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Langmuir 1998, 14, 1272-1277
Synthesis and Langmuir Film Formation of N-(E-Maleimidocaproyl)(dilinoleoylphosphatidyl)ethanolamine Tapani Viitala,*,† Willem M. Albers,‡ Inger Vikholm,‡ and Jouko Peltonen† Department of Physical Chemistry, Åbo Akademi University, Porthansgatan 3-5, 20500 Turku, Finland, and Technical Research Centre of Finland, Chemical Technology, P. O. Box 14021, FIN-33101 Tampere, Finland Received September 30, 1997. In Final Form: November 28, 1997 N-(-Maleimidocaproyl)(dilinoleoylphosphatidyl)ethanolamine (DLiPE-EMC) has been synthesized, representing a new polymerizable lipid with a linking group for covalent attachment of proteins for immunosensor applications. The linker lipid could be prepared by performing both the synthesis and separation steps under argon and avoiding heating. The product was characterized by 600 MHz 1H NMR, mass, and UV-vis spectrometry. Monomolecular films were successfully prepared on a Langmuir trough, and the characteristic surface pressure-area curves were measured on different subphases. A condensation of the monolayer could be achieved by introducing a micromolar concentration of uranyl ions in the subphase. Surface pressure-area isotherms of pure and mixed DLiPE/DLiPE-EMC monolayers were also measured. UV irradiation experiments were performed to demonstrate the polymerizability of the mixed monolayers.
Introduction The synthesis of lipids with terminal linking groups for immobilization of proteins and in particular Fab′-fragments has been studied intensively in the context of liposome preparation and recently also in the development of selective thin molecular films for immunosensor devices.1,2,3 Egger et al.2 coupled Fab′-fragments to small unilamellar vesicles and allowed these to fuse at the airwater interface. The direct coupling of Fab′-fragments to floating lipid monolayers is expected to lead in improved orientation of the antibodies. This is of great importance for increasing the antigen response in immunosensors, where the detection occurs by measuring the surface concentration. Our current interest lies in the preparation of linker lipids which are polymerizable in order to produce films with enhanced stability.4,5 The linker lipids should retain the amphiphilicity necessary for producing Langmuir (L) and Langmuir-Blodgett (LB) films. These lipids will in the future be used for direct coupling of Fab′-fragments to monolayers formed at the air-water interface. The first step toward our aim was to synthesize and characterize the monolayer behavior of a new linker lipid. We have generally prepared (dipalmitoylphosphatidyl)ethanolamine derivatives by reaction of the lipid with different commercially available succinimido ester reagents, using methods described in the literature.6 In this work, N-(-maleimidocaproyl)(dilinoleoylphosphatidyl)ethanolamine (DLiPE-EMC) was prepared from (dilinoleoylphosphatidyl)ethanolamine (DLiPE) and N-[(-ma* Author to whom correspondence is addressed. E-mail: tviitala@ abo.fi. † Åbo Akademi University. ‡ Technical Research Centre of Finland. (1) Vikholm, I.; Peltonen, J.; Teleman, O. Biochim. Biophys. Acta 1995, 1233, 111. (2) Egger, M.; Heyn, S. P.; Gaub, H. E. Biophys. J. 1990, 57, 669. (3) Stelzle, M.; Weissmu¨ller, G.; Sackmann, E. J. Phys. Chem. 1993, 97, 2974. (4) Markowitz, M.; Singh, A. Langmuir 1991, 7, 16. (5) Peltonen, J.; He, P.; Linde´n, M.; Rosenholm, J. B. J. Phys. Chem. 1994, 98, 12403. (6) Heath, T. D. Methods Enzymol. 1987, 149, 111.
