Langmuir 1995,11, 1257-1260
1257
Formation of Poly(diacety1ene) Thin Films with Uniform Fluorescence Shirley J. Johnson, Ralf W. Tillmann, Tom A. Saul, Ben L. Liu, Paul M. Kenney, Jay S. Daulton, Hermann E. Gaub,+ and Hans 0. Ribi* Biocircuits Corporation, 1324 Chesapeake Terrace, Sunnyvale, California 94089 Received June 27, 1994. I n Final Form: November 21, 1994@ This paper presents a novel way to produce poly(diacety1ene)thin films of uniform fluorescence on a solid support. The method involves film formation at the air-water interface from a modified diacetylene monomer using innovative spreading, crystallization, and transfer techniques. Pressure-area isotherms for the monomer N-(2,3-dihydroxypropane)pentacosa-l0,12-diynoicamidereveal the optimum lateral pressure (10mN/m) and temperature (40 "C) for film production. To prepare films, we apply a 2 mM solution ofthe monomer in chloroform to the air-water interface, either dropwise or as a nebulized spray. Irradiation with W light polymerizes the film to its red, fluorescent form. Pressurized nitrogen directed at the subphase surface creates turbulence in the interfacial layer, causing break-up and randomization of crystal domains. After stopping the surface disruption, we transfer the film to silanized glass slides held at 40 "C. Heating the transfer slides eliminates holes in the film attributed to condensation of water vapor onto the slides during transfer. The resulting film consists of uniformly distributed crystals of 5 10 pm, and it is free of the large cracks and defects commonly observed in solid-supported poly(diacety1ene) films. The transferred film has very uniform fluorescence with a coefficient of variance of only 5% in fluorescence intensity based on measurements 1 mm in diameter covering the entire slide (2.5cm x7.5 cm).
Introduction Poly(diacetylene1thin films are of interest for application in biosen~ors,'-~ as well as for use in electronic and nonlinear optical device^.^-^ For devices relying on electronic or polarimetric properties of poly(diacetylenes), orientation ofthe crystal domains is of utmost importance. Several groups have reported ways to influence domain orientation.s-ll Devices that rely on the fluorescence or absorbance properties of the film are not so critically dependent on domain orientation, and the crystal morphology does not affect the optical properties of the film.12 Nevertheless, films of good uniformity in fluorescence and absorbance on a macroscopic scale are required to
* To whom correspondence should be addressed. t Physikdepartment E22, Technische Universitat Munchen,
W-8046 Garching, Germany. Abstract published in Advance ACS Abstracts, J a n u a r y 15, 1995. (1)Ribi, Hans 0.Novel Lipid-Protein Compositions and Articles and Methods for their Preparation, US.Patent No. 4,859,538, 1989. (2) Ribi, Hans 0. Biosensors Employing Electrical, Optical, and Mechanical Signals, U.S. Patent No. 5,156,810, 1992. (3) Ribi, Hans 0.;Guion, T. A,; Murdoch, J. R.; Scott, J. C.; Pan, V.; Choate, G. L. Multioptical Detection System. U S . Patent No. 5,268, 305, 1993. (4)Charych, D. H.; Nagy, J. 0.;Spevak, W.; Bednarski, M. D. Direct Colorimetric Detection of a Receptor-Ligand Interaction by a Polymerized Bilayer Assembly. Science 1993,261,585. ( 5 )Laschewsky, A.; Ringsdorf, H.; Schmidt, G. Polymerization of Hydrocarbon and Fluorocarbon Amphiphiles in Langmuir-Blodgett Multilayers. Thin Solid Films 1985,134,153. (6)Ogawa, K.;Tamura, H.; Hatada, M.; Ishihara, T. Study of Photoreaction Processes of PDA Langmuir Films. Langmuir 1988,4, 903. (7) Suzuoki, U.; Kimura, A.; Mizutani, T. Electrical and Nonlinear Optical Properties of Polydiacetylene Langmuir-Blodgett Film. Proceedings-International Symposium on Electrets, 