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J. Phys. Chem. B 2010, 114, 5736–5741
A Fluorescence Correlation Spectroscopy Study of the Diffusion of an Organic Dye in the Gel Phase and Fluid Phase of a Single Lipid Vesicle Subhadip Ghosh, Aniruddha Adhikari, Supratik Sen Mojumdar, and Kankan Bhattacharyya* Physical Chemistry Department, Indian Association for the CultiVation of Science, JadaVpur, Kolkata 700 032, India ReceiVed: December 18, 2009; ReVised Manuscript ReceiVed: March 4, 2010
The mobility of the organic dye DCM (4-dicyanomethylene-2-methyl-6-p-dimethyl aminostyryl-4H-pyran) in the gel and fluid phases of a lipid vesicle is studied by fluorescence correlation spectroscopy (FCS). Using FCS, translational diffusion of DCM is determined in the gel phase and fluid phase of a single lipid vesicle adhered to a glass surface. The size of a lipid vesicle (average diameter ∼100 nm) is smaller than the diffraction limited spot size (∼250 nm) of the microscope. Thus, the vesicle is confined within the laser focus. Three lipid vesicles (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)) having different gel transition temperatures (-1, 23, and 41 °C, respectively) were studied. The diffusion coefficient of the dye DCM in bulk water is ∼300 µm2/s. In the lipid vesicle, the average Dt decreases markedly to ∼5 µm2/s (∼60 times) in the gel phase (for DPPC at 20 °C) and 40 µm2/s (∼8 times) in the fluid phase (for DLPC at 20 °C). This clearly demonstrates higher mobility in the fluid phase compared with the gel phase of a lipid. It is observed that the Dt values vary from lipid to lipid and there is a distribution of Dt values. The diffusion of the hydrophobic dye DCM (Dt ∼ 5 µm2/s) in the DPPC vesicle is found to be 8 times smaller than that of a hydrophilic anioinic dye C343 (Dt ∼ 40 µm2/s). This is attributed to different locations of the hydrophobic (DCM) and hydrophilic (C343) dyes. 1. Introduction In recent years, fluorescence correlation spectroscopy (FCS) has been developed as an extremely powerful technique to study biological systems at the single molecule level with high spatial resolution.1 FCS is frequently used to determine the local concentration, diffusion coefficient of biomolecules, and various other physical parameters that can generate fluorescence fluctuations.1 Fluorescence correlation spectroscopy (FCS) has recently been applied to study the binding of ligands to a protein,2a,b protein-protein interactions,3a,b DNA hybridization,4 polymer chain reactions,5a,b DNA-protein interactions,6a,b interactions with receptors,7a,b and supramolecular complexes.8 Zettl et al. studied the formation of micelles by FCS.9 They showed that FCS could be used for determination of cmc in ionic surfactant systems provided attractive ionic interactions exist between the surfactant and the noncovalent dye molecule. Very recently, Lioi et al. employed FCS to study the interaction of DNA with catanionic vesicles as a means of electrostatic molecular sequestration.10 Mueller et al. investigated the scope of encapsulating hydrophobic solutes in block copolymer vesicles.11 Most recently, we have studied the region dependence of Dt in the gel phase of a triblock copolymer.12 The gel consists of interpenetrating micellar aggregates of ∼20 nm size.13 The spatial resolution of a microscope is 0.61λ/NA (NA, numerical aperture ) 1.2, in our case), which is ∼250 nm for excitation at 470 nm. Thus, in principle, it is not possible to spatially resolve different regions of a micellar aggregate of ∼20 nm size. To overcome this, we used three probes with different polarities.12 These three probes (DCM, C343, and C480) exhibit similar Dt values in water. In the gel phase of the triblock copolymer, Dt of the hydrophobic DCM (1 µm2/s) is found to * To whom correspondence should be addressed. E-mail: pckb@ iacs.res.in.
be 29 times smaller than that of the hydrophilic probe C343 (Dt ∼ 29 µm2/s). To explain this, it is proposed that the highly hydrophobic DCM “walks” through the interpenetrating polymer chains.12 The highly polar probe C343 diffuses through the water filled “void” space inside the gel and hence exhibits a higher value of Dt.12 In this work, we will show that Dt values in different locations of a lipid also vary. A lipid vesicle, i.e., a “water pool” surrounded by a bilayer membrane, is a simple model of a biological cell. Thus, study of diffusion in a lipid has fundamental implications in cell biochemistry.14 In general, a lipid vesicle exhibits a well-defined phase transition temperature (Tm). Above Tm, the lipid remains in the “fluid” phase with enhanced mobility. Below Tm, a “gel” or “solid” phase is obtained with markedly reduced mobility. Hochstrasser and co-workers studied the interaction of Nile Red molecules with lipid vesicles using high spatial resolution (“single-molecule-like”).15 The lipid vesicles used in their study were of a size (∼100 nm) smaller than the focal spot of the excitation source.15 They investigated the bimolecular association and dissociation reactions of single Nile Red molecules with lipid vesicles from an analysis of the on-and-off fluctuations in the fluorescence signal.15 They, however, did not study diffusion of Nile Red in the lipid by FCS.15 Korlach et al.16 previously applied FCS to determine Dt of an organic molecule in different phases of giant unilamellar vesicles of size >25 µm. They used different ratios of DLPC/ DPPC to obtain the different phases (fluid or ordered) of the lipid bilayer at the same temprature. In the DLPC (no DPPC), only a single fluid phase (low viscosity) was observed. On increasing the DPPC content (i.e., DLPC/DPPC ) 0.6/0.4), an ordered phase (high viscosity) was shown to coexist with the fluid phase. FCS studies reveal that, at a high DLPC/DPPC (>0.8/0.2) ratio, only the fluid phase is present and the
10.1021/jp911971p 2010 American Chemical Society Published on Web 04/13/2010
FCS Study of the Diffusion of an Organic Dye SCHEME 1: Schematic Representation of (A) DCM, (B) C480, and (C) C343
autocorrelation curve is marked by fast diffusion (Dt ∼ 4 × 10-8 cm2/s). Though this study indicated a variation of mobility of different phases, they did not generate a gel phase, exclusively. At a low DLPC/DPPC (