leimidocaproyl)oxy]succinimide (EMCS), using a slightly modified procedure in which the synthesis and purification steps were carried out under argon. Also, heating of the reaction mixture was avoided. The compression isotherms of pure and mixed DLiPE and DLiPE-EMC monolayers were determined. The mixed monolayer isotherms indicate a high miscibility between the DLiPE and DLiPEEMC molecules. This is highly preferable when aiming at a matrix monolayer of homogeneously distributed linker units. Materials and Methods Materials. All chemicals used in this study were used as received. 1,2-(Dilinoleoylphosphatidyl)ethanolamine (DLiPE, >99% purity) was purchased from Avanti Polar Lipids; N-([maleimidocaproyl)oxy]succinimide (EMCS, >98% purity) was obtained from Fluka and triethylamine (98% purity) from J. T. Baker B.V. The chloroform used was of HPLC grade, and methanol was of PA grade. Sodium chloride (NaCl, PA grade) was obtained from Fluka; HEPES and uranyl acetate (UAc, PA grade) were obtained from Sigma. The 1 M NaOH solution used to adjust the pH for the saline/buffer was prepared from Titrisols. The water used for the monolayer studies was purified by a Millipore Milli-Q system, yielding a water resistance larger than 18 MΩ cm. Synthesis of N-(E-Maleimidocaproyl)(dilinoleoylphosphatidyl)ethanolamine (DLiPE-EMC). A total of 2 mL of DLiPE in chloroform (14.7 mg/mL) and 2 mL of EMCS in methanol (10 mg/mL) were added to a 10 mL Schlenk-type reaction vessel. A total of 5 µL of triethylamine in 2 mL of chloroform was then added, and the reaction was allowed to proceed in the dark for 18-20 h at 20 °C. Hereafter, the solvents were evaporated slowly on a rotation evaporator under argon at 0-4 °C. The reaction mixture was redissolved in 2 mL of chloroform and applied to a silica gel column (Si-60) equilibrated in chloroform. The silica column and fraction collection tubes were fitted to a vacuum manifold, enabling the purification step to be performed under argon. The column was washed with about 15 mL of chloroform and 10 mL of 5 vol % methanol in chloroform and the product finally eluted with 5 mL of 15 vol % methanol in chloroform. After addition of 5 µL of triethylamine, the chloroform was evaporated, again under argon at lowered temperature. About 1 mL of dry ether was added and evaporated, and this was repeated until the final product attained a constant
S0743-7463(97)01075-5 CCC: $15.00 © 1998 American Chemical Society Published on Web 02/13/1998
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Figure 1. Molecular structure and 1H NMR assignments of DLiPE-EMC. weight. The final yield was around 55%. The structure of the end product is shown in Figure 1. Characterization of the Lipids. The synthesized DLiPEEMC was characterized with 1H NMR, mass, and UV-vis spectroscopy. 1H NMR spectra of DLiPE-EMC were measured with a Varion Unity 600 MHz spectrometer. Both normal 1H NMR and 1H-COSY spectra were recorded. Mass spectra of DLiPE-EMC were recorded on a Micromass Quattro II in the ES mode. UV-vis spectra of EMCS, DLiPE, and DLiPE-EMC were obtained with a Perkin-Elmer UV-vis spectrometer with 1 cm quartz cells. Isotherms of Pure and Mixed DLiPE/DLiPE-EMC Monolayer. DLiPE and DLiPE-EMC were dissolved in chloroform at a concentration of 1 mg/mL. The solutions were protected against light and stored under argon in a refrigerator until spreading. The mixtures were freshly prepared prior to spreading. The surface pressure was measured with a Pt-Wilhelmy balance. All surface pressure-area isotherms were recorded with computerized LB instruments (KSV 5000 and KSV 2000, KSV Instruments, Helsinki, Finland) at 20 ( 0.5 °C. Monolayers were obtained by spreading the solutions onto various subphases. The solvent was allowed to evaporate 10 min prior to compression. The barrier was compressed at a speed of 3.3 Å2/molecule‚min. The isotherms of pure DLiPE and DLiPE-EMC were measured on three different subphases: pure ion-exchanged water with pH 5.6 (IEW), 20 mM HEPES + 0.9 wt % NaCl with pH 6.8 (buffer 1), and 20 mM HEPES + 0.9 wt % NaCl + 10-5 M uranyl acetate with pH 6.8 (buffer 2). The IEW subphase was used as a reference to show that DLiPE-EMC is amphiphilic enough to form a monolayer at the air-water interface, even though the polar linker group EMC has been attached to the phosphatidylethanolamine (PE) moiety. The saline/HEPES (buffer 1) subphase was used because it is a common immunoassay buffer, which will be of interest in future studies on immobilization of Fab′-fragments. To obtain a more condensed monolayer than that formed on the buffer 1 subphase, a small amount of uranyl acetate (UAc) was added (buffer 2). Surface pressure and surface potential isotherms of pure DLiPE and DLiPE-EMC were measured as a function of UAc concentration. The surface potential was measured simultaneously with the surface pressure by the vibrating plate method. The upper, vibrating electrode was perforated to minimize the noise. Mixed monolayers of DLiPE/DLiPE-EMC were finally obtained on the buffer 2 subphase. UV Irradiation of the Monolayer. The UV irradiation of the 90:10 DLiPE/DLiPE-EMC mixed monolayer on saline/buffer and saline/buffer/UAc was carried out using a 30 W low-pressure mercury lamp (maximum emission at 254 nm) placed at ca. 0.2 m above the monolayer. The UV-irradiation experiments were performed at a constant surface pressure of 25 mN/m.
