pubs.acs.org/Langmuir © 2009 American Chemical Society
Polymer Dynamics in Layer-by-Layer Assemblies of Chitosan and Heparin Maria Lundin,† Eva Blomberg,†,‡ and Robert D. Tilton*,§ †
Surface and Corrosion Science, Department of Chemistry, Royal Institute of Technology, Drottning Kristinas v€ ag 51, SE-10044 Stockholm, Sweden, ‡Institute for Surface Chemistry, P.O. Box 5607, SE-114 86 Stockholm, Sweden, and §Center for Complex Fluids Engineering, Department of Chemical Engineering and Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 Received August 10, 2009. Revised Manuscript Received October 21, 2009 The layer-by-layer deposition method has been used to build a multilayer thin film with two polysaccharides, chitosan CH (weak polycation) and heparin HEP (strong polyanion), on planar quartz surfaces. The film structure and dynamics in aqueous solution were studied with fluorescence resonance energy transfer (FRET) and total internal reflection fluorescence (TIRF). Particular emphasis was placed on the effect of deposition conditions, i.e., pH and salt concentration, on the out-of-plane (vertical) diffusion of fluorescence labeled chitosan in the chitosan/heparin (CH/ HEP) film. FRET analysis showed that CH molecules diffused within the film with a diffusion coefficient that was not significantly sensitive to the deposition pH and solution ionic strength. A pH-sensitive label bound to CH embedded within the CH/HEP film was sensitive to the charge of the outermost polymer layer even when buried under 14 alternate layers of CH and HEP. A consideration of the results obtained with both fluorescence techniques showed that the structure of the CH/HEP thin film was highly interpenetrated without clear boundaries between each layer. These results are consistent with the hypothesis that the previously observed exponential-like film growth of CH and HEP in terms of layer thickness and deposited amount versus deposition cycle can be attributed to out-of-plane diffusion of CH molecules in the multilayer.
Introduction The layer-by-layer (LbL) deposition technique was originally proposed for colloidal particles,1 and in the early 1990s Decher et al. showed that this method also could be used for multilayer polyelectrolyte film formation.2,3 LbL films are formed through the sequential adsorption of oppositely charged polyelectrolytes with rinsing by a polymer free solution in between depositions. The buildup is facilitated by overcompensation of the surface charge in each adsorption step, as has been measured experimentally.4-6 Mainly two types of film growth have been reported in the literature: film thickness and adsorbed amount increase either linearly or exponential-like with the number of deposited layers. This topic has been considered in recent reviews by von Klitzing7 and Picart.8 In general, a linear growth is observed for strongly charged synthetic polyelectrolytes whereas an exponential-like growth can be found for weak natural polyelectrolytes, such as polypeptides or polysaccharides.9,10,7 An advantage with weak polyelectrolytes is that their charge density in solution and in the adsorbed film can be adjusted by the solution pH. *Corresponding author: Tel 1-412-268-1159; Fax 1-412-268-7139; e-mail
[email protected]. (1) Iler, R. K. J. Colloid Interface Sci. 1966, 21, 569–594. (2) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210, 831–835. (3) Decher, G. Science 1997, 277, 1232–1237. (4) Blomberg, E.; Poptoshev, E.; Claesson, P. M.; Caruso, F. Langmuir 2004, 20, 5432–5438. (5) Picart, C.; Lavalle, P.; Hubert, P.; Cuisinier, F. J. G.; Decher, G.; Schaaf, P.; Voegel, J. C. Langmuir 2001, 17, 7414–7424. (6) Caruso, F. Chem.;Eur. J. 2000, 6, 413–419. (7) von Klitzing, R. Phys. Chem. Chem. Phys. 2006, 8, 5012–5033. (8) Picart, C. Curr. Med. Chem. 2008, 15, 685–697. (9) Lavalle, P.; Gergely, C.; Cuisinier, F. J. G.; Decher, G.; Schaaf, P.; Voegel, J. C.; Picart, C. Macromolecules 2002, 35, 4458–4465. (10) Elbert, D. L.; Herbert, C. B.; Hubbell, J. A. Langmuir 1999, 15, 5355–5362. (11) Ruths, J.; Essler, F.; Decher, G.; Riegler, H. Langmuir 2000, 16, 8871–8878.
3242 DOI: 10.1021/la902968h
Exponential-like growth previously has been attributed to various phenomena: an increased film surface roughness11 with a fractal-like growth, complexation of polyelectrolytes above the film surface,10 or more recently to diffusion5,9 of at least one of the two polyelectrolytes within the film. The diffusion hypothesis states that the adsorbed amount in a given deposition step depends not only on the amount of oppositely charged polymers in the outermost layers but also on the amount of free, diffusing polyelectrolytes available for complexation within the film. The amount of free polyelectrolytes is proportional to the multilayer thickness (volume) prior to deposition. For some polyelectrolyte pairs, such as chitosan/hyaluronan,12 poly(L-lysine)/ hyaluronan,13 poly(L-lysine)/poly(glutamic acid),14 and polyethylenimine/poly(acrylic acid),15 the confocal laser scanning microscopy (CLSM) technique has been used to visualize the out-of-plane (vertical) diffusion of fluorescence labeled polyions within very thick films during buildup. In the present work two polysaccharides, chitosan (CH) and heparin (HEP), were used in the LbL film formation on planar quartz silica surfaces. Chitosan is a weak cationic polysaccharide, obtained by N-deacetylation of chitin, which can be found in shells of shrimps, crabs, and insects.16 The degree of ionization of chitosan depends on the solution pH (pKa = 6.0-6.5).17 Two pH values are considered in the present work: pH 4.2 and 5.8. At pH 4.2 chitosan is highly charged, whereas approximately half of the amine groups are protonated at pH 5.8. Heparin is well-known (12) Richert, L.; Lavalle, P.; Payan, E.; Shu, X. Z.; Prestwich, G. D.; Stoltz, J. F.; Schaaf, P.; Voegel, J. C.; Picart, C. Langmuir 2004, 20, 448–458. (13) Picart, C.; Mutterer, J.; Richert, L.; Luo, Y.; Prestwich, G. D.; Schaaf, P.; Voegel, J. C.; Lavalle, P. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 12531-12535. (14) Lavalle, P.; Vivet, V.; Jessel, N.; Decher, G.; Voegel, J. C.; Mesini, P. J.; Schaaf, P. Macromolecules 2004, 37, 1159–1162. (15) Fu, J.; Ji, J.; Shen, L.; Kuller, A.; Rosenhahn, A.; Shen, J.; Grunze, M. Langmuir 2009, 25, 672–675. (16) Shahidi, F.; Abuzaytoun, R. Adv. Food Nutr. Res. 2005, 49, 93–135. (17) Rinaudo, M.; Pavlov, G.; Desbrieres, J. Polymer 1999, 40, 7029–7032.
