J . Phys. Chem. 1993, 97,2928-2932
2928
Monomeric and Polymeric Molecular Films from the Diethylene Glycol Diamine Pentacosadiynoic Amide Ralf W. Ti”,
Manfred Radmacber, and Hermann E. Gaub’
Physikdepartment E22, Technische UniversitBt Miinchen, W-8046Garching, Germany
Paul Kenney sad Hans 0. Ribi Biocircuits Corporation, 1450 Rollins Road, Burlingame, California 94010 Received: October 6, 1992; In Final Form: January 4, I993
A new positively charged polymerizable single chain surfactant was synthesized and analyzed. Monomolecular films made from this surfactant were investigated. Their physical characteristics were evaluated using standard film balance techniques and studied as a function of the subphase pH. The pK of these films was determined to be 6.8 f 0.5. At room temperature these films exhibit a pronounced fluid/solid coexistence with a transition enthalpyof 35 f 3 kJ/mol for high subphase pH values. A strong rate dependent hysteresis between compression and expansion was found. The crystal morphology of the solid domains was investigated by microfluorescence at the air-water interface. Although the molecules themselves are achiral, the solid domains exhibit a distinct chirality. After UV polymerization the solid domains showed an intense intrinsic fluorescence due to the polymer backbone. Polymeric monomolecular films were transferred onto silicon oxide by standard LangmuirBlodgett tecbtziqrws, The structural properties of these films were investigated using the atomic force microscope. At intermediate magnifications t h films appeared rippled; at high magnifications molecular resolution was obtained.
Introduction Ultrathin lipid films in combination with polymers such as polydiacetylenes and proteins can be used to create new classes of material with broad industrial and academic application^.^-^ Molecular polydiacetylene films have unique optical, electrical, mechanical, and chemical pr~perties.~J-*.~,* For example, they exhibit an extremely high optical density (monomolecular layers are visible by eye); they are intensely fluorescent9J0(typically several orders of magnitude more fluorescent than monolayers of commonly used fluorophores such as fluorescineor rhodamine), and they are highly anisotropic due to their crystallinity and polymer packing (certain classesof film have been shownto exhibit linear and circular dichroismand birefringence;11polydiacetylene films are photosemiconductm, and due to the stable nature of the polydiacetylenebackboneetrdorganicreactivity,a widevariety of surface functionalities can be introduced.’-’ Ligands, drugs, antibodies, receptors, oligonucleotides, and enzymes have been attached to ultrathin polymer films for a variety of immunodiagnostic and DNA testing purposes. The combined optical and chemical properties of the films provide the basis for a sensitive, rapid, and low cost detection system.’Jl Similarly, ultrathin films of this nature could be used as surface coatings including protective coatings for implantable devices and biologically active coatings for bioanalysis. For example, lipid monolayers have been used as substrates for the twodimensionalcrystallizationand structural determinationof protein moiec~les.l~-~~ Due to their diverse nature and the ability to control them both chemically and thermodynamically, polymerized molecular films can be rationally designed and applied to specific areas of interest. Their unique mechanical properties make them ideally suited test substrates for novel near field microscopes1s-20andascustom designable substrates for the immobilization of macromolecules to be imaged.16J8s21Chain lengths, linking groups, hydrogen bonding groups, and charged moieties can be introduced at the point of chemical synthesis.’-’ Pure or mixed lipid compositions 0022-3654/93/2097-2928S04.00/0
DPDA
Figure 1. Schematics of the surfactant diethylene glycol diamine pentacosadiynoicamide (DPDA)and the polymerization reaction of the diacetylenic group.