Results and Discussion Physical Chemistry Properties of DLiPE and DLiPE-EMC. 1H NMR of DLiPE-EMC (600 MHz, CDCl3) gave the following proton assignments (cf. Figure 1): δ 0.90 (t, J ) 6.5 Hz, 6H, ω), 1.30 (br m, 42H, h + y + γ), 1.60 (m, 6H, g + β), 1.65 (br q, J ) 7.5 Hz, 2H, i), 2.05 (br q, J ) 7.0 Hz, 8H, δ), 2.20 (t, J ) 7.0 Hz, 2H, f), 2.30 (br m, J ) 7.0 Hz, 4H, R), 2.77 (t, J ) 7.0 Hz, 4H, φ), 3.09 (q, J ) 7.0 Hz, 6H, x), 3.45 (br, J ) 7.0 Hz, 2H, e), 3.50 (t,
Figure 2. UV-vis spectra of EMCS in methanol (s), and DLiPE (- -), and DLiPE-EMC (‚‚‚) in chloroform.
J ) 7.0 Hz, 2H, j), 3.97 (br m, 4H, c + d), 4.16 (br dd, 1H, a′), 4.38 (br dd, 1H, a), 5.22 (br dd, 1H, b), 5.33-5.39 (m, 8H, ), 6.69 (s, 2H, k), 7.23 (br s, 1H, CONH). The calculated molecular weight for DLiPE-EMC (C51H84N2O11P) is 932.2; the molecular weight observed from the mass spectra is 931.6. Figure 2 shows the UV-vis spectra of EMCS in methanol and of DLiPE and DLiPE-EMC in chloroform. The spectra for EMCS and DLiPE derivatives were measured in different solvents because the molecules were dissolved in these particular solvents when they were used. The spectrum for EMCS shows a very broad band with a maximum absorbance at 295 nm. This band is assigned to the maleimidocaproyl group of EMCS, which in our case will be the most interesting part of EMCS because this part can combine with a thiol group via its double bond.7 The spectrum for EMCS also shows three bands located at 203, 210, and 215 nm (not shown) with a very high absorbance. These bands are attributed to the succinimide part of EMCS, which after the synthesis of DLiPE-EMC should have disappeared. The spectrum for DLiPE shows one broad absorbance band at 278 nm and one distinct band at 240 nm. The broad band at 278 nm is due to the carbonyl groups near the glycerol backbone, while the band at 240 nm has its origin from the double bonds located in the hydrocarbon chains. DLiPE-EMC gives one distinct absorbance band at 240 nm which is the same as that for pure DLiPE and another very broad band with a maximum at 283 nm which is due to the maleimide group of the linker attached to the polar headgroup of DLiPE. This, and the absence of any absorbance bands below 220 nm, clearly supports the data obtained from 1H NMR and mass spectra which all indicate that the synthesis of DLiPE-EMC has been successful. (7) Smyth, D. G.; Nagamatsu, A.; Fruton, J. S. J. Am. Chem. Soc. 1960, 86, 1839.