Published on Web 11/18/2009
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for its anticoagulant activity and for having the highest negative charge density of all biological polyanions18 due to its carboxyl and sulfonate groups. It is highly charged at both pH values used in this study. LbL film formation of CH and HEP at the solid-liquid interface has been investigated in a few earlier studies.19-21 For example, it has been shown that a multilayer film of CH and HEP has the ability to reduce bacterial adhesion on medical implants where the antimicrobial efficiency depends on the solution conditions used during buildup.21 The number of bacteria that adhered to the surface was found to decrease with decreasing pH of the polymer assembly solution in the pH range 2.9-6.0. This was attributed to the increased hydrophilicity of the polymer film at low pHs. However, the film with the highest amount of CH in the outermost layer (at pH 3.8) was most efficient in killing bacteria that did adhere. On silica surfaces an increase in solution ionic strength and pH was found to increase the CH/HEP film thickness.19 The same observation was made in a recent study using identical solution conditions as in the present work.22 Further, the average film thickness and adsorbed amount measured with the dual polarization interferometry (DPI) technique increased exponential-like with the number of deposited layers. The aim of this study was to determine whether the observed exponential-like growth in thickness can be attributed to out-ofplane diffusion of CH molecules within the CH/HEP film. We further evaluate if the diffusion coefficient depends on the polymer deposition solution conditions and, hence, on the multilayer film structure. Limited by the out-of-plane resolution of CLSM, films must be built up to thicknesses as high as several micrometers in order to use that technique.13 The thickness and adsorbed amount of alternate depositions of CH and HEP, for the solution conditions used here, grow exponential-like in films less than 70 nm in thickness.22 Therefore, an attractive, alternate approach to CLSM for thin films is to study the out-of-plane polyelectrolyte diffusion using the fluorescence resonance energy transfer (FRET)23,24 technique. With a characteristic length scale of several nanometers set by the F€orster radius, FRET makes it possible to monitor polymer motion on much finer length scales and in much thinner films. In FRET measurements CH molecules were labeled by either a donor dye or an acceptor dye. Donor-acceptor energy transfer is extremely sensitive to the distance between the fluorophores and therefore serves as a reporter of changes in proximity of labeled polymers as they diffuse. Both dyes (succinimidyl esters in the Alexa family of commercially available dyes) showed low sensitivity to local changes in pH. By varying the initial distance, i.e., the number of intermediate layers, between the donor- and acceptor-labeled CH molecules in the film, the diffusion path was varied. Complementary qualitative information about how the film relaxes to accommodate additional layers was obtained with the total internal reflection fluorescence (TIRF) technique using either a highly pH-sensitive FITC dye or a relatively pHinsensitive Alexa dye on chitosan molecules adsorbed onto or embedded within the multilayer film. (18) Salmivirta, M.; Lidholt, K.; Lindahl, U. FASEB J. 1996, 10, 1270–1279. (19) Boddohi, S.; Killingsworth, C. E.; Kipper, M. J. Biomacromolecules 2008, 9, 2021–2028. (20) Serizawa, T.; Yamaguchi, M.; Akashi, M. Biomacromolecules 2002, 3, 724– 731. (21) Fu, J. H.; Ji, J.; Yuan, W. Y.; Shen, J. C. Biomaterials 2005, 26, 6684–6692. (22) Lundin, M.; Solaqa, F.; Thormann, E.; Blomberg, E., manuscript in preparation. (23) F€orster, T. Ann. Phys. 1948, 2, 55–75. (24) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983.
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Materials and Methods Chemicals. Chitosan (CH) was purchased from Fluka (cat. no. 50494, low-viscosity grade) with a molecular weight of ∼150 000 g/mol and a 90% degree of deacetylation. The stock solution of CH was prepared with a concentration of 1 wt % in 1 wt % aqueous acetic acid (10 000 ppm) and stirred for at least 48 h before use in order to ensure complete dissolution. This solution was diluted to 100 ppm prior to measurements. Heparin, sodium salt, from porcine intestinal mucosa, with a molecular weight of ∼14 000 g/mol, was purchased from Merck Bioscience (cat. no. 375095). A stock solution of 1 wt % was prepared in aqueous solution and diluted to 1000 ppm for use in experiments. The fluorescent dyes fluorescein 5-isothiocyanate (FITC), Alexa Fluor 488, and Alexa Fluor 555 protein labeling kits were purchased from Molecular Probes (Eugene, OR, cat. no. F143, A10235, and A20174, respectively). NaCl (Suprapur grade, >99.99% purity) was obtained from Merck. All chemicals were used as received. All water used for solution preparation was of Milli-Q grade (18 MΩ 3 cm resistivity). LbL depositions were carried out at either pH 4.2 or pH 5.8. For all sample solutions used during LbL deposition (including rinsing solutions), the pH was adjusted to 4.2 or 5.8 by acetic acid and sodium hydroxide prior to each use. The pH of all solutions was stable for at least 1 day. Dye Labeling of Chitosan. For TIRF and FRET measurements, CH was fluorescence labeled using three different aminereactive dyes: FITC, Alexa 488, or Alexa 555. Labeling with FITC was done according to the procedure suggested by Richert et al.,12 except that CH was dissolved in 1 wt % acetic acid, since it was insoluble in pure Milli-Q water. FITC was dissolved in dimethyl sulfoxide (DMSO) and mixed with the pre-equilibrated CH solution (1 wt % CH in a 1 wt % acetic acid solution) at the molar ratio 14 mol of FITC/mol of CH. The mixed solution was adjusted to pH 6.0 and stirred with a magnetic bar for 24 h at room temperature. Chitosan labeling with the Alexa succinimidyl ester dyes was carried out according to the amine labeling protocol suggested by the supplier (www.probes.com), with one exception: the reaction was carried out overnight at pH 6.0 instead of for 1 h at pH 8.5. All dye-CH solutions were passed through a Sephadex G-25 size exclusion column (PD-10, Amersham Bioscience, Sweden). Thus, the buffer was exchanged to the desired final ionic strength, and any unbound dye was separated from the final reaction mixture. UV-vis spectrophotometry (Cary 300 Scan, Varian, Inc., Palo Alto, CA) was used to prepare reference curves of the absorbance, A, of chitosan, FITC, Alexa 488, and Alexa 555 at known bulk concentrations, c. Extinction coefficients, ε, were calculated using the Beer-Lambert law. By measuring the absorbance of the purified CH-dye solution at two different wavelengths, the concentrations of both CH and dye were estimated from eq 1, and the molar labeling ratio between the attached dye and CH could be calculated via Aλ ¼ εCH, λ cCH þ εdye, λ cdye
ð1Þ
Labeling ratios were measured in concentrated (∼10 000 ppm) solutions to ensure accuracy. Prior to measurements, the chitosan was diluted to 100 ppm in a solution with a predetermined background electrolyte concentration. Labeled stock solutions were refrigerated in aliquots and used within 4 weeks of preparation according to manufacturer guidelines. Substrates. Measurements were performed on quartz substrates (Bioelectrospec, now TIRF Technologies, Inc., Morrisville, NC), which were first cleaned by rinsing with RBS detergent (containing sodium hypochlorite) (Pierce Biotechnology, Thermo Fisher Scientific, Rockford, IL) followed by an excess amount of Milli-Q water. The surfaces were then soaked in Chromerge solution, mixed in the same manner as suggested in a previous paper,25 for 30 min. After an extensive Milli-Q water rinse, the (25) Daly, S. M.; Przybycien, T. M.; Tilton, R. D. Langmuir 2003, 19, 3848– 3857.