can be introduced during the process of film formation.22 Chemical and biological modification can also be accomplished once the polymer-lipid layer has been f ~ r m e d . ~ . ~ ’ The following report discusses the monolayer characteristics of the new polymerizable lipid, diethylene glycol diamine pentacosadiynoic amide (DPDA). The molecule represents one in a series of molecules currently under investigation. We describe here several of the physical and chemical properties of pure DPDA monolayers. Materials and Metbods Lipid Synthesis. A solution of 1.87 g (5.0 mmol) of 10,12pentacosadiynoic acid from Farchan Laboratories and 0.675 g (1 .O equiv, 6.0 mmol) of I-hydroxybenzotriazolefrom Fluka and 1.24 g (1.2 equiv, 5.0 mmol) of 1,3-dicyclohexylcarbiimide from Aldrich were added to a 250-mL round bottom flask with
8 1993 American Chemical Society
Monomeric and Polymeric Molecular Films from DPDA 100 mL of chloroform and stirred a t room temperature for 0.5 h. A neat solution of 2.6 g (5.0 equiv, 25.0 mmol) of diethylene glycol diamine from Texaco was added and stirred at room temperature for 1 h. The reaction was monitored for completion by thin layer chromatography (2: 1 ch1oroform:methanol). The solution was filtered through Whatman no. 2 filter paper to remove polymer and dicyclohexylurea. The filtered solution was diluted to 500 mL with chloroform and washed two times with brine, dried using sodium sulfate, and then concentrated to give 1.1 g of a light sensitive, blue tinted solid material. Purification by flash chromatography (50 X 150 mm silica gel 230-400 mesh Merck silica gel; 1500 mL of 5:l followed by 700 mL of 2:l ch1oroform:methanol) gave 0.8 g (34%) of white solid material which rapidly turned blue upon exposure to UV light (254 nm). Analytical results were as follows: Rf 0.16 by thin layer chromatography (2:1 ch1oroform:methanol); mp 9 1-93 OC. Anal. Calcd. for C29H~202N2: C, 75.6; H , 11.3; N, 6.08. Found: C, 72.14; H, 11.17; N , 5.78. Other Chemicals. Texas Red-DPPE (Molecular Probes) was used to label the fluid phase for the monomer microfluorescence investigations. Additional chemicals were purchased from Sigma unless otherwise noted. HEPES buffer consisted of 20 mM HEPES and 20 mM NaCl titrated with NaOH. Citric acid buffer consisted of 20 mM citric acid (Aldrich) and 20 mM NaC1, titrated with Na2HP04. Substrates. Silicon (100) wafers with a thermally grown oxide layer of 180-nm thickness (a kind gift of Wacker Burghausen) were cleaned following the standard procedures given in ref 24. Square centimeter sized pieces of the wafers were then glued to the metal sample holders with an epoxy resin and stored in a desiccator. Wafer sections were checked routinely for cleanliness by atomic force microscopy (AFM) prior to use. Microfluorescence Film Balance. A custom built microfluorescence film balance was used for this study. It is described in detail e l s e ~ h e r e .The ~ ~ pressure area ( s A ) diagrams were recorded on a trough with a surface area of 400 cm2, The fluorescence micrographs were recorded using a small Langmuir trough with 30-cmZsurface area. UV polymerization was carried out at constant pressure (30 mN/m) by irradiating the surface with a Hg pen ray lamp (Oriel) kept at a distance of approximately 5 cm for 30 s. Polymerized films were transferred by standard Langmuir-Blodgett (LB) techniques at transfer rates of 0.1 mm/ S.
Atomic Force Microscopy. The combined fluorescence/force microscope (see ref 26 for details) allows the simultaneous imaging of the fluorescence and surface relief of the sample. Preselection of certain areas of interest and the coarse positioning of the AFM cantilever were accomplished using the instrument's fluorescence imaging mode. This combination additionally allowed the determination of the orientation of the polymer backbone by measuring the fluorescence anisotropy of the polymer backbone. All AFM images were recorded with silicon nitride cantilevers from Digital Instruments (spring constant: 64 mN/m) with electron beam deposited carbon tips.27 Results and Discussion Because the amine head group of DPDA can be titrated using different subphase pH, DPDA films were expected to exhibit a pronounced pH dependence in their thermodynamic properties. Figure 2a shows the pressure-area isotherms of DPDA for subphases with different pH levels. In each case the first compression scans were plotted. DPDA films exhibit a pronounced rate dependent hysteresis between compression and expansion. The fluid phase starts at a molecular area of approximately 0.9 nm2. At high pH the s A isotherms exhibit a well pronounced plateau starting at a molecular area of 0.7 nm2 and ending at a molecular area of approximately 0.27 nm2. Film collapse occurs at pressures above 40 mN/m and molecular areas
The Journal of Physical Chemistry, Vol. 97, No. 12, 1993 2929 40
E
t
HEPES buffer (T
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ea
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Figure 2. Monomeric DPDA films at the air-water interface. (a) Pressure-area isotherms as a function of the subphase pH. (b) Fluidsolid coexistence pressure plotted as a function of the pH.