7th; GerhardMulthaupt, R., Ed.; IEEE: New York, 1991; p 850. ( 8 )Grunfeld, F.; Pitt, C. W. Diacetylene Langmuir-Blodgett Layers for Integrated Optics. Thin Solid Films 1983,99, 249. (9) Thakur, M.; Meyler, S. Growth of Large-Area Thin-Film Single Crystals of Poly(diacety1enes). Macromolecules 1985,18, 2341. (10) Miyano, K.; Hasegawa, T. Pressurehisotropy and Orientational Order of a Polydiacetylene Monolayer and its Use as a Template for Vacuum Deposition. Thin Solid Films 1991,205,117. (11)Arisawa, S.; Yamamoto, R. Control of Molecular Orientations by a Pulsating Electric Field Applied to a Monolayer at the Air-Water Interface. Langmuir 1993,9, 1028. @
minimize spatial variation during detection. Formation of such films is not a trivial problem, as poly(diacety1ene) thin films are quite fragile and crack easily. Crystal domain size and shape at the air-water interface can be conveniently controlled through use of additives and choice of spreading solvent13J4but stress induced during transfer of the polymerized film onto a solid support generally causes defects.14 In addition, polymerization of the monomer is accompanied by shrinkage of the monolayer,15J6 and stress from polymerization can cause fragmentation of the film leading to surface areas devoid of poly(diacetylene1 and its accompanying fluorescence. The dificulty of reproducibly preparing and transferring high-quality films has hindered the development of devices incorporating poly(diacetylene) thin films. Others have studied the polymerization behavior of poly(diacetylenes) and have discussed the significance of monomer s t r ~ c t u r e . ~ J ~ ,We l ~ -have ' ~ also investigated different monomer surfactants to produce poly(diacetylene) films and have previously reported on the phase (12) Miyano, K.; Maeda, R. Photoluminescence, Absorption, and Raman Spectra of a Polydiacetylene Monolayer. Phys. Rev. B 1986,33, 4386. (13)Hui, S. W.; Yu, H. Microdomain Structures in Polymerizable and Nonpolymerizable Diacetylenic Phosphatidylcholine Monolayers. Langmuir 1992,8, 2724. (14) Tieke, B.; Weiss, K. The Morphology of Langmuir-Blodgett Multilayers of Amphiphilic Diacetylenes: Effects of the Preparation Conditions and the Role of Additives. J. Colloid Interface Sci. 1984, 101, 129. (15) Meller, P.; Peters, R.; Ringsdorf, H. Microstructure and Lateral Diffusion in Monolayers of Polymerizable Amphiphiles. Coll. Polym. Sci. 1989,267,97. (16) Day, D.; Ringsdorf, H. Polymerization of Diacetylene Carbonic Acid Monolayers at the Gas-Water Interface. J. Polym. Sci., Polym. Lett. 1978,16, 205. (17) Tieke, B.; Leiser, G. Influences of the Structure of Long-chain Diynoic Acids on their Polymerization Properties in Langmuir-Blodgett Multilayers. J . Colloid Interface Sci. 1982,88, 471. (18)Hupfer, B.;Ringsdorf, H. Spreading and Polymerization Behavior of Diacetylenic Phospholipids at the Gas-Water Interface. Chem. Phys. Lipids 1983,33, 263. (19) Tieke, B.; Weiss, K. Amphiphilic Diacetylenes with Pyridine and 2,Z'-Bipyridine Headgroups-Polymerization Properties in the Crystalline State, in LB-Multilayers,and in Complexes with Transition Metal Salts. Morphology of Polymerized Multilayers. Colloid Polym. Sci. 1985,263,576.
0743-7463/95/2411-1257$09.00/00 1995 American Chemical Society
1258 Langmuir,
Vol.11,No.4,1995
Johnson et al.
behavior and polymerization properties of two of them.20*21 This paper describes another novel surfactant, N-(2,3dihydroxypropane)pentacosa-lO,l2-diynoicamide(hereafter referred to as propanediol) suitable for producing red poly(diacety1ene)film. Our innovative use of alternate spreading methods, surface disruption, and temperature control of the subphase and solid support significantly improves macroscopic fluorescence uniformity of the transferred film.