DOI: 10.1021/la902968h
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Table 1. UV-Vis Spectrophotometry Analysis of the Dye to CH Labeling Ratio and the Extinction Coefficient, ε, of CH and Dyes at Various Wavelengths ε(λ=210 nm) (M-1 cm-1) chitosan 3.0 10 FITC 3.7 104 Alexa 488 4.9 104 Alexa 555 2.8 104 a Data obtained from the manufacturer. 5
ε(λ=494 nm) (M-1 cm-1)
ε(λ=555 nm) (M-1 cm-1)
0 6.4 104 7.4 104 2.7 104
0
quartz slides were left in 6 M hydrochloric acid for 20 min and rinsed again with water before the final cleaning step where the surfaces were soaked in a 10 mM sodium hydroxide solution for 20 min. After this treatment the surfaces were completely wettable by water. Substrates were used immediately after cleaning. The final procedure before mounting a substrate into the TIRF instrument was a three-step ethanol/water/ethanol rinse and drying by a nitrogen stream. Substrates cleaned by this procedure can be reused many times for adsorption experiments without any detectable difference in results. Total Internal Reflection Fluorescence (TIRF). TIRF is a spectroscopic technique capable of detecting fluorescent molecules adsorbed at or in the vicinity of an interface between two materials with different refractive indices. The basic principles of the technique can be found elsewhere.26 For this study, we used a modular Spex-Fluorolog-3 fluorescence spectrometer (Horiba Jobin Yvon, Edison, NJ) with unpolarized excitation light. An incident light beam is directed into a 73 dove-type prism, which is optically coupled to a quartz slide via 2 μL of glycerol. A rectangular slit flow cell (Bioelectrospec, now known as TIRF Technologies, Inc.) was used with dimensions of 16 mm 24 mm 400 μm. The flow rate was set to 2 mL/min. Excitation and emission slit widths were 3 nm for CH-FITC and 1 nm for CH-Alexa 488. The fluorescence excitation and emission of both FITC and Alexa 488 were monitored at λex = 494 nm and λem = 520 nm. A baseline was established for at least 10 min prior to the injection of CH for 10 min under flow. The TIRF cell was then rinsed for 5 min with a polymer-free background solution of equal ionic strength and pH as the chitosan and heparin solutions. Next, HEP was injected into the flow cell, and adsorption proceeded for 10 min under flow followed by rinsing with background solution for 5 min again at the same constant pH. This procedure was then repeated to form a multilayer film. For the experiments described below, dye-labeled polymers were placed at different positions in the film, and only the dye-labeled polymers contributed to the TIRF signal. All measurements were carried out at least twice to demonstrate reproducibility. Fluorescence Resonance Energy Transfer (FRET). The fluorescence (or F€ orster) resonance energy transfer technique (FRET) is based on the energy transfer between two fluorophores. Detailed descriptions of the technique are provided by Lakowicz24 and Stryer.27 In brief, a donor dye in its excited state may transfer energy through dipole-dipole interactions to an acceptor dye in close proximity (∼1-10 nm). The efficiency of the FRET process decreases with the sixth power of the interfluorophore separation distance. Experimentally, FRET is observed as a decrease in donor emission intensity and an increase in acceptor emission intensity, when the system is excited at the donor excitation wavelength. The steady-state FRET technique was used to monitor changes in the distance between CH molecules that originated in different layers within the CH/HEP multilayer film. For this purpose CH molecules were labeled with a donor dye (Alexa 488) or an acceptor dye (Alexa 555). The CH donor dye was always placed in the fifth layer from the quartz substrate whereas the position of the CH acceptor was varied in the film. (26) Lok, B. K.; Cheng, Y. L.; Robertson, C. R. J. Colloid Interface Sci. 1983, 91, 87–103. (27) Stryer, L. Annu. Rev. Biochem. 1978, 47, 819–846.
3244 DOI: 10.1021/la902968h
1.5 105
Mw (Da)a
labeling ratio (ndye/nchitosan)
150000 389 884 1250
2 2 2
The maximum initial dye separation distance is equal to the sum of the average thickness of the intervening CH/HEP layers that separate the donor and acceptor dyes. The average layer thickness within the CH/HEP film was estimated from previous dual polarization interferometry measurements using the same solution conditions.22 For a given donor-acceptor separation distance, r, the FRET efficiency, E, follows24 E ¼
1 1 þ ðr=R0 Þ6
ð2Þ
where R0 is the F€ orster radius, the distance at which E is 50%. The F€ orster radius for the Alexa 488/Alexa 555 FRET pair is 7 nm, a value provided by the supplier (Molecular Probes). In the Results section, data are presented as the efficiency of energy transfer calculated from the relative steady-state fluorescence emission intensity of the donor dye in the presence, Fda, and in the absence, Fd, of the acceptor dye:24 E ¼ 1-
Fda Fd
ð3Þ
The FRET measurements were carried out using the TIRF instrument with the same flow cell and an identical adsorption procedure for the LbL film buildup. The fluorescence emission intensities of Alexa 488 (donor) and Alexa 555 (acceptor) were monitored simultaneously with the excitation and emission wavelengths set to the following pairings: λex = 494 nm (donor excitation maximum) and λem = 520 nm (donor emission maximum), λex = 555 nm (acceptor excitation maximum) and λem = 565 nm (acceptor emission maximum), and λex = 494 nm (donor excitation maximum) and λem = 565 nm (acceptor emission maximum).
Results Chitosan, FITC, and Alexa in Solution. In general, dye labeling of primary amine groups on polymers and proteins is considered a straightforward procedure. However, some difficulties were encountered when labeling CH molecules. It was impossible to carry out the reaction at pH 8.5, as suggested by the manufacturer, since the pre-equilibrated CH solution (1 wt %) began to phase separate when the solution pH was raised above pH 6.4. Further, the FITC dye dissolved in DMSO was not soluble in solution below pH 6.0. However, at exactly pH 6.0 both CH and FITC were soluble in a mixed solution, and the reaction could proceed. Table 1 shows the extinction coefficients, ε, of CH and the fluorescence dyes and their final labeling ratios. The low pH used during dye labeling of CH resulted in relatively low labeling ratios. This is in fact an advantage as long as the emission intensity is high enough to monitor because the adsorption affinity of the sparsely labeled CH will not be significantly altered compared to unlabeled CH. The final labeling ratio was 2 mol of dye/mol of CH, which is equivalent to 2 mmol of dye/mol of CH saccharide unit. Hence, only 0.2% of the amine groups in CH were covalently bound to a dye molecule. Langmuir 2010, 26(5), 3242–3251
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Figure 1. Change in fluorescence emission intensity due to addition of CH molecules labeled with FITC (a) or Alexa 488 (b) from a 150 mM NaCl solution at pH 5.8 to a quartz substrate precoated with five bilayers of unlabeled CH and HEP. The emission intensity is measured at λem = 520 nm. Arrows indicate the beginning of a rinse with polymer-free electrolyte solution. The inset in (a) shows that the FITC emission had started to decrease before the rinse started.