less than 0.23 nm2. The latter value is roughly the value expected for close packed hydrocarbon chains with a tilt imposed by the diacetylenic groups.15,28The plateau was therefore assigned to a fluid-solid coexistence. The plateau pressure was determined to be a function of the subphase pH. At high pH values the plateau pressure starts a t approximately 2 mN/m and increases with decreasing pH. Below pH 6 no plateau was detectable at room temperature (20 "C). Therefore the temperature was decreased to 7 OC,and a similar set of ?rAisotherms was recorded for lower pH values (curves not shown). Titration experiments with a similar headgroup had shown that this temperature shift does not alter the pK within our experimental error.29 The onset values of the coexistence pressure were plotted as a function of the pH in Figure 2b for both sets of data. The pressure scales were shifted with respect to each other such that the coexistence pressure measured at low and at high temperature on the pH 6.5 subphase were superimposed. This plot shows that the pH dependence of the coexistence pressure levels off both at high and at low pH values. The pK was assigned to the inflection point of the roughly sigmoidal curve at 6.8 f 0.5. Salt free subphases are desirable for many technical applications of such polymeric films because the transfer process may result in residual contaminations due to the salt. Figure 3 shows the ?rA diagrams of DPDA on Millipore water as a function of the temperature. It is interesting to note that a t room temperature a DPDA film on the unbuffered water surface exhibits the same s A isotherm as a DPDA film on a Hepes subphase at pH 8.7, which corresponds to a virtually uncharged molecule. Assuming
2930
Tillmann et al.
The Journal of Physical Chemistry, Vol. 97, No. 12, 1993
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a
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Figure 3. Pressure-area isotherms of monomeric DPDA as a function of the temperature. Insert: Clausius-Clapeyron analysis of the fluidsolid transition of the DPDA monolayer.
that pure water in equilibrium with atmospheric C02 has a pH of 5.5, this finding suggests that the DPDA monolayer has a significant buffering capacity. The temperature dependence of the fluid-solid transition of the DPDA film was analyzed with the two-dimensional ClausiusClapeyron equation.30 The resulting heat of transition is plotted as a function of the temperature in the insert of Figure 3. The plot shows a comparably weak but significant temperature dependence suggesting that the critical temperature of DPDA must be unusually high compared to its length of the hydrocarbon chains. The value of 35 f 3 kJ/mol at room temperature is higher than the corresponding value for the same molecule with a saturated hydrocarbon chain. Other data indicate that the heat of transition would in this case be about 20 kJ/m01.~' This discrepancy may be caused by the sterical hindrance of the chain mobility due to the rather stiff diacetylenic group. This interpretation is also supported by the finding that other lipids with diacetylenic hydrocarbon chains exhibit high values for the heat of transition too.32 When observed by fluorescence microscopy at the air-water interface, films from DPDA exhibit a distinct pattern in the fluidsolid coexistence plateau (see Figure 4a). At the beginning of the plateau, the microfluorescenceshows fernlike domains which exclude the fluorophor Texas Red-DPPE. It is generally thought, that such domainsare densely packed two-dimensional solid^.^^-^' These domains grew with increasing film compression. After UV polymerization the fluorescence contrast was inverted and thedomains exhibited a highly polarized and intense fluorescence due to the polymer backbone (Figure 5b-d). The polymerization of the diacetylenes is known to be a topochemical reaction which occurs only when the reactive groups have a certain relative distance and orientation with respect to each other (see Figure 1). Generally such conditions are fulfilled only when the monomers are arranged in a crystalline array. This corroborates the previous assumptions that the formerly dark domains had been crystalline and that the plateaus in the ?rAdiagrams were fluid-solid coexistence regions. The DPDA crystals exhibit a distinct chirality even though the monomeric molecules are not chiral. Some of the domains are single crystals, but most of the domains have multiple single crystallinebranches with different chiral orientation. A symmetry breaking element in the crystalline packing is required to explain
Figure 4. Fluorescence micrograph of a DPDA monolayer at the airwater interface. (a) Macroscopic crystal morphology of the monomeric DPDA film at an average molecular area of 0.6 nm2. The bright areas represent the fluid phase stained with the fluorescent lipid probe Texas Red. Dark areas represent solid lipid domains. (b) Same film as above after polymerization by UV light. Here the intrinsic fluorescenceof the polymer backbone is much brighter than that of the fluorescent label in the fluid phase. (c,d) DPDA monolayer polymerized at a molecular area of 0.3 nm2, imaged with linear polarized fluorescence excitation. The analyzator was rotated by 90° between images c and d. (Imaging parameters: room temperature; subphase, pure water; image size, 300 pm X 500 pm; Lxc= 520 nm; L,,, > 540 nm.)