fcr
H&tcd Platc
Side View
Methods Monomer Synthesis. To synthesize the propanediol monomer, we added 2.47 g (12 mmol) of dicyclohexylcarbodiimide (Aldrich Chemical Co., Milwaukee, WI) to a solution of 3.7 g (10 mmol) of 10,12-pentacosadiynoic acid (Farchan Laboratories, Gainesville, FL) and 1.35 g (10 mmol) of 1-hydroxybenztriazole (Fluka Chemical Corp., Ronkonkoma, NY)in 200 mL of room temperature chloroform and then stirred for 0.5 h. To this mixture, we added a solution of 1.0 g (11 mmol) of (&I-3aminopropanediol (Fluka) dissolved in 6 mL of 1:l chloroform: ethanol and then stirred for 72 h at room temperature. Filtration removed precipitated dicyclohexylurea and residual polymer, and applied vacuum evaporated the chloroform. Crystallization of the residue 2 times from 100 mL of methanol yielded 2.18 g of white solid, and flash chromatography (50x 150 mm silica gel, 230-400 mesh (Merck, Darmstadt, Germany) 1O:l chloroform: methanol eluant) further purified this material. Crystallization ofthe pure fractions 2 times from ethanol yielded 1.3g(2.9 mmol) of white solid, which slowly turned blue on exposure to light (yield 29%; mp 95 "C; TLC Rr 0.25, 1O:l ch1oroform:methanol silica). The structure of the propanediol monomer follows.
Phase Characterizationof the Monomer. We prepared a 2 mM solution of propanediol in chloroform to measure pressure-area isotherms a t the air-water interface using a custom-built Langmuir trough with a movable barrier. A Wilhelmy plate coupled to a tensiometer (Nima Technology, Ltd., Coventry, U.K.) measured lateral surface pressure. The trough was constructed of Teflon-coated aluminum with dimensions 11 cm x 31cm. The compression speed was 400pds. The subphase consisted of pure water, filtered through a reverse osmosis deionization system (Continental Water Systems Co., Palo Alto, CA). Film Production Conditions. We compared two application techniques to spread the monomer at the air-water interface. The first technique was the traditional dropwise application of monomer solution, applied until a lens formed on the surface. The second technique involved nebulization of monomer solution through an ultrasonic nozzle (Digispense 2000, Ivek Corp., North Springfield, VT) at a flow rate of 17 p u s at 4 W ultrasonic dispensing power. During dispensation, the nozzle traversed slowly across the length of the trough. W irradiation for 5 s from a xenon lamp (RC-5008 pulsed U V curing system, Xenon Corp., Woburn, MA) positioned 22 cm above the surface polymerized the monolayer on the air-water interface. In a subset of experiments, pressurized filtered nitrogen directed at the interface through nozzles caused disruption of the surface layer during both monomer application and subsequent polymerization. The nozzles were located at two diagonal corners of the trough and directed along the trough length, thus creating turbulent vortex flow ofthe surface layer. After polymerization, disruption of the surface layer continued for 1 min. We then stopped the disruption and transferred the polymerized film to silanized glass microscope slides by Schaefer transfer, i.e., by gently touching the slides horizontally to the surface. The silanization procedure utilized dimethyloctadecylchlorosilane,and the contact angle of (20) Tillmann, R. W.; Radmacher, M.; Gaub, H. E.;Kenney,P.; Ribi, H. 0. Monomeric and Polymeric Molecular Films from the Diethylene Glycol Diamine Pentacosadiynoic Amide. J.Phys. Chem. 1993,97,2928. (21) Sullivan,B.;Ribi, H. O.;Tillmann,R. W.;Hofmann, U.G.;Gaub, H. E. Molecular Films from the Polymerizable Lipid Ethyl Morpholin Pentacosadiynoic Amide. J. Vac. Sci. Technol. A 1994, 12, 2975 .
Suction
S
Too View Figure 1. . Schematic diagram of experimental apparatus showing the transfer arm assembly.
"T
0
0.2
0.3
0.4
0.6
0.6
0.7
o m por m d r m k (nm*/mokeub)
Figure 2. Pressure-area isotherms for propanediol on a subphase of pure water. the silanized glass slides was approximately 80". For the transfer, ten slides were held by vacuum to a heated mechanical transfer arm, shown in Figure 1. A stepping motor controlled the movement of the arm, and the speed of transfer was adjustable. We photographed the transferred film through a fluorescence microscope, exciting the film at 480 nm and collecting the emitted fluorescence light above 525 nm. An epifluorescence instrument (excitation filter at 550 nm with 40 nm full width at half maximum (fwhm)and emission filter at 650 nm with 40 nm fwhm) measured fluorescence intensity by scanningthe film with an interrogation area 1mm in diameter. We calculated the coefficient of variance in fluorescence intensity from 234 measurements evenly spaced across the entire slide (2.5 cm x 7.5 cm).