CH molecules absorb a significant amount of light in the wavelength range 190-230 nm, whereas no increased absorbance relative to the background electrolyte occurs at or above 400 nm (data not shown). The ε value for CH is therefore relatively high, 3.0 105 M-1 cm-1, at an excitation wavelength of 210 nm whereas it is 0 at 494 nm. The dyes have absorbance maxima at approximately 494 nm (FITC and Alexa 488) and 555 nm (Alexa 555), but they also absorb light of shorter wavelength. Their extinction coefficients were also measured at 210 nm in order to account for the absorbance contribution from the dye when evaluating the concentration of CH in dye-labeled solutions by eq 1. The fluorescence emission intensity from FITC molecules is 66% weaker in solution at pH 4.2 compared to pH 5.8, whereas the intensity decrease from the Alexa dye is only 8% (see Supporting Information). The diminished emission intensity of fluorescein in acidic solutions (pKa ∼ 6.5) is well-established.28-31 Such pH-sensitive dyes also show emission intensities that are strongly dependent on local electrostatic potential and thus proximity to a charged surface.28,25 The low pH sensitivity for the Alexa dye is therefore critical for FRET experiments in a multilayer film, where the local electrostatic potential fluctuates across layers. In order to test the stability of the covalent bond between CH and the dye at low solution pH, a solution of Alexa-labeled CH was acidified from pH 6 to pH 4. After 4 h of equilibration, the solution was passed through a size exclusion column. A single yellow band of Alexa-labeled CH was visible, and there was no trace of unbound dye in the column. The final sample contents after the acid equilibration and column passage were analyzed with UV-vis spectrophotometry and found to have the same labeling ratio as the initial stock solution had at pH 6.0. This proves that the covalent bond between chitosan and the dye is stable in low-pH solutions down to at least pH 4. Fluorescence Emission from Labeled CH Adsorbed in/on the CH/HEP Film. TIRF was used to study the adsorption and possible desorption of labeled CH on a CH/HEP multilayer film. It is important to note that in all TIRF measurements (28) (29) (30) (31)
Robeson, J. L.; Tilton, R. D. Langmuir 1996, 12, 6104–6113. Chen, R. F. Arch. Biochem. Biophys. 1969, 133, 263–279. Klugerman, M. R. J. Immunology 1966, 95, 1165. Emmart, E. W. Arch. Biochem. Biophys. 1958, 73, 1.
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Figure 2. Change in emission intensity due to sequential adsorption of CH and HEP on top of a CH-FITC layer from a 150 mM NaCl solution at pH 5.8. A precursor of two CH/HEP bilayers was preadsorbed onto the quartz substrate before CH-FITC solution was added. After rinsing, indicated by r, the first injection of HEP was made. Subsequent additions of unlabeled CH (black line) or HEP (gray line) produced an increase or decrease in the fluorescence intensity, respectively, even though neither CH nor HEP was labeled. For reference, the inset shows the film thickness and deposited amount measured separately by DPI after each deposition step.22
(Figures 1-3) only one CH layer is adsorbed from a solution of dye-labeled CH. All other layers were deposited from unlabeled CH solutions or HEP solutions. The measured changes in emission intensity during the buildup were therefore due to the response of those labeled CH molecules to subsequent microenvironmental changes induced by further LbL deposition. Since the pH was constant during all deposition and rinse steps, observed changes cannot be ascribed to any bulk pH effects. Figure 1 shows the adsorption of CH-dye molecules on a quartz surface that had been precoated with five bilayers of CH and HEP. The emission intensity increased immediately upon the DOI: 10.1021/la902968h
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Figure 3. Change in emission intensity due to sequential adsorption of CH and HEP on top of a CH-Alexa 488 layer from a 150 mM NaCl solution at pH 5.8. A precursor of two CH/HEP bilayers was preadsorbed onto the quartz substrate before the CH-Alexa solution was added. After rinsing, r, the first injection of HEP was made. Subsequent additions of unlabeled CH (black line) and HEP (gray line) produced small changes in fluorescence intensity, with a general tendency to increase with each added bilayer.
addition of CH-FITC (Figure 1a) or CH-Alexa (Figure 1b). For adsorbed CH-FITC the signal passed through a maximum and began decreasing before the start of a rinse step. The signal continued decreasing at the same rate after rinsing with a polymer-free background electrolyte for 5 min followed by exposure to the electrolyte solution for 10 h. In contrast, the CH-Alexa intensity decreased only slightly and remained stable over 10 h. To determine whether this loss of CH-FITC intensity was caused by photobleaching, a repeat measurement was conducted with an increased number of measuring points and longer excitation exposure times. This did not significantly change the intensity decay rate (data not shown), ruling out photobleaching. It is unlikely that the decay in emission intensity with time is caused by desorption of preadsorbed CH-dye molecules, since the emission intensity from adsorbed CH-Alexa 488 dye molecules remained nearly constant during rinsing and the 10 h exposure to electrolyte solution (Figure 1b) and since the CH-FITC intensity decrease actually started before rinsing. It likely indicates a change in the CH-FITC local environment to be discussed below. The sequential adsorption of CH and HEP to a preadsorbed CH-FITC (Figure 2) or CH-Alexa (Figure 3) layer was further evaluated with the TIRF technique. In Figure 2, the addition of CH-FITC to a preadsorbed two-bilayer film of CH and HEP on quartz at pH 5.8 with 150 mM NaCl caused a rapid increase in the emission intensity as the labeled CH adsorbed. Rinsing decreased the measured emission intensity slightly, whereas subsequent addition of HEP drastically decreased the emission intensity. The deposition of additional layers of unlabeled CH caused the emission intensity to return approximately to its original value prior to the next addition of HEP. This is noteworthy since no extra dye had been added to the adsorbed film. Upon subsequent depositions of HEP (gray lines) and CH (black lines), with rinsing in between, the intensity showed maxima and minima when CH or HEP was adsorbed in the outermost layer, respectively. Further, the difference between the intensity maxima and minima decreased slightly after each CH/HEP deposition cycle. A repeat 3246 DOI: 10.1021/la902968h
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measurement was carried out in solution at pH 4.2 instead of pH 5.8, but the emission intensity from CH-FITC molecules was on the limit of instrumental detection (data not shown) and could not be reliably studied at that pH. Figure 3 shows the adsorption of CH-Alexa 488 molecules and the change in emission intensity due to subsequent addition of unlabeled CH and HEP. The measurement conditions were identical to those used for the buildup with CH-FITC in Figure 2, except that an Alexa 488 dye was used instead of FITC. The major difference between these measurements is that there was no significant decrease in the emission intensity from preadsorbed CH-Alexa molecules upon subsequent adsorption of HEP (gray line). Instead, there was a small but continuous intensity increase with the number of deposited layers that was most pronounced upon addition of CH (black line). A measurement of the LbL deposition using the Alexa 488 dye was also carried out in solution at pH 4.2. The emission intensity evolution in this measurement was very similar to the measurement performed at pH 5.8 (see Supporting Information). Diffusion of Chitosan-Dye Molecules within Multilayers. The out-of-plane diffusion of CH molecules within the CH/HEP multilayer was studied with FRET. CH molecules were labeled with one of two Alexa dyes: CH-Alexa 488 or CH-Alexa 555. The donor dye (Alexa 488) was in all measurements (Figures 4 and 5) placed in the 5th layer from the quartz substrate, and the acceptor dye (Alexa 555) was placed in the 7th or 19th layer. In all other layers, CH was unlabeled. The maximum initial separation distance between the dyes in this study is assumed to be equal to the sum of the average thicknesses of the intermediate layer or layers. Table 2 shows these values obtained from previous measurements of the film thickness using DPI.22 The film thickness, and hence the separation distance between the dyes, increases when the deposition solution has high ionic strength and pH. According to eq 3, only the donor emission intensity is required to determine the FRET efficiency E. Nevertheless, the other combinations of excitation and emission were recorded for completeness to ensure that the loss in donor emission was due to energy transfer and not to some other quenching process or simply loss of dye from the film. The energy transfer was confirmed since the donor Alexa 488 emission (λex = 494 nm, λem = 520 nm) decreased simultaneously with the increase in acceptor Alexa 555 emission (λem = 565 nm) upon addition of Alexa 555 (illustrative raw data are presented in Supporting Information). Figure 4 shows the efficiency of energy transfer in a 150 mM NaCl solution at pH 5.8 as a function of time. An arrow marks the addition of the acceptor dye. The acceptor-labeled CH was the last layer deposited in these experiments. Independent of whether the dyes were initially separated by one HEP layer (open diamonds) or by 13 CH and HEP layers (filled diamonds), there was an instant transfer of energy upon addition of the acceptorlabeled CH. The FRET efficiency showed an almost linear increase during the initial 30-60 s with a slope of approximately 55 and 6 min-1 for the single intermediate layer and 13 intermediate layers, respectively. In the latter case, E increased continuously with time without reaching a plateau on the measurement scale. After 10 min of acceptor-CH adsorption followed by 5 min rinsing the FRET efficiency was 21%. This value increased when the polymer film was equilibrated in the background electrolyte solution for an additional 30 min (24%) without flow. An even longer equilibration time, 14 h, resulted in a FRET efficiency of 27% (data not shown). Figure 5 shows the FRET efficiency as a function of time for three independent measurements conducted in the same manner Langmuir 2010, 26(5), 3242–3251
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Figure 4. Energy transfer efficiency between CH-Alexa 488 and CH-Alexa 555 dye molecules adsorbed in/on a CH-HEP multilayer film prepared in a solution of 150 mM NaCl at pH 5.8. Donor and acceptor were initially separated either by (1) 1 HEP layer (open diamonds) or (2) 13 layers of CH and HEP (filled diamonds). These two situations are illustrated schematically at right. CH-Alexa 488 was present in layer 5. Arrows mark the addition of CH-Alexa 555 (acceptor dye) and the beginning of a rinse with polymer-free electrolyte solution. Lines are drawn to guide the eye.