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Figure 3. Compression isotherms of (a) DLiPE and (b) DLiPEEMC on IEW (s), buffer 1 (- •• - •• - •• -), and buffer 2 (- - -). Table 1. Aext and Ai Values for DLiPE and DLiPE-EMC on Three Different Subphases DLiPE subphase
IEW
Aext 82 ( 2 molecule) 2 Ai (Å / 111 ( 4 molecule) (Å2/
DLiPE-EMC
buffer 1 buffer 2 63 ( 3
58 ( 1
86 ( 3
80 ( 2
IEW
buffer 1 buffer 2
83 ( 3 110 ( 4
76 ( 1
149 ( 9 167 ( 6 155 ( 6
Pressure-Area Isotherms of Pure DLiPE and DLiPE-EMC. Parts a and b of Figure 3 show the behavior of respectively DLiPE and DLiPE-EMC on three different subphases. Table 1 shows the values of the extrapolated mean molecular area, Aext, of the high-pressure phase and the mean molecular area where the surface pressure starts to increase significally, AI, both obtained from the isotherms. The isotherms measured on IEW show that both lipids are able to form monolayers at the air-water interface. The larger AI value of DLiPE-EMC indicates that the polar headgroup, (-maleimidocaproyl)phosphatidylethanolamine (EMC-PE), is not vertically oriented in the subphase at large molecular areas. However, the same Aext value for DLiPE and DLiPE-EMC monolayers indicates that the different polar headgroups do not contribute to the final mean molecular area of a compressed monolayer, which is, at this particular degree of condensation, mainly determined by the dilinoleoyl double chain unit. On the buffer 1 subphase, the DLiPE monolayer showed a condensation whereas the monolayer of DLiPE-EMC showed an expansion as compared with the monolayers formed on an IEW subphase. The PE headgroup of DLiPE is a zwitterion in the pH range 2.3-8,8 and the negative and positive charge centers are readily accessible to ions in the subphase, since the PE headgroup is oriented (8) Tocanne, J.-F.; Teissie´, J. Biochim. Biophys. Acta 1990, 1031, 111.
parallel to the subphase surface.9 This enables the Na+ and Cl- ions of buffer 1 to interact with the PE groups, leading to a formation of two opposite dipoles which compensate each other, thus decreasing the repulsion between adjacent molecules. A condensing effect is hence observed. For DLiPE-EMC, the negatively charged phosphate group is only forming one dipole in the polar headgroup. Thus, we explain the expansion to arise from the repulsive interaction of parallel dipoles between adjacent molecules. Furthermore, the expanding effect becomes more dominant in buffer 1 as compared to the IEW since the pH is increased from 5.6 to 6.8, respectively. The introduction of a very small amount of UAc (10-5 M) to the subphase caused a condensation and a higher fracture pressure of the monolayer for both DiLPE and DLiPE-EMC as compared with the monolayers measured on buffer 1 subphase. It has been suggested that (UO2)2(OH)22+ is the main adsorbing uranyl complex at pH ≈ 7 to a lipid monolayer from a UAc-containing subphase, forming a bridge between the charged phosphate groups.8,10,11 On buffer 2, the most pronounced relative condensation was observed for the monolayer of DLiPE-EMC. This may be a result of a higher affinity of the dinuclear uranyl acetate complex to the negatively charged phosphate group in comparison with Na+ ions. The resulting interaction between the divalent uranyl complex and two DLiPEEMC molecules gives a more densely packed monolayer. The enhanced condensation is also seen in the isotherm measured for DLiPE on the buffer 2 subphase. An increased fracture pressure further indicates a closepacked structure and a monolayer of high elasticity. However, it is obvious that the linker unit affects the packing properties of the condensed monolayer, giving a value of Aext of 76 Å2, which is clearly larger than the corresponding value of DLiPE, 58 Å2. Pressure-Area Isotherms of Pure DLiPE and DLiPE-EMC as a Function of UAc Concentration in the Subphase. Parts a and b of Figure 4 show a selected set of isotherms measured for DLiPE and DLiPE-EMC on subphases (0.9 wt % NaCl + 20 mM HEPES, pH 6.8) with various concentrations of UAc. An increasing amount of UAc in the subphase caused a condensation of the monolayer, seen as a decrease