RESULTS AND DISCUSSION Pressure-Area Isotherms. Figure 2 shows pressure-area isotherms for a monolayer of propanediol monomer. The isotherms demonstrate that the monomer undergoes a phase transition at higher temperatures. The plateau representing the two-phase region is clearly visible at 40 "C and above, where the heat of transition is 40 kJ/mol. At 20 "C, no two-phase region exists, and the monomer condenses directly into a solid-condensed phase as the pressure is increased. The aredmolecule in the solid-condensed state is 0.260 f 0.015 nm2. The phase behavior of propanediol is comparable to other diacetylene
Langmuir, Vol. 11, No. 4, 1995 1259
Poly(diacety1ene) Thin Films
m-
20 T
E 15t
Y
i 0.0
0.2
0.4
0.6
0.8
1.0
monomer dispensed per area ( pL/cm 2,
Figure 3. Lateral pressure of propanediol applied by ultrasonic dispensation of a 2 mM solution in chloroform at a flow rate of 17pUs.Error bars represent the standard deviation of six trials.
Monomer Application. The topochemical polymerization of diacetylenes is subject t o geometric constraints, so the diacetylene groups must be an appropriate distance from one another.6 Polymerization of propanediol occurs more effectively when the monomer is in the solidcondensed state compared with the two-phase region. Rather than using mechanical compression to achieve the desired lateral pressure, for the sake of simplicitywe apply the monomer dropwise until a lens forms on the surface. For a subphase temperature of 40 "C, dropwise application until lens formation produces a lateral pressure of 10 mN/ m, conditions at which propanediol adopts the solidcondensed state. The phase transition to the solidcondensed state at 40 "C occurs just below 10 mN/m (see Figure 2), thus permitting the propanediol to readily spread as it is applied. Examination of polymerized film formed by dropwise application of propanediol to the surface reveals cracks and ring-shaped artifacts at the point where the final drop of solution was placed. Other investigators report that the crystal morphology and domain structure of a solidsupported poly(diacety1ene)film result from the structure at the air-water i n t e r f a ~ e ,further ~ ~ ? ~ ~emphasizing the importance of spreading conditions on final polymer morphology. This led us to investigate dispensing the monomer using an ultrasonic nozzle, which disperses the solution in micrometer-sizedroplets. Figure 3 shows the increase in lateral pressure with amount of applied propanediol per trough surface area. As excess monomer is applied, the lateral pressure approaches an asymptotic value, because the monomer already on the surface comprises a closed monolayer. Polymerized film made with ultrasonic dispensation of propanediol to 10 mN/m as no visible application artifacts and no macroscopic cracking at the air-water interface. Heated Transfer Slides. Transferring the polymerized film to a solid support at room temperature results in a film filled with small holes visible under the fluorescence microscope, as shown in Figure 4. The holes are due to condensation of water vapor onto the transfer slide as the slide moves closer t o the heated subphase during film transfer. The size and number of holes directly relate to the speed at which the film is transferred, with slower transfer speed resulting in more holes. By heating (22) Day, D.; Lando, J. B. Morphology of Crystalline Diacetylene Monolayers Polymerized a t the Gas-Water Interface. Macromolecules 1980,13,1478. (23) Tieke, B.; Lieser, G. Weiss, K. Parameters Influencing the Polymerization and Structure of Long-chain Diynoic Acids in Multilayers. Thin Solid Films 1983,99, 95.
Figure 4. Fluorescence micrograph of polymerized propanediol on a solid support. The film was made on a water subphase maintained a t 40 "C, while the transfer slide was at room temperature. The holes are due to water condensing on the slide as the film was being transferred.