as above but using different solution deposition conditions. The donor and acceptor labeled CH dyes were separated by 13 layers of CH and HEP in these experiments. An arrow in the figure indicates the addition of the acceptor CH. The rate of increase of the FRET efficiency was higher in LbL films that were prepared in solutions of low ionic strength and pH. Figure 5 shows the following order in terms of the rate of the linear increase in FRET efficiency within the first minute of acceptor Ch deposition at t = 5 min: 30 mM NaCl at pH 4.2 > 30 mM NaCl at pH 5.8 > 150 mM NaCl at pH 5.8. In Figure 5b, these initial rates were 31 min-1 (open diamonds), 14 min-1 (circles), and 6 min-1 (filled diamonds). The final value of E also depended on deposition conditions in a similar manner, but this limiting value depends on the concentrations of donor and acceptor deposited in the film, which vary for different conditions as discussed below. A small overshoot was observed in the FRET efficiency in the 30 mM NaCl, pH 4.2 experiment.
Discussion Film Structure. From TIRF measurements some important observations were made about the structure of the CH/HEP film. These measurements were carried out with two different dyes, Alexa 488 and FITC. The dyes have similar absorbance and emission spectra but differ in that Alexa is more photostable, brighter, and less pH-sensitive compared to FITC.32 The fact that FITC exhibits pH sensitivity and therefore also electrostatic potential sensitivity can be advantageous since monitoring the changes in fluorescence emission provides additional information about the local environment of the dye. On the other hand, interpretation of data may be misleading when only a sensitive dye is used. For example, comparing the decreased emission intensity with time observed for CH-FITC molecules adsorbed on a CH/HEP film with the emission intensity of the CH-Alexa dye showed that the loss of signal was not due to desorption of preadsorbed CH-FITC molecules (Figure 1a). In solution, the fluorescence emission intensity of the FITC dye showed a strong dependence on pH in the interval pH 4-6. (32) Panchuck-Voloshina, N.; Haugland, R. P.; Bishop-Stewart, J.; Bhalgat, M. K.; Millard, P. J.; Mao, F.; Leung, W.; Haugland, R. P. J. Histochem. Cytochem. 1999, 47, 1179–1188.
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Similar observations have been made previously and explained by the well-known fact that a FITC molecule is titrated among its prototropic forms that give different emission intensities.29,28 Even at a constant bulk pH, the protonation state and therefore emission intensity of a FITC molecule confined in a film are governed by the local electrostatic potential. Thus, proximity to a negatively charged substrate or polyelectrolyte favors protonation, which diminishes the fluorescence emission intensity.28 Bearing in mind that the bulk pH was constant throughout these experiments, the change in FITC protonation state in response to local electrostatic potential can explain the observed variations in emission intensity depending on whether CH or HEP is adsorbed in the outermost layer onto a preadsorbed CH-FITC layer (Figure 2). Clearly, the decreased intensity upon adsorption of anionic HEP to the CH-FITC layer is not caused by desorption of preadsorbed CH-FITC molecules since the intensity increases again upon further addition of cationic, unlabeled CH. The charge nature of the last adsorbed polymer evidently plays a decisive role in the fluorescence of the embedded CH-FITC molecules. The intensity variations persist even after adsorption of seven HEP/CH bilayers, where the distance between the initial position of the embedded CH-FITC molecules and the outermost layer by far exceeds the Debye screening length, κ-1 (κ-1 = 8 A˚), in an aqueous 150 mM NaCl solution. The continuing influence of layer deposition after so many steps can be explained by the diffusion of CH-FITC throughout the film, so that some FITC always remains in close proximity to the newly deposited layers. The decreasing intensity observed when CH-FITC was deposited in the outermost layer with no subsequent deposition (Figure 1) was also likely due to the diffusion of CH-FITC into and throughout the underlying negatively charged HEP layer. The time scale for the FITC emission decay (several hours) was notably longer than the time scale for the FRET experiments, which showed significant increases in FRET efficiency after just 30 min. This could point to a cause other than diffusion for the observed decrease in CH-FITC emission, but as noted above, the FRET efficiency was still increasing slightly even after 14 h of donor deposition, pointing to continuing slow evolution of the chitosan distribution in the film. DOI: 10.1021/la902968h
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Figure 5. Energy transfer efficiency between the CH-Alexa 488 and CH-Alexa 555 dye molecules adsorbed in/on a CH-HEP multilayer
film. Three independent measurements are shown where 13 layers of CH and HEP, namely (HEP-CH)6-HEP, were adsorbed between the layers containing the dyes. Film deposition was carried out from a solution at 150 mM NaCl, pH 5.8 (filled diamonds), 30 mM NaCl, pH 5.8 (solid line), or 30 mM NaCl, pH 4.2 (open diamonds). Arrows mark the addition of CH-Alexa 555 (acceptor dye) and rinsing in (a). Lines are drawn to guide the eye in the figure. The first minute after CH-Alexa 555 deposition is depicted in (b). Table 2. Layer Thickness and Adsorbed Amount of the CH and HEP Layers That Separate the Donor and Acceptor Dyes
solution deposition conditions
surface thickness of 13 CH concentrationa and HEP of 13 CH and HEP layers (nm) layers (mg/m2)
150 mM NaCl, pH 5.8 49 35 150 mM NaCl, pH 4.2 34 23 30 mM NaCl, pH 4.2 20 13 a Values calculated from DPI measurements using the de Feijter equation using dn/dc = 0.18 mL/g and a refractive index of 1.335 for the surrounding media.