in the Aext value. However, at a certain concentration of UAc the monolayer became very rigid and further increase in the UAc concentration resulted in monolayers so viscous that the surface pressure could not be properly measured. This effect could clearly be seen in the isotherms as a decrease in the fracture pressure and also as a sudden increase in the Aext values. The UAc concentration at which this effect was initially seen was 2 × 10-5 M for DLiPE and 10-4 M for DLiPEEMC. The difference in this concentration is attributed to the differences in the polar headgroup as discussed in the previous section. Figure 5 shows ∆Vmax for both lipids as a function of UAc concentration. It can be seen that the trends are opposite to each other. One striking feature is, however, the fact that the surface potential reaches approximately the same value for both lipids in the high UAc concentration region, indicating that the dipolar contributions at the most condensed state are equal for both lipid monolayers. (9) Gorwyn, G.; Barnes, G. T. Langmuir 1990, 6, 222. (10) Pen˜acorada, F.; Reiche, J.; Katholy, S.; Brehemer L.; Rodrı´guezMe´ndez, M. L. Langmuir 1995, 11, 4025. (11) Pen˜acorada, F.; Reiche, J.; Dietel, R.; Zetzsche, T.; Stiller, B.; Knobloch, H.; Brehmer, L. Langmuir 1996, 12, 1351.
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Figure 4. Compression isotherms on subphases containing 0.9 wt % NaCl + 20 mM HEPES (pH 6.8) and various amounts of UAc for (a) DLiPE, 10-7 M UAc (s), 10-6 M UAc (- -), 5 × 10-6 M UAc (- - -), 10-5 M UAc (-‚-), 2 × 10-5 M UAc (- •• - ), and 10-4 M UAc (‚‚‚) and (b) DLiPE-EMC, 10-6 M UAc (s), 10-5 M UAc (- -), 2.5 × 10-5 M UAc (- - -), 5 × 10-5 M UAc (-‚-), 7.5 × 10-5 M UAc (- •• -), and 10-4 M UAc (‚‚‚).
Figure 5. ∆Vmax as a function of UAc in a 0.9 wt % NaCl + 20 mM HEPES (pH 6.8) subphase for DLiPE (9) and DLiPEEMC (O).
The surface potential for a monolayer at the air-water interface can in its very simplest scheme be expressed by the Helmholtz equation as
∆V ) µn/0A
(1)
where µn is the normal component of the molecular dipole moment, is the relative permittivity of the monolayer, and 0 is the permittivity of a vacuum. µn may be split up into different components associated with different regimes of the interface. According to Vogel and Mo¨bius,12 µn can simply be divided into two parts, µR and µω. All contributions associated with the polar headgroup includ(12) Vogel, V.; Mo¨bius, D. J. Colloid Interface Sci. 1988, 126, 408.
ing the reorganization of water molecules near the interface are represented by µR, whereas µω represents the apparent dipole moment of the hydrophobic part of the monolayer. Since µn represents the normal component of the effective dipole moment, the value of ∆V should be sensitive to changes in the polar region of the monolayer and/or to changes in the orientation of the chains. Thus, the changes in the ∆Vmax values in Figure 5 can mainly be attributed to the changes in the polar region, because ∆Vmax is measured at a point where the monolayer is in its most condensed state. At pH 6.8 the PE headgroup of DLiPE is in a zwitterionic form, and an increase in salt concentration has the same effect on an ionizable monolayer as an increase in pH.13 When the UAc concentration in the subphase is increased from 10-7 to 5 × 10-6 M, the ∆Vmax value remains unchanged, while it suddenly starts to drop at a concentration of 10-5 M. This is propably due to the deprotonization of the amine group, which leads to a decreasing and finally a vanishing contribution of its dipole moment to µR, which in turn lowers the ∆Vmax value because the dipole directed upward gives a positive contribution to the measured ∆Vmax value. This is also supported by the fact that the amine part of the PE headgroup has an intrinsic pK value of 10,7 in combination with the realistic assumption that the deprotonization of the amine group starts about two pH units below the intrinstic pK. The almost monotonic increase of ∆Vmax for DLiPEEMC monolayers with increasing UAc concentration is interpreted to be dominated by an ion-exchange reaction between Na+ and (UO2)2(OH)22+, with