Figure 5. Fluorescence micrograph of'polymerized propanediol on a solid support. The film was made on a subphase maintained at 40 "C,and the transfer slide was also heated to 40 "C before transferring the film. The cracks are typical of solid-supported poly(diacetylene) films.
the transfer slide to the same temperature as the subphase, i.e., 40 "C, we eliminate condensation ofwater vapor onto the transfer surface,and the resulting film shown in Figure 5 does not contain holes. Surface Disruption. Ultrasonic dispensation of the monomer solution successfully eliminates macroscopic cracks in the polymerized film, but many cracks and defects are still visible under the fluorescence microscope that affect the fluorescence uniformity of the transferred film. Cracks and defects are considered characteristic of supported poly(diacety1ene)films and have been observed by many investigator^.^^*^^-^^ The film in Figure 5 contains representative examples of microscopic cracks. To improve fluorescence uniformity of the film, we disrupt the subphase surface during both monomer application and (24) Berrbhar, J.; Lapersonne-Meyer, C.; Schott, M.; Villain, J. Formation of Periodic Crack Structures in Polydiacetylene Single Crystal Thin Films. J. Phys. 1989,50,923. (25) Wiesenhorn, A. L.; Gaub, H. E.; Hansma, H. G.; Sinsheimer, R. L.; Kelderman, G. L.; Hansma, P. K. Imaging Single-Stranded DNA, Antigen-AntibodyReaction and Polymerized Langmuir-Blodgett Films with an Atomic Force Microscope. Scanning Mic (26) Putnam, A. J.; Hansma, H. G.; Gaub, H Polymerized LB Films Imaged with a Combined At scope-Fluorescence Microscope. Langmuir 1992,8,
1260 Langmuir, Vol. 11, No. 4, 1995
Johnson et al.
t
E o~ down-slide position
Fi uorescence micrograph of polymerized propanediol on support. The film was made on a subphase maintained at and the interfacial surface was disrupted during monomer application as well as during polymerization. The disruption was stopped before the film was transferred to a slide heated to 40 "C. The film consists of many small crystals and no large cracks.
subsequent UV irradiation by directing streams of pressurized nitrogen at the water surface to create turbulence. Turbulence at the surface causes the film to disintegrate into many small individual crystals of random orientation. Motion of the crystals on the turbulent surface randomizes defects caused by polymerization-induced shrinkage of the film. Furthermore, a to relieve stress
:
cracks commonly introduced during polymerization and/ or the transfer process. Monomer application artifacts are also eliminated, as surface disruption yields a film with the same appearance whether we apply the monomer dropwise or via ultrasonic nebulization. Figure 6 displays a typical micrograph of a transferred film, showing that the film consists of randomly oriented and evenly distributed crystals of polymerized material of 510 pm in size. At millimeter resolution, the transferred film appears very uniform in fluorescence, with a coefficient of variance of approximately 5%in fluorescence intensity across the entire slide. Figure 7 exemplifies the improvement in fluorescence uniformity accomplished through surface disruption. To our knowledge, this is the first report of the use of surface disruption on a Langmuir trough to improve macroscopic film uniformity.
Conclusions Past efforts to produce poly(diacetylene) thin films transferred to solid supports have been hindered by the fragility ofthe polymerized film at the air-water interface. The transferred films inevitably contained many cracks, thus leading to nonuniformity in fluorescence. We developed a new method to produce poly(diacety1ene)thin films of improved fluorescence uniformity. Our method simplifies film production relative to the LangmuirBlodgett technique by eliminating the need for a movable
crossslide position
b)
Q
down-slide position
crossslide position
Figure 7. Maps of normalized fluorescence intensity of polymerized propanediol films on a solid support: (a) a film made without surface disruption; (b) a film made with surface disruption. The slide dimensions are 7.5 cm x 2.5 cm, and each intensity reading comprises an area 1mm in diameter. Readings are normalized by the slide average. See text for further discussion.
barrier. The monomer propanediol is particularly suited for the production of fluorescent poly(diacetylene) film. The pressure-induced transition from liquid-expanded to solid-condensed phase is conveniently controlled by titration of the surface with monomer solution to an appropriate pressure. Disruption of the air-water interface during monomer spreading, crystallization, and polymerization results in randomly oriented and evenly distributed individual crystals of 510 pm. Transfer of the film t o a solid support held at the same elevated temperature as the subphase eliminates holes attributed to condensation of water vapor onto the support during transfer. Films produced with these techniques have superior fluorescence uniformity and thus simplify the development of devices relying on changes in optical properties of poly(diacety1ene)films.
Acknowledgment. We thank Todd Ard for his assistance in performing the experiments. We also acknowledge Dr. Joel Blatt and Jonathan Lull for helping with the figures. LA9405096
