0 Copyright 1994 American Chemical Society
The ACS Joumal of
Surfaces and C o l l o i OCTOBER 1994 VOLUME 10,NUMBER 10
Letters Lateral Diffusion of Lipoidal Spectroscopic Probes in Langmuir-Blodgett Films at the Solid/LiquidInterface Frank Caruso, Peter J. Thistlethwaite, and Franz Grieser* School of Chemistry, University of Melbourne, Parkville, Victoria 3052, Australia
D. Neil Furlong CSIRO Division of Chemicals & Polymers, Private Bag 10, Bayview Avenue, Clayton, Victoria 31 68, Australia Received January 5, 1994. In Final Form: June 3, 1994@ The lateral diffusion of the spectroscopic probe-quencher pair N-(1-pyrenesulfony1)dipalmitoyl-L-aphosphatidylethanolamine, triethylammonium salt (pyrene-DPPE) and 4-(NJV-dimethyl-N-hexadecylammonio)-2,2,6,6-tetramethylpiperidine-l-oxyl, iodide (CAT-16)has been measured in Langmuir-Blodgett (LB) films in contact with aqueous solution. The mutual lateral diffusion coefficients were determined to be in the range (6-8) x cm2s-l by analyzing the time-resolved fluorescence decay of the excited pyrene-DPPE in the presence of CAT-16. A comparison of diffusion in “wet”LB films with “dry“ films and air-water monolayers shows the diffusion in the “wet”film is considerably faster than in the ‘‘dry“ film, but approximately half to 1order of magnitude slower than in an air-water monolayer. In addition, it is shown that the state of dispersion of pyrene-DPPE in the deposited film is highly sensitiveto the number of LB layers deposited and the composition of the film.
Introduction Fundamental information on the factors that affect the dynamic motion of small molecules in ordered arrays, such as vesicles,bilayers, and monolayers, has long been sought to help in understanding the motion of the constituent molecules in biological membranes. In more recent years ordered films such as LangmuirBlodgett (LB)films have been revisited as “carriers” for specific functional molecules as components for chemical and biochemical sensors.1p2 In large part, these types of biochemicalhhemical sensors will be required to make
* To whom correspondence should be addressed. Abstract published in Advance ACS Abstracts, September 1, 1994. (1) Schuhmann, W.; Heyn, S.-P.;Gaub, H. E. Adu. Muter. 1991,3, 388. (2)Moriizumi, T. Thin Solid Films 1988,160,413. @
contact with an aqueous medium containing the molecule of interest. Fundamental information on the dynamics of molecules in “wet”ordered arrays is required in order to help describe the physicochemical properties of such films. Mostly, diffusionin biomimetic films has been measured by the fluorescence recovery after photobleaching ( F W ) m e t h ~ d . ~We - ~present here an alternative method which allows the diffusion of probe molecules in a film to be measured. The method has the distinct advantage of indicating whether or not aggregation of the probes has (3)Peters, R.;Beck, K. P m .Nutl. Acud. Sci. U S A . 1983,80,7183. (4)Kim, S.;Yu, H. J. Phys. Chem. 1992,96,4034. (5)Tamada, K.;Kim, S.; Yu, H. Langmuir 1993,9,1545. (6)Tamm, L. K.; McConnell, H. M.Biophys. J. 1985,47,105. (7)Merkel, R.;Sackmann, E.; Evans, E. J.Phys. (Paris) 1989,50, 1535. (8)Wu, E.-S.;Jacobson, K.; Papahajopolous,D. Biochemistry 1977, 16,3936. (9)von Tschamer, V.;McConnell, H. M.Biophys. J. 1981,36,421.
