Langmuir 1996, 12, 4921-4928
4921
Influence of Structural Heterogeneity on the Fluorimetric Response Characteristics of Lipid Membranes Containing Nitrobenzoxadiazolyldipalmitoylphosphatidylethanolamine Jason D. A. Shrive, Sabeshan Kanagalingam, and Ulrich J. Krull* Chemical Sensors Group, Erindale College, University of Toronto, 3359 Mississauga Road North, Mississauga, Ontario L5L 1C6, Canada Received July 13, 1995. In Final Form: June 21, 1996X
Structural alterations in lipid membranes that are induced by changes in pH or by selective enzymesubstrate or antibody-antigen interactions can provide changes in fluorescence intensity from membranes containing nitrobenzoxadiazolyldipalmitoylphosphatidylethanolamine (NBD-PE). For example, fluorimetric responses of vesicles consisting of mixtures of phosphatidic acid (PA) and phosphatidylcholine (PC) prepared either from egg yolk or as synthetic, dipalmitoyl-substituted moieties and containing 2.3 mol % NBD-PE consist of either an increase or decrease in total fluorescence yield upon alteration of supporting electrolyte pH. This response was due to changes in NBD-PE self-quenching and the local environment of the fluorophore. The present work attempts to identify the factors that determine the fluorescence sensitivity of these lipid membranes. The impact of phosphatidic acid loading in vesicular bilayer lipid membranes and microscopic structural heterogeneity in monolayers at the air-water interface were explored. The results indicated that monolayers that exhibited laterally-separated phase domains on the microscopic scale provided enhanced sensitivity to perturbations of bulk pH and membrane surface charge redistribution through the alteration of the degree of headgroup ionization. Variations in the surface potential upon compression of either egg PA/egg PC or DPPA/DPPC monolayers in the liquid-expanded state were observed in order to facilitate the qualitative correlation of trends in the fluorimetric response of structurally homogeneous and structurally heterogeneous vesicular membranes with the existence of laterally-separated phase domains. The results indicated a strong correlation between sensitivity and structurally heterogeneity. The results also suggest the existence of ordered sub-microscopic domains in otherwise structurally homogeneous egg PA/egg PC membranes.
Introduction Previous work in our group has demonstrated the utility of phospholipid monolayers and bilayer lipid membranes (BLMs) as experimental models for the development of fluorimetric and electrochemical transducers for biosensor development.1-3 BLMs provide a framework which may be exploited for sensitive, generic transduction of selective binding events or pH alterations at the membrane surface. A localized event may affect a large number of lipids in the membrane, providing intrinsic amplification. More recently, we have determined that a surface concentration of the fluorescent membrane probe nitrobenzoxadiazolyldipalmitoylphosphatidylethanolamine (NBD-PE) of 2.3 mol % yields an optimum fluorescence sensitivity to alterations of membrane surface charge density induced by altering the supporting electrolyte pH for structurally homogeneous and heterogeneous lipid membranes. This optimum was determined for membranes containing acidic headgroups alone or together with phosphatidylcholine in vesicular BLMs.4 It was observed that significant improvements in the sensitivity of the self-quenching response of NBD-PE to structural changes in these membranes were achieved when the labeled membranes existed as laterally separated phase domains. The goal of the present work was to determine the structural basis of the previously observed enhancement of sensitivity. * Author to whom correspondence should be addressed. Fax: (905) 828-5425. X Abstract published in Advance ACS Abstracts, August 15, 1996. (1) Brennan, J. D.; Brown, R. S.; McClintock, C. P.; Krull, U. J. Anal. Chim. Acta 1990, 237, 253. (2) Brennan, J. D.; Brown, R. S.; Della Manna, A.; Kallury, K. M. R.; Piunno, P. A.; Krull, U. J. Sens. Actuators, B 1993, 11, 109. (3) Krull, U. J.; Seethaler, S. L.; Brennan, J. D.; Nikokelis, D. P. Thin Solid Films 1994, 244, 917. (4) Shrive, J. D. A.; Brennan, J. D.; Brown, R. S.; Krull, U. J. Appl. Spectrosc. 1995, 49 (3), 304.