Variation in ionization state for a weak polyelectrolyte embedded in a multilayer polyelectrolyte film has been observed previously by Fourier transform infrared spectroscopy.33 Its ionization oscillated with the net charge of the outermost layer, changing in the direction that would maintain electroneutrality of the multilayer. The same is expected of the carboxyl groups on the fluorescein molecule. Addition of a negatively charged polyelectrolyte to the film moves the overall electrostatic potential in a more negative direction, favoring protonation of the fluorescein with the resulting decrease in fluorescence emission. In a previous study von Klitzing and M€ohwald reported variations in the emission intensity when changing the bulk pH over LbL multilayer films of two flexible synthetic polyelectrolytes, poly(allylamine hydrochloride) (PAH) and poly(styrenesulfonate) (PSS), containing a buried PAH-FITC layer.34 They observed that a pH change in the surrounding solution caused a change in emission intensity from the buried PAH-FITC in the manner expected;decreasing pH decreased FITC emission intensity. This sensitivity to the external pH was attributed to the permeability of the PAH/PSS film to protons. This is consistent with our results. Analysis of the pH dependence of the emission intensity indicated that the pKa of the FITC label shifted to higher pH when the outermost layer was the polyanion PSS. When PSS was added to the film protonation of FITC was favored, consistent with our findings. Another interesting finding when comparing the fluorescence intensity evolution during the CH/HEP and PAH/PSS buildup is that the difference between the maximum and minimum emission (33) Xie, A. F.; Granick, S. Macromolecules 2002, 35, 1805–1813. (34) von Klitzing, R.; Mohwald, H. Langmuir 1995, 11, 3554–3559.
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intensities decreased with increasing number of deposited layers in both systems. However, after deposition of seven layers of PSS and PAH on top of the PAH-FITC layer, these changes were evened out and for the remaining film formation there were no further intensity changes.34 This observation was attributed to a gradual densification of the film, whereby the FITC molecules lost their sensitivity to changes in the outermost layer. In contrast, for the CH/HEP buildup not even 14 layers of HEP and CH was sufficient to reach a constant emission intensity value. The difference between films prepared from PAH/PSS and CH/HEP is probably related to their internal film structure. The film formed with synthetic flexible polymers, such as PSS/PAH, shows a linear buildup in thickness with the number of deposition cycles9 whereas CH/HEP films have an exponential-like growth. Because of the stronger complexation of flexible PAH/PSS, we can expect this film to be rigid with a compact layer structure in comparison to a highly hydrated and poorly organized CH/HEP film. Additionally, layers of PAH/PSS are interpenetrated only by the nearest neighbors.35 This has been demonstrated by the observations of Bragg peaks in X-ray reflectivity measurements. The polymer distribution width of PAH/PSS has been estimated to be approximately 1.5-2.5 bilayers.36 No Bragg peaks have to our knowledge ever been reported for films of natural polyelectrolytes that exhibit the exponential-like growth in terms of thickness. In CH/HEP films, the polyelectrolytes are more free to interdiffuse so some dye is always near the top and sensitive to the electrostatic potential changes, and concomitant proton diffusion, caused by the outermost layer being deposited. The increased emission intensity from the Alexa 488 dye caused by subsequent addition of HEP and CH to the preadsorbed CH-Alexa layer in Figure 3 is puzzling, and currently we merely rule out one possible cause. Although the Alexa dye is less pHsensitive than FITC, it is still not completely insensitive to environmental changes. A variety of factors affect fluorescence emission, including the polarity, pH, and refractive index of the surrounding media.24 The effects that changes in these parameters have on the emission intensity vary from one dye to another, which makes it difficult to make a general assumption. We first hypothesized that the increased emission intensity was caused by a (35) Schmitt, J.; Grunewald, T.; Decher, G.; Pershan, P. S.; Kjaer, K.; Losche, M. Macromolecules 1993, 26, 7058–7063. (36) Schonhoff, M. J. Phys.: Condens. Matter 2003, 15, R1781–R1808.
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gradual densification of the film as the layers became less hydrated with increasing depositions. A decreased relative water content in the film during buildup has been observed previously for films of CH/HEP22 using the same solution conditions as in this study as well as for the polypeptides, PLL/PGA.37 These results were obtained from a combination of two techniques that measure the adsorbed amount including trapped water (quartz crystal microbalance with dissipation) or the “dry” adsorbed amount (ellipsometry or DPI). The decreased polarity of the film due to a densification with the number of deposited layers could increase the emission intensity from preadsorbed dye molecules. One measurement was carried out with CH-Alexa 555 dissolved in solutions with different dielectric constants, either Milli-Q water or a 50 wt % methanol in Milli-Q water solution. The emission intensity was higher in the solvent of higher polarity (Milli-Q water). Thus, the observed increase in emission was not likely due to decreasing environmental polarity accompanying a gradual dehydration of the film. Out-of-Plane Diffusion. The time-dependent FRET efficiencies demonstrate that CH chains in CH/HEP multilayers are mobile in the direction perpendicular to the polymer film (i.e., out-of-plane diffusion). A significant energy transfer occurs immediately upon CH acceptor dye deposition when the CH acceptor dye and the CH donor dye are separated by a single ∼2 nm thick HEP layer (Figure 4). This is expected considering that the HEP layer thickness is less than the F€orster radius (7 nm). According to eq 2, E depends on the inverse sixth power of the separation. With an increase to 13 layers of HEP and CH between the initial CH donor placement and the CH acceptor deposition, the FRET efficiency should drastically decrease, if the CH chains were immobile. If the dyes were in fact separated by a distance of 50 nm, which can be assumed from previous thickness measurements,22 E would have been only 7.5 10-4%. In fact, E reached 25% as shown in Figure 4. The refractive index of the film does affect the F€orster radius; R0 scales as n-2/3.24 For a multilayer film refractive index of 1.47,22 which is the highest value measured for the CH/HEP film, this would decrease R0 from 7 nm in water to 6.5 nm in the film. This cannot explain the efficient FRET in these films. Clearly, the donor- and acceptor-labeled chitosan molecules diffuse closer to each other than the distance predicted based on an organized film of immobile polymers. These FRET results showing that CH-dye molecules diffuse within thin CH/HEP LbL films are consistent with previous reports for several polymer pairs using CLSM in thick films.13-15,38 Those studies have demonstrated the diffusion of fluorescence labeled polyelectrolytes “in-plane” and “out-ofplane” inside the LbL film. One potential, but not critical, concern with those studies, except for ref 14, is the use of the pH-sensitive FITC label, which may have affected the interpretation of the outof-plane polymer diffusion based on changes in the emission intensity from FITC labels. Out-of-plane diffusion of CH-dye molecules was observed for all deposition conditions considered here. The diffusion rate, judged by the rate of increase of the FRET efficiency, was dependent on ionic strength and pH (Figure 5). The FRET efficiency increased most rapidly in low ionic strength and low solution pH deposition conditions. The key effect of the solution deposition conditions, as summarized in Table 2, is that the layers are thinner and denser in the low ionic strength, low pH condition. (37) Halthur, T. J.; Elofsson, U. M. Langmuir 2004, 20, 1739–1745. (38) Jourdainne, L.; Arntz, y.; Senger, B.; Derby, C.; Voegel, J. C.; Schaaf, P.; Lavalle, P. Macromolecules 2007, 40, 316–321.