0743-7463/94/2410-3373$04.50/00 1994 American Chemical Society
3374 Langmuir, Vol. 10, No. 10, 1994
Letters
occurred. It also has an advantage over F W measurements in that local heating, hence temperature gradients leading to surface ~ ~ o w , ~ is , ~virtually JO eliminated. The present letter describes the use of time-resolved fluorescence quenching profiles in "wet" LB films onsilica to determine the mutual lateral diffusion coefficiqt of the probe-quencher pair pyrene-DPPE and CAT-16. The method has already been successfully applied to air-water m ~ n o l a y e r s l ' - ' ~but has not previously been extended to LB films. The mutual lateral diffusion coefficients obtained are compared with those obtained at t h e air-
m-'. In all cases the deposition ratios were 1.0 f 0.1. The LB films were transferred to a fluorimeter cell, remaining submerged in the aqueous subphase solution, and stored in darkness. All deposition experiments were conducted at 20 & 1 "C. Lapgmuir-Blodgett Fluorescence Measurements. Steady-state fluorescence excitation and emission spectra of Langmuir-Blodgett films containing probe were measured on a Perkin-Elmer LS-5B luminescence spectrometer or a Spex Fluorolog 1680 spectrofluorimeter. The fluorescence emission was registered in the direction perpendicular to the excitation beam. Background fluorescence excitation and emission spectra were measured for bare silica plates submerged in the aqueous subphase solution. The fluorescence excitation and emission water interface. spectra of the mixed monolayers were obtained by subtracting the appropriate background spectrum from the spectra of the Experimental Section silica substrates with the mixed monolayers. Fluorescence decay curves were measured by the timeMaterials. N-(1-pyrenesulfony1)dipalmitoyl-L-a-phosphaticorrelated single-photon counting technique, employing a dylethanolamine, triethylammonium salt (pyrene-DPPE) and 4-(NJV-dimethyl-N-hexadecylammonio)-2,2,6,6-tetramethylpip- Rhodamine 6G dye laser synchronouslypumped by a mode-locked argon ion laser, or a DCM dye laser synchronously pumped by eridine-1-oxyl,iodide (CAT-16)were purchased from Molecular a mode-lockedNd:YAG laser, as the excitation source. The dye Probes Inc. Dimyristoyl-L-a-phosphatidylcholine (DMPC) was lasers were operated at a repetition rate of 4 MHz. Complete purchased from the Sigma Chemical Co. Sodium perchlorate, details of these systems are given el~ewhere."-'~J7 The subcadmium chloride, and sodium hydroxide were all analytical strates were rotated until the maximum fluorescencesignal was grade and were purchased from either Merck or Ajax Chemicals. observed. The fluorescencephotons were isolated using an Oriel Hydrogen peroxide, ammonia, and nitric acid were all analytical 320 nm cutoff filter and a monochromator. grade reagents and were supplied by BDH Chemicals. 1-DodeThe bare silica plate showed a short-lived background fluocanol (>99.5%pure) was purchased from Fluka. Unless othrescence emission. In order to correct for the emission from the erwise stated, all chemicals were used as supplied. "Milli-Q" bare silica substrates, the background decay was gathered with water was used in the preparation of subphase solutions. the same overall excitationintensity as the substrates containing Langmuir-Blodgett Film Preparation. The substrates the mixed monolayer and subsequently subtracted to yield the used for LB deposition of the monolayer films were made from decay curves of the mixed monolayers. Instrument response Suprasil silica. The silica plates were cleaned in the usual functions were measured by separately collectingthe excitation manner in order to obtain hydrophilic surface^.'^ The substrates light scattered and/or reflected by a bare silica plate. The were made hydrophobiceither by esterificationwith 1-dodecanol'5 maximum channel of the response function was taken as zero or by depositing a layer of cadmium arachidate (CdA) using the time in the fluorescence decay curves. The fluorescence decay Langmuir-Blodgett technique. All monolayers were spread from data obtained from the LB films in the absence of quencher were 1mM stock chloroform solutions in the conventional manner.I6 analyzed using the nonlinear least squares method based on the The monolayers were compressed at a rate of 0.04 & 0.01 n m 2 Marquardt algorithm.'