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The relationship between fluorimetric signal magnitude and structural heterogeneity was investigated using phospholipid monolayers spread at the air-water interface as models for the vesicle BLMs studied using fluorescence techniques. In order to qualitatively identify structural phenomena, fluorescence microscopy of lipid monolayers spread at the air-electrolyte interface was employed to study the microscopic structural features associated with each loading of acidic phospholipid in the dipalmitoylphosphatidic acid/dipalmitoylphosphatidylcholine (DPPA/ DPPC) system. As the egg-derived phosphatidylcholine (egg PC) and phosphatidic acid (egg PA) contained mixtures of saturated and unsaturated acyl substituents on their phosphoglycerol headgroups, this system was expected to be structurally homogeneous on a microscopic scale. The structurally homogeneous egg-derived phospholipid monolayers were studied as a control group in order to isolate the effects of structural heterogeneity on the fluorescence sensitivity of lipid membranes. The surface potential of a lipid monolayer may be used to determine its structural heterogeneity. The existence of laterally-separated phase domains in the monolayer provides a mechanism by which lipids and their associated dipoles may be redistributed upon compression or expansion of the monolayer. This mechanism is unavailable in structurally homogeneous membranes and was used to qualitatively indicate the existence of microscopic and submicroscopic structural features in lipid membranes. The surface potential of monolayers of polar phospholipids spread at the air-electrolyte interface has been discussed at length in the literature, and several models for surface potential have been developed from the original Helmholtz equation: © 1996 American Chemical Society
4922 Langmuir, Vol. 12, No. 20, 1996
∆V )
µ⊥ A0
Shrive et al.
(1)
where ∆V is the surface potential, µ⊥ is the average component of the phospholipid molecule dipole moment which is perpendicular to the monolayer surface, A is the average area per molecule, is the relative permittivity of the monolayer, and 0 is the permittivity of free space.5 As a simplification, the value of is often assumed to be unity, yielding large inaccuracies in the predictions of this model. Several more complete models have been successfully fitted to experimental data from a variety of systems.6-8 Improvements in the modeling of the surface potential of lipid monolayers spread at the air-water interface have been achieved through the use of composite terms to account for the variable profile of environmental polarity through a monolayer and the subphase.9,10 Further corrections to account for the presence of ionized headgroups at the air-electrolyte interface and the presence of a Gouy-Chapman electrical double layer have also been shown to improve the fit of experimental data to predictions. An example is the approach used by Demchak and Fort.6,9,11 The general form of the modified Demchak-Fort model was presented by Oliveira et al.6:
∆V )
(
)
1 µ1 µ2 µ3 + + + ψ0 A0 1 2 3
(2)
where µ1, µ2, and µ3 are the dipole contributions from the water dipoles at the interface, the dipoles at the filmwater interface, and the dipoles at the air-film interface, with their associated dielectric constants 1, 2, and 3, respectively. ψ0 represents the contribution of the GouyChapman double layer. The importance of this term and its calculation have been reviewed in a comprehensive paper by Tocanne and Tessie´.12 Similarly, the lateral surface potential profile of phase-separated phospholipid monolayers has been studied using scanning surface potential microscopy techniques.13,14 It is apparent that several factors may influence the observed average surface potential of a monolayer, including lipid reorganization, subphase composition, and the nature of the lipids which comprise the monolayer. As a result, no comprehensive and widely accepted model of the surface potential profile of a phase-separated monolayer exists at this time. In this investigation, the sensitivity of the surface potential (∆V) to the area per molecule (A) as a function of the loading of acidic headgroups was studied. The mole ratio of two differing molecular dipoles in a spread phospholipid monolayer was studied after normalizing the surface potential change with respect to the area per molecule (∆(∆V)/∆A) for differences in monolayer compressibility (∆π/∆A), where π is the surface pressure. This normalized parameter, F, was calculated using eq 3: (5) Taylor, D. M.; DeOliveira, O. N., Jr.; Morgan, H. J. Colloid Interface Sci. 1990, 139 (2), 508. (6) Oliveira, O. N., Jr.; Riul, A., Jr.; Leal Ferreira, G. F. Thin Solid Films 1994, 242, 239. (7) Vogel, V.; Mo¨bius, D. J. Colloid Interface Sci. 1988, 126 (2), 408. (8) Smaby, J. M.; Brockman, H. L. Biophys. J. 1990, 58, 195. (9) Demchak, R. J.; Fort, T., Jr. J. Colloid Interface Sci. 1974, 46 (2), 191. (10) Schumann, D. J. Colloid Interface Sci. 1990, 134 (1), 152. (11) Oliveira, O. N., Jr.; Taylor, D. M.; Morgan, H. Thin Solid Films 1992, 210/211, 76. (12) Tocanne, J.-F.; Tessie´, J. Biochim. Biophys. Acta 1990, 1031, 111. (13) Heckl, W. M.; Baumga¨rtner, H.; Mo¨hwald, H. Thin Solid Films 1989, 173, 269. (14) Inoue, T.; Yokoyama, H. Thin Solid Films 1994, 243, 399.