our finding. Either a uniform tilt of the hydrocarbon chains or the relative arrangementof the polymerizablegroups with respect to the crystal axis may provide such a symmetry break. In this case both of these factors might contribute. The relatively high molecular area of 0.23 nm2/moleculestrongly suggests a tilt of the chains, and the high polarization degree of the fluorescence indicates a strong correlation of the polymer backbone with the monomer crystal axis. DPDA filmswere polymerized at ?r = 30 mN/m and transferred by standard LB techniques onto amorphoussilicon oxide surfaces. The positively charged head groups undergo a strong Coulomb interactionwith the negatively charged silicon oxide surface.The interaction results in an extremely small transfer meniscus which is comparableto that of Cd-ara~hidate.~After transfer the films exhibited a fluorescence pattern comparable to those at the airwater interface. Transferred films were investigated with the AFM. Figure 5a shows massive mechanical defects in an otherwise homogeneous and flat film. Mechanical distortion during the transfer may have torn the film into flat ribbons. The step height of the ribbons was equivalent to that of a single monolayer. Fluorescence polarization of the DPDA film was measured to be along the direction of the cracks indicating that the polymer backbone extends along the length of the ribbons. The macroscopically flat areas like the one in the right upper corner of Figure 5a show at higher magnification a well pronounced stripe pattern (see Figure 5b) which is parallel to the orientation of the polymer backbone. The contrast of the stripe pattern increases with the applied force and depends on the scan direction relative to the stripes. Parts b and c of Figure 5 were recorded quasi simultaneously during forward and backward scanning. Although both images were recorded from the same area, the stripe pattern was not superimposable under any conditions. This observation suggests that the pattern represents other properties of the surface than merely its relief. As shown on similar LB films,*this kind of contrast may be caused by local variations of lateral forces rather than height variations of the sample. This interpretation oversimplified would mean, that at higher forces, the tip interactspartially with the polymer backbone. If this interpretation is right, it would quite generally mean that the surface topology of similar samples may only be addressed
Monomeric and Polymeric Molecular Films from DPDA
The Journal of Physical Chemistry, Vol. 97, No. 12, 1993 2931
Image Site :6.6 Nm
Figure 5. AFM images of a polymeric DPDA Langmuir-Blodgett monolayer on silicon oxide a t low magnifications. (a) Area where the film was mechanically distorted during transfer. (b,c) Close-up view of the flat area in the upper right corner of a, recorded during forward (b) and backward (c) scanning. (d) Image at high magnification. The insert was Fourier filtered. Due to technical reasons this image is slightly rotated with respect to a+. The arrow indicates the direction of the polymer backbone. (Imaging parameters: raw data; constant force in air a t 10 nN; room temperature, carbon tip, 64 mN/m; scan speed, 10 lines/s.)
at very low forcesand that at higher forces,the image is determined by “bulk properties” of the film. At high magnification (see Figure 5d) this wavelike pattern was still faintly visible; however, the image was dominated by a more or less well ordered lattice structure. In our raw data, the periodicity of the lattice was in most cases clearly pronounced in one direction and only locally visible in the other two directions. This disorder may stem from the roughness of the underlying amorphous silicon oxide. It may also be inherent in the film and may be caused by local disturbances of the lattice during polymerization or transfer from the air-water interface. After Fourier filteringthe underlying oblique lattice becomes prominent (see insert in Figure 5d). The orientation of the polymer backbone (see arrow) coincides with one of the crystal axes. Within the accuracy of our instrument (about 10%) the unit cell area of the lattice correspondsto the molecular area of the film as measured at the air-water interface. This value also agrees well with the lattice measured on films from molecules with the same hydrocarbon chain region but with different headgroups.I5J7 This indicates that the molecular arrangement of these polymerizable single chain surfactants is, as long as no sterical hindrance is imposed by the headgroup, dominated by the packing of the hydrocarbon chains and the stiff diacetylene group. Acknowledgment. This work was supported by the Deutsche Forschungsgemeinschaft. We kindly thank Janice Sich for preparing the DPDA monomer and David Keller for the carbon tips.
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