* molecule-l min-I to the depositing pressure. The air-water monolayerswere deposited onto the silica substrates using either a Lauda FL-1 E Filmlift or a KSV 2200 AF'CDFC LB deposition Results and Discussion system. Prior to deposition,the monolayer was left to equilibrate at the depositing pressure for 15 min. In all LB experiments the The steady-state fluorescence spectra of a 2 mol % substrate was placed parallel to the advancing barrier. The pyrene-DPPE/DMPC monolayer with various amounts of depositing speed, for both the upward and downward stokes, CAT-16 deposited at 25 mN onto 1-dodecanol-treated was 5 mm min-l. During deposition of the monolayers the surface substrates in an aqueous solution are shown in Figure 1. pressure was maintained at a constant value. The fluorescence spectrum of each deposited film exhibits A water subphase solution containing 1mM CdClz was used monomer emission only with peak maxima at 378, 398, to prepare the CdA monolayers. The subphase pH was maintained in the range 6.0-6.5 by addition ofNaOH. The CdAlayers a n d 419 nm (shoulder). The absence of excimer emission were deposited onto the cleaned hydrophilic silica plates at a indicates that pyrene-DPPE is homogeneously distributed surface pressure of 20.00 k 0.02 mN m-' by raising the plates in the deposited monolayer films. vertically through the monolayer films. The CdA monolayers Figure 2 shows the fluorescence decay behavior of a 2 were allowed to dry in air for 30 min before subsequent layers % pyrene-DPPE/DMPC monolayer deposited at 25 mol were deposited. The deposition ratios ofthe CdA layers were 1.0 mN m-l onto a 1-dodecanol-treated substrate in the ?c 0.1. presence a n d absence of CAT-16 quencher in an aqueous The mixed monolayers containing the probe were deposited solution. In t h e absence of CAT-16, (curve a), the onto the hydrophobic plates in order to separate the monolayer fluorescence decay curve follows single-exponential decay containing the probe from the silica substrate so as t o eliminate kinetics, which confirms that pyrene-DPPE is not agpossible interference of the silica surface with the fluorescence of the probe. The mixed monolayers containing the probe were gregated in the deposited monolayer film. The solid line deposited on the downward stroke and were submerged in the is a fit to a single-exponential decay function a n d yields aqueous subphase solution following deposition. These monoa value of 21.5 f0.5 ns for the decay time. The quenched layers were deposited at a surface pressure of 25.00 & 0.02mN decays (curves b and c) are clearly nonexponential a n d are well described by the theory of diffusion-influenced (10)Axelrod, D. Biophys. J . 1977,18, 129. fluorescence quenching in a two-dimensional environ(11)Caruso, F.; Grieser, F.; Murphy, A,; Thistlethwaite, P. 3.; ment.19$20The solid lines are the fitted curves, and t h e Urquhart, R.; Almgren, M.; Wistus, E. J . Am. Chem. Soc. 1991,113, 4838. mutual lateral diffusion coefficients determined from the (12)Caruso, F.;Grieser, F.; Thistlethwaite, P. J.; Almgren, M. fittings to the equations for a two-dimensional s y ~ t e m ' ~ ~ ~ ~ Lanemuir 1993.9. 3142. - ~ ~ a r e shown in Table 1. (r3)CaGso, F.; Grieser, F.; Thistlethwaite, P. J.; Almgren, M.; - - I
?
Biophys. J . 1993,65, 2493. (14)Caruso, F.; Grieser, F.; Thistlethwaite, P. J.; Furlong, D. N. Macromolecules 1994,27,77. (15)Trau,M.; Murray, B.S.;Grant, K.; Grieser,F.J. ColloidZnterfme Sci. 1992,148, 182. (16) Gaines, G. L. Insoluble Monolayers at Liquid-Gas Interfaces; Wiley: New York, 1966; Chapter 3.
(17)Scholes,G. D.; Wilson, G. J.; Ghiggino, K. P. Chem.Phys. 1991, 155,127. (18)Marquardt, D. W. J. SOC.Ind. Appl. Math. 1963,11,431. (19)Medhage, B.;Almgren, M. J . Fluoresc. 1992,2,7 . (20)Medhage, B.Ph.D.Thesis,UppsalaUniversity, Uppsala, Sweden, 1993.
Letters
Langmuir, Vol. 10, No. 10,1994 3375
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Wavelength (nm) Legend
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= DMPC,
[ = pyrene-DPPE, 1= CAT-16.
Only one side of the substrate surface is shown.