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The greater density could be expected to hinder polymer diffusion due to a stronger complexation between CH and HEP at these deposition conditions. The stronger complexation stems from the stronger electrostatic attraction between heparin and fully charged chitosan at low solution pH. Previous measurements of the lateral (i.e., in-plane) diffusivity using fluorescence recovery after photobleaching (FRAP) showed a correlation between the strength of interactions between neighboring polymer layers and the lateral diffusion coefficient.39 There it was concluded that a low polymer charge density, high solution ionic strength and high intrinsic backbone stiffness promoted diffusion. Zacharia and coworkers also showed, using FTIR, that a low polymer charge density also promoted the out-of-plane interdiffusion of polycations in a preadsorbed multilayer film.40 For four different polyamines, having different size and structure, interdiffusion occurred if the deposited polyamine had a degree of ionization less than 70% in solution. Nevertheless, analysis of the diffusion process underlying the FRET measurements, discussed below, will indicate that the solution conditions in fact did not significantly alter the CH diffusion coefficient in CH/HEP films. By controlling the layer thicknesses, the solution conditions also affect the initial separation distance between donor- and acceptor-labeled CH. This affects the evolution of the energy transfer efficiency. From the results in Table 2, it is evident that thick intermediate polymer films initially separating the two dyes result in a decreased rate of change of the energy transfer efficiency as expected (Figure 5), simply because diffusion must occur over longer distances. FRET experiments were modeled according to the diffusion of donor- and acceptor-labeled CH molecules initially placed in separate slabs bounded by a finite rectangular space of total thickness L, the value of which depends on film deposition conditions (see Supporting Information for coordinates). The donor-labeled species were placed in a slab of thickness h = 2 nm at x = 0. The acceptor-labeled species were placed in a slab of thickness h = 2 nm at x = L. No flux is allowed through the planes at x = 0 or x = L, capturing the experimental observation that polymers do not desorb from the film. For these conditions, the time-dependent concentration profile Cp(x,t) of the diffusing polymers is given by41 ¥ X 1 h þ 2nL -x h -2nL þ x pffiffiffiffiffiffi þ erf pffiffiffiffiffiffi Cp ðx, tÞ ¼ C0 erf 2 2 Dt 2 Dt n ¼ -¥ ð4Þ where D is the diffusion coefficient and C0 is the initial concentration of polymer deposited in the slab. The latter is determined from the surface concentration Γ, layer thickness h, and polymer molecular weight M as C0 = Γ/Mh. In the case of pH 5.8, 150 mM NaCl deposition conditions, Γ = 1.1 and 5.4 mg/m2 for CH donor in layer 5 and CH acceptor in layer 19, respectively, and L = 49 nm. In the case of pH 4.2, 30 mM NaCl deposition conditions, Γ = 0.6 and 2.6 mg/m2 for CH donor in layer 5 and CH acceptor in layer 19, respectively, and L = 20 nm.22 The local concentration of fluorescent donor Cd(x,t) or acceptor Ca(x,t) is the product of the local polymer concentration Cp(x,t) and the labeling ratio (2 dyes/CH chain). (39) Nazaran, P.; Bosio, V.; Jaeger, W.; Anghel, D. F.; von Klitzing, R. J. Phys. Chem. B 2007, 111, 8572–8581. (40) Zacharia, N. S.; Modestino, M.; Hammond, P. T. Macromolecules 2007, 40, 9523–9528. (41) Crank, J. The Mathematics of Diffusion, 2nd ed.; Oxford University Press: New York, 1975; p 16.
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The evolution of the FRET efficiency is dictated by the changing proximity of the donor- and acceptor-labeled CH molecules as they diffuse inward from opposite edges of the rectangular space. The concentration profiles for donor- and acceptor-labeled CH are calculated separately by eq 4. The local rate of energy transfer varies with time and position across the space. To calculate the local rate of energy transfer, the average volume occupied by a single donor label residing at x was calculated as a function of time t from the local donor concentration Cd(x,t), assuming it occupies a spherical volume of radius Rd: Rd ðx, tÞ ¼
3 4πCd ðx, tÞNav
1=3 ð5Þ
The energy transfer rate kt from a single donor, in a region of acceptor concentration Ca, is obtained by integrating the rate between that donor and each individual acceptor that is located within a spherical shell spanning from some minimum radial approach distance Rmin out to Rd, giving42 kt ¼
6 4π R0 Ca Nav pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðRd 3 -Rmin 3 Þ 3τd Rmin Rd
ð6Þ
where τd is the donor lifetime in the absence of acceptor. Equation 6 is used to calculate the local energy transfer rate kt(x,t) based on the local acceptor concentration Ca(x,t) and donor occupied radius Rd(x,t). Rmin represents the minimum distance within which an acceptor can approach a donor and is treated as a fitting parameter. At any position x within the film, the local FRET efficiency is24 Eðx, tÞ ¼
kt ðx, tÞ τd -1 þ kt ðx, tÞ
ð7Þ
and the local emission intensity from the donor in the presence of the acceptor is Fda ðx, tÞ ¼ RCd ðx, tÞf1 -Eðx, tÞg
ð8Þ
where R is a proportionality constant. Referring to eq 6, one notes that τd, which is unknown in a steady-state fluorescence measurement, cancels in eq 7. Following eq 3, the overall time-dependent FRET efficiency E is then RL EðtÞ ¼ 1 -
0
Cd ðx, tÞf1 -Eðx, tÞg dx RL 0 Cd ðx, tÞ dx
ð9Þ
When using eq 9 to model the multilayer FRET experiments, the time t = 0 marks the time when the acceptor-labeled chitosan was deposited. The donor-labeled CH was deposited at a time tlag = 85 min earlier, so all donor concentrations corresponding to any time t are evaluated by allowing the donor-labeled chitosan to diffuse over a time t þ tlag from its initial condition. The F€orster radius was set to R0 = 6.5 nm, and the value of Rmin was varied to match the long-time asymptote in E. The diffusion coefficients D were varied until the calculated E(t) best matched the timedependent energy transfer efficiencies plotted in Figures 4 and 5 for the two deposition conditions. Thus, Rmin was empirically set to 7.8 nm for the 150 mM pH 5.8 condition and to 7.1 nm for the 30 mM pH 4.2 condition. (42) Halpert, J. E.; Tischler, J. R.; Nair, G.; Walker, B. J.; Liu, W.; Bulovic0 , V.; Bawendi, M. G. J. Phys. Chem. C 2009, 113, 9986–9992.
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Figure 6. FRET efficiency time dependence predicted for CH having D = 8 10-16 cm2/s and Rmin = 7.1 nm for CH/HEP multilayer films prepared at 30 mM NaCl and pH 4.2 (open diamonds) or D = 1.0 10-15 cm2/s and Rmin = 7.8 nm for 150 mM NaCl at pH 5.8 (filled diamonds). Donor-labeled CH is placed in layer 5, and acceptor-labeled CH is placed 185 min later in layer 19. The difference in FRET evolution rate is caused by the smaller thickness of the 13 intervening CH/HEP bilayers between the donor and acceptor layers: 20 nm for 30 mM pH 4.2 and 49 nm for 150 mM pH 5.8 conditions. Start times are offset to 5 min to match the deposition time of the acceptor-labeled CH as presented in Figure 5.