Na being initially bound (before addition of any UAc) to the phosphate group of the PE unit carrying a negative charge. Thus, Na+, being monovalent, only binds to one lipid molecule, while (UO2)2(OH)22+ is attached to two molecules (soap formation), resulting in dipolar components being inclined from the subphase surface normal and hence giving a smaller negative contribution to ∆Vmax than Na+. The smaller initial ∆Vmax value of DLiPE-EMC than that of DLiPE most obviously is because of the lacking amino group in DLiPE-EMC which gives a positive contribution to ∆Vmax for DLiPE. Pressure-Area Isotherms of Mixed DLiPE/DLiPEEMC on thec Buffer 2 Subphase. The buffer 2 subphase for the study of the mixed DLiPE/DLiPE-EMC films was chosen in order first of all to get as condensed monolayers as possible and secondly to avoid an initially too viscous monolayer which would be very difficult to measure. Surface pressure-area isotherms of the mixed monolayers are shown in Figure 6. The miscibility of the two components can be determined by analyzing the fracture pressures and/or molecular areas obtained from the isotherms.14 According to the surface phase rule developed by Crisp and presented by Gaines,14 two immiscible components in a mixed film are characterized by a constant equilibrium or fracture pressure regardless of the composition, while if the two components are miscible, the fracture pressure will vary with composition. For pure and mixed DLiPE/ DLiPE-EMC monolayers, the differences in fracture pressures are too small to make any use of the surface phase rule; therefore, we need another method, i.e., the additivity rule for studying the miscibility behavior. This rule was also presented by Gaines, and it states that the average molecular area in a mixed film of two components (13) Linde´n, M.; Rosenholm, J. B. Langmuir 1995, 11, 4499. (14) Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interfaces; Wiley-Interscience: New York, 1966.
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Figure 6. Compression isotherms of DLiPE/DLiPE-EMC mixtures on the buffer 2 subphase: 100:0 (s), 90:10 (- -), 80:20 (- - -), 70:30 (-‚-), 50:50 (- •• -), 25:75 (- - -) and 0:100 (‚‚‚).
Figure 7. Mean molecular area for the mixed monolayers as a function of the molar fraction of DLiPE-EMC at different points. Ai: π0 mN/m (9), π ) 5 mN/m (0), π ) 25 mN/m (b), π ) 40 mN/m (O); Aext (4).
at any surface pressure will follow a linear dependence if the components are immiscible, i.e.
A12 ) x1A1 + x2A2
(2)
where A12 is the average molecular area in the mixed film, xi is the mole fraction of component i, and Ai is the average molecular area of the pure film of component i at the same surface pressure. The criterion that only immiscible components conform to the additivity rule is not definitive, because it is also possible that mixed films with completely miscible components follow the linear dependence according to eq 2. In such a case ideal behavior might result from a homogeneous surface mixture of noninteracting molecules. Figure 7 shows the average molecular area at surface pressures of 5, 25, and 40 mN/m and the extrapolated area of the mixed DLiPE/DLiPE-EMC monolayers as a function of the molar fraction of DLiPE-EMC. A linear correlation for the average molecular areas according to the additivity rule is observed within the experimental error for all molar fractions at each surface pressure. This is in our opinion an indication of nearly ideal miscibility of the two components. This conclusion is supported by the fact that both lipids have identical bulky hydrophobic chains, and therefore no kind of new interaction is introduced to the hydrocarbon chain region when they are mixed; i.e., the cohesive forces in the hydrocarbon region are the same in mixtures as those for pure components. At the studied experimental conditions both lipids are mainly negatively charged (due to the conclusions drawn in the previous chapter) and the (UO2)2-
Figure 8. Barrier speed as a function of UV irradiation time of the 90:10 DLiPE/DLiPE-EMC monolayer on buffer 1 (-‚-) and buffer 2 (s). UV light switched on at t ) 5 min.