Figure 1. Steady-state fluorescence spectra of a 2 mol % pyrene-DPPEDMPC monolayer with various amounts of CAT16 deposited onto l-dodecanol-treated substrates in aqueous solution: (a)0 mol % CAT-16; (b) 10 mol % CAT-16; (c) 20 mol % CAT-16. Excitation wavelength = 350 nm. Temperature = 20 f 1 "C.
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aqueaussolution
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Legend
4
= DMPC,
[ = pyrene-DPPE, 7 = CAT-16.
Only one side of the substrate surface is shown.
Figure 2. Fluorescence decays of a 2 mol % pyrene-DPPE/ DMPC monolayer with various amounts of CAT-16 deposited onto l-dodecanol-treated substrates in aqueous solution: (a)0 mol % CAT-16; (b) 10 mol % CAT-16: (c) 20 mol % CAT-16. Excitation wavelength = 350 nm, emission wavelength = 400 nm. Temperature = 20 f 1"C. All fluorescence decays have been normalized to the same maximum intensity. Table 1. Mutual Lateral Diffusion Coefficient of Pyrene-DPPEICAT 16 in a DMPC Air-Water Monolayer and LB Films at 20 f 1 "C D,, cm2s-l CAT-16, CAT-16, system zf(pyrene-DPPE),ns 10 mol % 20 mol % monolayela 11.2 5 f 2 x 10-8 4 f 2 x 10-8 LB filmb 21.5 6 f 1x 8 f 1x *
a
Monolayer pressure of 25 mN m-l. Cast monolayer pressure
of 25 mN m-l.
The results presented in Table 1clearly show that the molecules in the deposited films in contact with aqueous solution are highly mobile when compared to "dry"
deposited films which have diffusion coefficients -= cm2 s-1.6$21t22 Their mobility, however, is significantly lower than in an air-water monolayer state (see Table l), for similar temperature and deposition conditions. A limited number of lateral diffusion measurements have been made on "wet" LB Merkel et u Z . , ~ using the F W technique, obtained a value of 1 x cm2 s-l (at 20 "C) for the diffusion of N-(7-nitrobenz-2oxa-l,3-diazol-4-yl)-~-a-dimyristoylphosphatidylethanolamine (NBD-DMPE) in DMPC monolayers deposited at 20 mN m-l onto a hexadecyltrichlorosilane monolayer immersed i.n water. Tamm and McConnell,6 also using the F W technique, reported a value of (4 f 1) x cm2 s-l (at 21 "C) for the diffusion of N-(7-nitro-2,1,2benzoxadiazol-4-yl)egg phosphatidylethanolamine(NBDPE) in a deposited bilayer of DMPC in an aqueous environment. These values are in good agreement with the mutual lateral diffusioncoeficients of the amphiphiles obtained in this work. It should be noted that the lateral diffusion coefficients obtained in this work stem from a fluorescence quenching technique which only measures diffusion over a short-range, compared with the FRAP technique. An important comparison is that of the magnitudes of the mutual lateral diffusioncoefficientsof the amphiphiles in a monolayer at the air-water interface and those in deposited monolayer films submerged in aqueous solution. Examination of Table 1shows that the lateral diffusion coefficients obtained for the mixed monolayer of pyreneDPPEDMPC deposited at 25 mN m-l onto a l-dodecanoltreated substrate and immersed in aqueous solution are approximately half to an order to magnitude lower than those measured for the same mixed monolayer at the airwater interface at a surface pressure of 25 mN m-l. This difference is in excellent agreement with the work of Lindholm-Sethson et al.,23 who reported the lateral diffusion coefficient of a octadecylferrocene monolayer deposited onto an octadecyltrichlorosilane monolayer immersed in water to be approximately an order of magnitude lower than at the air-water interface, for similar deposition and surface pressures. The above results suggest that the extent of interactions between the alkyl chains of the dodecanol layer and the layer containing 2 mol % pyrene-DPPE, DMPC, and 10 or 20 mol % CAT-16 affects the value of the mutual lateral diffusion coefficients observed. This agrees well with literature r e p 0 r t s ~ 9that, ~ ~ for substrate-coupled bilayers, diffisivity a t the monolayer-monolayer interface may be