduced a higher binding energy shoulder in the C 1s spectra for the PDA-alaninamide film. A computer-assisted peak separation for the C 1s signal shows two overlapping peaks (Figure 1A). The shoulder at 289.3 eV correspondsto the heterosubstituted carbons while the peak at 286.5 eV corresponds to the saturated carbon atoms of the lipid chain. In Figure lB, the C 1s signal is plotted as a function of takeoff angle. Decreasing takeoff angles are displayed from back to front, and lower values reflect shallower sampling. Note that the area of the saturated carbon peak at 286.5 eV decreases steadily with decreasing takeoff angle while the area of the shoulder at 289.3 eV is independent of the takeoff angle. A composite graph depicting the elemental peak areas as a function of takeoff angle is presented in Figure 2. The C ls, N ls, Si 2s, and Si 2p peak areas are plotted against the sine of the takeoff angle. Each peak is scaled independently for clarity, and the relative areas do not reflect the actual elemental composition. It can be seen that both the Si 2s and Si 2p peak areas increase smoothly with increasingtakeoff angle. The silicon peaks arise from the underlying glass support. In contrast, the N 1s peak area, due to the amide nitrogen atoms, remains nearly independentof takeoff angle. Finally, the saturated C 1s
peak also increases with greater takeoff angles while the C 1s carbonyl shoulder remains constant. The independence of peak area for the N 1s peak and C 1s shoulder indicates that the nitrogen and heterosubstituted carbon atoms are localized in the interfacial region. In contrast, an increase in the absolute intensity for the C 1s parent peak and Si 2s and Si 2p peaks indicates that these atoms are located deeper in the bulk. These data are consistent with the head groups being exposed at the film-ambient interfaceand the saturated carbon chains extending toward the glass surface.13 This interpretation of the XPS data is supported by contact angle measurements. The contact angle values of 4 4 O f 3" (PDA-ethanolamine),48O f 2 O (PDA-serinamide), !Xoi 3O (PDA-alaninamide) and 1 1 2 O f 3O (silylated CISglass support) illustrate that the wettability properties can be controlled with an appropriate choice of head g r 0 ~ p . lThese ~ values agree with those obtained by other researchers for similar ~ystems.1~ FluorescenceMicroscopy. The polydiacetylene mone (14) It is interesting to note that the contact anglee reflectthe polarity head groups despite the porosity of the films. The pores in the films expose a substantial area of the underlying hydrophobic support which is not reflected in the wetting measurements. For a more detailed discussion on the interpretationof contact angles, see: (a)N w , R. G.; Dubois, L. H.; Allara, D. L. J . Am. Chem. SOC.1990,212,558. (b) Whiteaides, G. M.; Leibinis, P. E.Langmuir 1990,6 (11, 87. (15) See for example: (a) Bain, C. D.; Evall,J.; Whitesides, G. M. J. Am. Chem. SOC.1989,12I,7155. (b) Bain, C.D.; Whitesides, G. M. Zbid. 7164 and references contained therein.
of the ex-
2364 Langmuir, Vol. 8, No. 10, 1992 layers appeared bright orange-red under magnifications of N O 0 to X500 using epifluorescence microscopy and rhodamine filters. The films were also strongly birefringent and could be studied using polarized-light microscopy. The morphology of the monolayers was close-packed,with microcrystalline domains as large as 1mm in diameter. Figure 3 shows fluorescence micrographs of a PDAethanolamine film (A) and a PDA-alaninamide film (B) supported on silylated glass slides. Both surfaces are presented at XlOO magnification and appear to be continuous with some cracks and imperfections due to the transfer process. Magnified views (X5O0) of the samefilms are shown in parts C and D of Figure 3, respectively. Small holes varying from 0.1 to 10pm in diameter are visible and presumably originate from contraction of the film during the polymerization reaction.16J7 We also observe directionality in the film fibers which is reflected in the birefringence of these materials. Such order may be anticipated, however, since the diacetylene polymerization has been shown to require an ordered, contiguous array of monomers.l6 (16)For references concerning the mechanism and properties of diacetylenepolymerizntions,see: (a) Fukuda, K.; Shih&, Y.;Nakahara, H. Thin Solid Film 1988,160,43. (b) Baughman, R. H. J. Chem. Phys. 1978,68, 3110. (c) Tieke, B.;Graf, H. J.; Wegner, G.; Naegele, D.; Ringsdorf, H.; Banerjie, A.; Day, D.; Lando, J. B. Colloid Polym. Sci. 1977,255,521. (d) Steinback, M.; Wegner, G. Makromol. Chem. 1977, 1978, 1671. (17) We have examined these 'dark spots" using scanning tunneling microscopy (STM) and verified that they are holes in the monolayers and not areas of nonfluorescent material. Wilson, T. E.; Ogletree, D. F.; Salmeron, M. 8.;Bednarski, M. B. J. Am. Chem. Soc., submitted for publication.
Letters
Conclusions. Polydiacetylene films functiondized with amino acids offer a means of modifying surfaceswith peptide head groups. Characterization of the films by angle-resolved XPS and contact angle measurements indicates that the peptide head groups are localized at the film-ambient interface. Furthermore, the quality and reproducibility of these films is rapidly assessed using fluorescence microscopy. In summary, polydiacetylene films may provide promising model systemsfor studies in areas such as molecular recognition, interfacial crystallization, and adhesion. Acknowledgment. This work was supported by the Director, Office of Energy Research, Basic Energy Science, Materials Science Division of the U.S. Department of Energy under Contract Number DE-AC03-76SF00098and by the National Institutes of Health Award R29 GM4303702. T.E.W. thanks the Howard Hughes Medical Institute and the Fannie and John Hertz Foundationfor Predocioral Fellowships. M.D.B. thanks the American Cancer Society for a Junior Faculty Award, 1990-1993 JFRA-261, and the Eli-Lilly Corp. for a Young Investigator Award. Supplementary Material Available: Experimental data for compounds 2 and 7-10 and detailed information on the glass silylation procedure (5 pages). Ordering informationis given on any current masthead page. Registry No. 7 (Homopolymer),137870-34-9;8 (homopolymer), 137870-36-1;9 (homopolymer), 137870-38-3;10 (homopolymer), 137870-40-7.