(OH)22+ concentration is already so significant that soap formation takes place in the monolayer. Thus, any repulsive contributions from an electrostatic point of view or from the polar headgroup parallel dipoles are not expected. Considering the steric effects, one DLiPE-EMC molecule is preferentially bridged by (UO 2)2(OH)22+ to one DLiPE instead of to a DLiPE-EMC molecule. Irradiation of DLiPE/DLiPE-EMC (90:10 mol %). The particular mixture of 90:10 mol % DLiPE/DLiPEEMC was chosen for the reactivity experiments because when the immunological measurements will be performed, the concentration of the linker molecules should not be too high, which in fact could lead to sterical hindrance of the Fab′-fragments to attach to the linker. The crosslinking reaction of a floating monolayer can be followed in real time by measuring the change in the mean molecular area and/or barrier speed during the irradiation under constant surface pressure.5,15 Figure 8 shows the results obtained when the monolayer was UV-irradiated at 25 mN/m on buffer 1 and buffer 2 subphases. It can be seen that the monolayer hardly reacts at all on the buffer 1 subphase when the UV light is turned on, which can be seen by the slow and small changes in the barrier speed corresponding to almost no change in the mean molecular area. An introduction of a small amount of UAc is however enough to induce a marked increase in the reactivity of the monolayer. This can be seen as a change of the barrier speed after the UV light has been turned on. When the reaction is initiated on buffer 2, the barrier directly responds by a quick expansion (negative speed) after which it increases suddenly (monolayer shrinkage) and reaches a maximum after about 2.5 min. After the maximum, the barrier speed decreases exponentially toward a constant value. The much higher reactivity obtained when UAc is added to the subphase is due to the condensing effect, as shown for the monolayers of the pure components in Figures 3 and 4. It has also been shown earlier that the reactivity of the monolayers of unsaturated amphiphiles is controlled by the orientation of and the distance between the individual monomer molecules.15,16 The reactivity curves obtained for the DLiPE/DLiPEEMC mixture are quite similar to those our group has earlier obtained for linoleic acid monolayers on a TbCl3 subphase.5 Other studies by UV-vis and FT-IR spectroscopy by our group also have shown that the changes (15) Ringsdorf, H.; Schupp, H. In Interfacial Synthesis; Carraher, C. E., Preston, J., Eds.; Marcel Dekker: New York, 1982; Vol. 3, p 335. (16) Viitala, T. J. S.; Peltonen, J.; Linde´n, M.; Rosenholm, J. B. J. Chem. Soc., Faraday Trans. 1997, 93 (17), 3185.
Synthesis and Formation of DLiPE-EMC
in the barrier speed are a clear indication of a polymerization reaction occurring in the monolayer.16,17 Conclusions The synthesis of DLiPE-EMC from DLiPE and EMCS was successful, which was evidenced by 1H NMR, mass, and UV-vis spectroscopy. The amphiphilic nature of the synthesized lipid DLPE-EMC was demonstrated with the film balance experiments. The phase behavior of the monolayers was strongly dependent on the subphase composition. The higher Aext and AI values for pure DLiPEEMC than for pure DLiPE indicate that the linker unit affects the molecular packing mostly in the beginning of the compression and the behavior at smaller molecular areas is attributed to the different charge distribution of the polar headgroups of DLiPE and DLiPE-EMC. UAc (17) Linde´n, M.; Gyo¨rvary, E.; Peltonen, J.; Rosenholm, J. B. Colloids Surf. A 1995, 102, 105.
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had a clear condensing effect on the monolayers of pure DLiPE and DLiPE-EMC. The mixed monolayers of DLiPE and DLiPE-EMC showed ideal miscibility behavior. An introduction of a minor amount of UAc to the subphase was enough to perform a polymerization reaction of a 90: 10 mol % mixture of DLiPE/DLiPE-EMC. At present we have work going on which aims at coupling Fab′-fragments directly to these monolayers formed at the air-water interface. Preliminary measurements on these kinds of films have already been done, and the results concerning antibody and subsequently antigen immobilization from the subphase by the EMC group are very promising. Acknowledgment. Financial support from the Finnish Academy of Sciences and VTT is gratefully acknowledged. We thank Anita Teleman and Jouni Enqvist, VTT Chemical Technology, for providing 600 MHz NMR spectra. LA971075S