dominated by frictional effects. Thus, the differences in the mutual lateral diffusion coefficients observed in the LB films and the air-water monolayer may be accounted for by hydrocarbon chain interdigitation between adjacent layers and/or the frictional drag exerted by amphiphiles in one layer against amphiphiles in the adjacent layer in the LB a s ~ e m b l i e s . ~ ~ ~ ~ , ~ ~ The sensitivityof clustering of deposited probe molecules is illustrated in Figures 3 and 4. Depositing the probe containing monolayers onto a cadmium arachidate (CdA) coated silica surface results in pyrene excimer emission (band with ,A = 485 nm; see Figure 3) and nonexponential decay of the pyrene fluorescence (data not shown), (21)Rabe, J. P.; Novotny, V.; Swalen, J. D.; Rabolt, J. F. Thin Solid Films 1988,159,359. (22) Honig, E. P.; Koning, B. R. Surf. Sci. 1976, 56, 454. (23) Lindholm-Sethson, B.; Orr, J. T.; Majda, M. Langmuir 1993,9, 2161. (24) Teissie, J.; Tocanne, J.-F.; Baudras, A. Eur. J. Biochem. 1978, 83, 77.
Letters
3376 Langmuir, Vol. 10,No.10,1994 I""
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h ._ m
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1= CdA, 1 DMPC, 8 = pyrene-DPPE. =
Only one side of the substrate surface is shown.
Figure 3. Steady-state fluorescence spectrum of a 2 mol % pyrene-DPPEDMPC monolayer with various amounts of CAT16 deposited onto a CdA layer, in aqueous solution. Excitation wavelength = 350 nm. Temperature = 20 f 1"C.
thus preventing the use of the fluorescence quenching approach to determine mutual lateral diffision coefficients for this film. Depositing two layers of cadmium arachidate on this film disperses the aggregated pyrene-DPPE (see spectrum a in Figure 4). These results are in agreement with those of Murphy,25 who also observed that the deposition of two additional layers of CdA on top of a mixed layer containing 10 mol % dansyldihexadecylamine (DDHA)in oleic acid (which is deposited onto a CdA layer) and submerged in aqueous solution causes the break up of DDHA aggregated species, and hence the presence of more DDHA monomer. In addition, for a film of pyreneDPPE in DMPC with CAT-16 deposited onto CdA-coated silica, with two CdA layers on top, pyrene-DPPE is again in cluster form (see spectrum b in Figure 4). These results illustrate the highly sensitive nature of the state of the molecules in the deposited film. It also shows the usefulness of the probe method in detecting variations in the film morphology with variations in film fabrication. (25) Murphy, A. Ph.D. Thesis, The University of Melbourne, Melbourne, Australia, 1992.
450
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Wavelength (nm)
Wavelength (nm) Legend
410
Legend
1= CdA, 1= DMPC, 1= pyrene-DPPE, 7 = CAT-16. Only one side of the substrate surface is shown.
Figure 4. Steady-state fluorescence spectra of a 2 mol % pyrene-DPPEDMPC monolayer without CAT-16 quencher (spectrum a) and with 10 mol % CAT-16 quencher (spectrum b) deposited onto a CdA layer, with two CdA layers on top, in aqueous solution. Excitation wavelength = 350 nm. Temperature = 20 f 1"C.
Conclusions The work presented demonstrates that the fluorescence quenching approach can also be extended to LB films immersed in aqueous solution. This work also confirms that there is a significant difference in lateral mobility of amphiphiles embedded in air-water monolayers and LB films immersed in aqueous solution. The aggregation behavior of pyrene-DPPE was found to be sensitive to the composition of the LB film and the number of LB layers deposited.
Acknowledgment. We are most grateful to Prof. M. Almgren and Dr. E. Mukhtar from the Department of Physical Chemistry, University of Uppsala, Sweden, for the use of equipment in their laboratory. F.C. acknowledges the receipt of a University of Melbourne Postgraduate Writing-Up Award. Financial assistance from the Australian Research Council is also gratefully acknowledged.