F)
∆(∆V)/∆A ∆π/∆A
(3)
where ∆V is the surface potential, π is the lateral surface pressure in the monolayer as measured with a Wilhelmy plate, and A is the area per molecule. Since the goal was to compare qualitative structural and electrostatic phenomena from systems which were expected to exhibit either structural homogeneity or heterogeneity in fluorescence micrographs, we studied changes in the relative contributions of the perpendicular component of each type of dipole moment (µ⊥) in a binary mixture of phospholipids. Variations in the compressibility of the monolayers studied would clearly affect the packing density and therefore the magnitude of µ⊥ at the surface of the monolayer by altering the ease with which lipids were mixed and redistributed in the monolayer during compression. For that reason, it was necessary to employ a compression-normalized indicator of surface potential and, thus, ∆µ⊥ during monolayer compression. Moreover, the normalization routine also minimized the impact of the film balance calibration error. The use of F allowed the heterogeneity of a monolayer to be qualitatively assessed through an alternate technique to imaging, as this parameter indicated the mean surface density of the perpendicular component of the phospholipid molecule dipoles, µ⊥. Changes in the loading of each phospholipid would be expected to result in a proportional increase or decrease in F, depending on the relative magnitudes of µ⊥ for each component of the binary mixture. Heterogeneity in the monolayer surface would cause deviations in the proportionality of F to the mole ratio of the two components, as lipids combined in domains of different molecular packing density would exhibit different changes in µ⊥ in response to monolayer compression or expansion. NBD attached to an amphiphile may modify interfacial stability and domain shape.15 In order to preserve any structural features or phenomena associated with the presence of NBD-PE, the previously predicted optimum concentration of fluorophore was used in all monolayers studied. This investigation determined that structural heterogeneity on a microscopic scale resulted in the enhanced fluorescence sensitivity of membranes that contained NBD-PE upon perturbations of surface electrostatics and membrane structure. Such membranes provide a mechanism by which local NBD-PE concentrations may vary rapidly and significantly. The results also established the existence of sub-microscopic domains of increased order in the microscopically homogeneous egg PA/egg PC monolayers studied. Materials and Methods Chemicals and Supplies. Lyophilized egg phosphatidylcholine, egg phosphatidic acid, DPPC, and DPPA were purchased from the Sigma Chemical Company (St. Louis, MO) and were used as received. The fluorescent probe molecule N-(7-nitrobenz2-oxa-1,3-diazol-4-yl)dipalmitoylphosphatidylethanolamine (NBD-PE) was purchased from Avanti Polar Lipids (Birmingham, AL). Water for all experiments was obtained from a Milli-Q 5 stage cartridge filtration system (Millipore Corp., Mississauga, ON) and had a specific resistance of not less than 18 MΩ cm. All other chemicals were of at least analytical reagent grade. Equipment. The apparatus used for fluorescence microscopy of phospholipid monolayers at an air-water interface, preparation of pressure-area isotherms, and determinations of the relative fluorescence emission intensity of vesicle suspensions was as described below (see Figure 1). Light from a Coherent (15) Muller, P.; Gallet, F. Phys. Rev. Lett. 1991, 67 (9), 1106.
Fluorimetric Response of Lipid Membranes
Figure 1. Schematic representation of the device used to determine the relative fluorescence yield of NBD-PE-labeled vesicle suspensions. The same inverted microscope was used for fluorescence microscopy, except that a Lauda film balance was situated under the microscope objective. Innova 70 CW argon ion laser (Coherent Laser Products, Palo Alto, CA) operated at 488 nm and 10 mW power was directed into a Zeiss IM microscope (Carl Zeiss, Oberkochen, FRG) which was mounted such that a 16× water-immersion objective was situated directly above the monolayer. The microscope, Lauda Model 1974 film balance (Sybron-Brinkman, Toronto, ON), and camera were mounted on a gas-damped vibration isolation table (Melles Griot, Rochester, NY). Laser radiation was directed down to the monolayer via reflection by a dichroic mirror. The fluorescence emitted from the monolayer was transmitted through the dichroic mirror to a low light level Dage-MTI SIT 66 video camera (Dage-MTI Ltd., Michigan City, IN) which generated a video image. The camera was connected to a Data Translation video grabber card (Data Translation, Marlborough, MA) for storage and processing by an IBM PC-AT microcomputer. A rotatable mirror allowed the fluorescent emissions from the monolayer or vesicle suspensions to be reflected into a Hamamatsu R928 PMT (Hamamatsu, Bridgewater, NJ) for the determination of average fluorescence intensity values. Determination of the intensity of fluorescence emissions from vesicle suspensions for optimization studies was accomplished by placing a 5 mL transparent sample cell under the objective of the microscope and placing a 5 cm length of plastic-clad quartz optical fiber with polished ends under the 16× objective while the distal end was immersed in the vesicle suspension so as to provide a light conduit for Ar+ ion laser emissions and fluorescence emissions from the vesicles. Alignment of the optical fiber was optimized for sensitivity prior to the beginning of each set of measurements. Surface potential determinations were made by suspending the probe housing of an Isoprobe noncontacting electrostatic voltmeter (model 162, Monroe Electronics, Lyndonville, NY) at a minimum distance (typically