Model calculations for D = 8 10-16 cm2/s for the 150 mM pH 5.8 condition and D = 1.0 10-15 cm2/s for the 30 mM pH 4.2 condition are shown in Figure 6. These diffusion coefficients satisfactorily capture the experimentally measured rate and longtime energy transfer efficiency values. It is evident that the FRET data for these deposition conditions are well modeled by very similar diffusion coefficients. One caveat regarding the use of this model is that it assumes that acceptor-labeled chitosan chains are deposited instantaneously, whereas ∼10 min was required for the deposition of each layer. The empirical minimum approach distance, Rmin, is comparable to the F€orster radius in these experiments. Polyelectrolyte chain stiffness may obstruct closer approach between most of the donor and acceptor dyes. The diffusion coefficients estimated here in the direction transverse to the multilayer plane can be compared to lateral (in-plane) diffusion coefficients for various polyelectrolyte pairs previously measured by FRAP.39 The smallest lateral diffusion coefficient reported was less than 10-15 cm2/s (below the detection limit) for poly(styrenesulfonate)/poly(allylamine hydrochloride) multilayers. Diffusion coefficients for poly(styrenesulfonate) multilayers with poly(diallyldimethylammonium chloride-stat-Nmethyl-N-vinylacetamide) of differing linear charge densities ranged from 0.3 10-14 to 5.0 10-14 cm2/s. The largest lateral diffusion coefficient, 5 10-12 cm2/s, was for hyaluronic acid/ poly(diallyldimethylammonium chloride) multilayers. The transverse diffusivity estimated for chitosan in chitosan/heparin multilayers, D = (0.8-1) 10-15 cm2/s, is comparable in order of magnitude to all but the largest of these lateral diffusion coefficients. It should be noted that these diffusion coefficients are very near the lower limit of detection by FRAP. In fact, the diffusion coefficients reported by Nazaran et al. required 5 days for one measurement.39 At the same time, there is uncertainty in the diffusion coefficient reported here by FRET due to the unavoidable violation of the assumption of instantaneous acceptor deposition noted above. Although the qualitative conclusion that Langmuir 2010, 26(5), 3242–3251
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CH is mobile across the CH/HEP multilayer would not be affected, complicating factors arise in the quantitative interpretation of the FRET measurement if the rotational mobility of the donor or acceptor dye is hindered. For D = 8 10-16 cm2/s, eq 4 indicates that the donor-labeled CH had effectively diffused everywhere throughout the CH/HEP multilayer film by the time the acceptor layer was deposited. This is consistent with the lack of any lag time in the FRET data;had there been any significant initial separation between the donor and acceptor layers, energy transfer could not have begun until enough time had elapsed for the acceptor to diffuse closer to the donor in these films that are significantly thicker than R0. Thus, the FRET experiments demonstrate that the polymers deposited within a CH/HEP multilayer freely interdiffuse in the transverse direction and destroy any discrete layer-by-layer organization within the film. The CH diffusion coefficient does not appear to depend significantly on the different deposition conditions used here, even though the multilayer films are thinner and denser for the low ionic strength, low pH conditions. Heparin based layer-by-layer films have previously been found to exhibit an exponential-like layer growth when using FTIR absorbance, and it was suggested that HEP was the diffusing species.43 In the current study, HEP molecules were not fluorescence labeled, as it proved to be very difficult to label. Its diffusion could therefore not be measured directly, but if chitosan could diffuse throughout the film, it would be difficult to conceive how heparin would not do the same. The molecular weight of HEP is low, ∼1 order of magnitude smaller than CH. Previous studies have shown that a low polymer molecular weight of the polycation44 or polyanion45 favors diffusion. A 20 kDa molecular weight poly(L-lysine) (PLL) diffused through an entire 16 μm HA/PLL film within a few minutes whereas high molecular weight PLL (M = 360 kDa) was restricted to the upper part of the film even 1 h after deposition.44 The results obtained from TIRF and FRET measurements indicate that the CH/HEP film is highly interpenetrated with no clear boundaries between the CH and HEP layers. This helps explain observations made in a previous study by Serizawa et al.20 where LbL films with CH and dextran sulfate were compared to those formed by CH and HEP. The anticoagulant versus procoagulant activity of these films against whole human blood was investigated based on the knowledge that CH is procoagulant whereas HEP and dextran sulfate are anticoagulant. After a fivelayer assembly, anticoagulation and procoagulation behavior was (43) Wood, K. C.; Chuang, H. F.; Batten, R. D.; Lynn, D. M.; Hammond, P. T. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 10207-10212. (44) Porcel, C.; Lavalle, P.; Decher, G.; Senger, B.; Voegel, J. C.; Schaaf, P. Langmuir 2007, 23, 1898–1904. (45) Sun, B.; Jewell, C. M.; Fredin, N. J.; Lynn, D. M. Langmuir 2007, 23, 8452– 8459.
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observed on the dextran sulfate and CH outer layers, respectively;the coagulant behavior reflected that of the outermost layer. In contrast, CH/HEP assemblies showed strong anticoagulant activity regardless of whether CH or HEP was in the outermost deposited layer. Penetration of the CH surface layer by HEP molecules was suggested to be the reason. Our results showing substantial out-of-plane diffusion of CH and the sensitivity of embedded CH-FITC toward the charge of widely spaced layers support that explanation.
Conclusions This work demonstrates that FRET can be a useful complement to the CLSM technique in order to measure interlayer diffusion of a polyelectrolyte in a layer-by-layer multilayer film, especially for thin films in the 10-100 nm range where CLSM is of limited use. In all measurements regardless of the deposition solution conditions, dye-labeled CH was found to diffuse in the transverse direction through 13 intermediate CH and HEP layers within minutes. The estimated transverse, or out-of-plane, diffusion coefficient for chitosan in a chitosan/heparin film, (8-10) 10-16 cm2/s, is comparable in order of magnitude to lateral diffusion coefficients measured previously for several other polyelectrolyte multilayer films. The exponential-like growth in thickness and adsorbed amount previously observed for CH/HEP films with the number of deposition cycles is therefore likely due to out-of-plane polymer diffusion within the film during deposition as hypothesized in the literature. A pH-sensitive FITC dye embedded in the CH/HEP film was highly sensitive toward the sign of charge of the outermost layer, even after a large number of additional layer depositions. This also shows that chitosan chains freely diffuse throughout the entire film, so that even at larger film thicknesses some CH-FITC chains can reside close enough to the outermost layer to respond to changes in local electrostatic potential. A better understanding of the film structure and the interlayer diffusion is of great relevance to the design of improved film coatings for medical implants or to control the interlayer diffusion for controlled drug release for pharmaceutical applications. Acknowledgment. M.L. acknowledges SSF-Colintech for financial support. E.B. gratefully acknowledges financial support from the Swedish Research Council, VR. Supporting Information Available: Comparison of the pH sensitivity of FITC and Alexa 488 emission intensity, TIRF measurement of LbL deposition using Alexa 488 dye at pH 4.2, sketch of the coordinates for diffusion analysis, and representative raw data for FRET experiments. This material is available free of charge via the Internet at http://pubs.acs.org.
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