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Subdiffraction-Resolution Optical Measurements of Molecular Transport in Thin Polymer Films Suman Pahal, Ashok M Raichur, and Manoj M Varma Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00527 • Publication Date (Web): 13 May 2016 Downloaded from http://pubs.acs.org on May 21, 2016

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Subdiffraction-Resolution Optical Measurements of Molecular Transport in Thin Polymer Films Suman Pahal1, Ashok M Raichur2, 4 and Manoj M Varma1, 3* 1

Center for Nano Science and Engineering, Indian Institute of Science, Bangalore 2

3

Dept. of Materials Engineering, Indian Institute of Science, Bangalore

Dept. of Electrical Communication Engineering, Indian Institute of Science, Bangalore 4

Nanotechnology and Water Sustainability Unit, University of South Africa, Florida 1710, Johannesburg

ABSTRACT

The measurement of molecular transport within polymer films yields information about the internal structural organization of the films and is useful in applications such as the design of polymeric capsules for drug delivery. Layer-by-Layer assembly of polyelectrolyte multilayer films has been widely used in such applications where the multilayer structure often exhibits anisotropic transport resulting in different diffusivities in the lateral (parallel to the film) and transverse (normal to the film) directions. While the lateral transport can be probed using techniques such as Fluorescence Recovery After Photobleaching (FRAP), it cannot be applied to probing transverse diffusivity in polymer films smaller than the diffraction limit of light. Here we present a technique to probe transport of molecules tagged with fluorphores in polymer films thinner than the optical diffraction limit using the modulation of fluorescence emission

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depending on the distance of the tagged molecules from a metal surface. We have used this technique to probe diffusion of proteins Biotin and Bovine Serum Albumin (BSA) in polyelectrolyte multilayer films. We also studied interdiffusion of chains in multilayer films using this technique. We observed a 3 order of magnitude increase in interdiffusion as a function of ionic strength of the medium. This technique, along with FRAP will be useful to study anisotropic transport in polymer films, even those thinner than the diffraction limit, as the signal in this technique arises only due to transverse and not lateral transport. Finally, this technique is also applicable to studying the diffusion of chromophore labelled species within a polymer film. We demonstrate this aspect by measuring the transverse diffusion of Methylene Blue in PAHPAA multilayer system.

INTRODUCTION Several emerging applications in multi-agent targeted drug delivery require the use of polymer capsules. The loading and release behaviour of drugs from such capsules strongly depends on the diffusivity of the drug molecules within these polymers whose thicknesses can range from tens of nanometers to microns.1-4 For instance, programmed release of a drug, with temporal control of the release rate would require detailed knowledge of the diffusive behaviour of the drug, which includes depth of penetration, concentration profile and so on. One might also be interested in the change in these parameters as a function of environmental variables. Layer-ByLayer (LbL) deposition of polyelectrolytes is a very versatile technique for the fabrication of devices, functionalization of biomaterials, designing reservoirs for protein and drugs in tissue engineering and drug delivery applications.5-10 Polyelectrolyte multilayer (PEM) capsules have been extensively explored for targeted drug delivery and programmed release.11,12 PEM films have also been used as scaffolds and reservoirs of growth agents in tissue engineering


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applications which too require a good understanding of diffusion through PEMs. In addition to the study of a diffusing species across PEM films, another interesting aspect of PEMs is the interdiffusion of their constituent PEs, the extent of which strongly influences the PEM growth regime, ranging from linear or exponential with respect to the number of layers.13-14 These examples illustrate the importance of techniques to probe diffusion of molecules in PEMs with thicknesses ranging from a few nanometres to a few microns. Measurement of diffusion phenomena in PEM systems can be broadly classified into two types; optical techniques based on fluorescence from the diffusing species and non-optical techniques such as X-ray Photoelectron Spectroscopy (XPS) and Secondary Ion Mass Spectrometry (SIMS) which have been extensively used to measure diffusion in inorganic materials. For example, the role of interdiffusion in exponential layer build-up was recognized by the measurement of interdiffusion profiles in poly(L-lysine)/poly(L-glutamic acid) (PLL/PGA) using confocal microscopy.15 However, optical measurements can only be made from films thicker than the optical diffraction limit. For e.g. the study of interdiffusion in PLL/PGA using confocal microscopy was possible because the PEM films, in this case, are thicker than a micron, well above the diffraction limit. Optical techniques cannot be used to probe transverse diffusion in PEM films with thickness smaller than about 0.5 microns due to diffraction limited optical resolution. Therefore, techniques such as X-ray photoelectron spectroscopy (XPS) and Secondary ion mass spectrometry (SIMS) are required to probe diffusion in ultra-thin PEM layers.16-17 However, the use of these techniques for studying diffusion in soft polymeric thin films suffers from several challenges. Firstly, access to these sophisticated instruments is a more limited than optical microscopes which are widely available. Secondly, XPS and SIMS can damage soft polymer layers which can potentially result in


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erroneous conclusions drawn from these studies. For example, C60 cluster ions have recently been used in depth-profiling XPS to minimize the damage to soft materials.16 Although similar in their destructive nature, SIMS has an approximately three orders of magnitude better LoD compared to XPS.17,18 However, the identification of secondary ions unique to the diffusing species could be a problem in polymer systems which are a lot more heterogeneous than inorganic materials for which these techniques were developed initially.18 Therefore, it is of interest to consider alternate measurement techniques, which do not suffer from the disadvantages of XPS and SIMS, to probe diffusion in sub-diffraction limit thin films. In this paper, we describe an optical method capable of extracting the concentration profile of a fluorescently labeled diffusing species in PEM films as thin as 150 nm, well below the 500 nm axial resolution of confocal microscopes.19 The basis of this technique is the distance dependent periodic modulation of fluorescence intensity of a fluorophore from a metal surface.20 This phenomena is described in greater detail in the Theoretical Modeling section. Although the discussion presented there is centred on fluorescence emission, it is equally applicable in the case of emission from chromophores from a metal surface. We illustrate this aspect of the technique by probing the transverse diffusion of the dye molecule Methylene Blue in a PEM system. Henceforth, we will describe the technique in terms of fluorescence emission with the understanding that the entire discussion is equally applicable in the case of chromophores. The fluorescence signal obtained from a sample as a function of time is a result of the cumulative fluorescence from fluorophores distributed at different distances from the metal surface as shown in Figure 1a. By measuring the fluorescence as a function of time or the PEM thickness, we can extract the diffusion profile based on the theoretical model described subsequently. This technique can be applied to study the diffusion of any fluorophore or


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chromophore labelled molecule, through a diffusing medium deposited on a metal surface although in this article we primarily focus on fluorescent labelled systems due to their wide applicability in biological studies.

Figure1. Distance dependent modulation of fluorescence intensity near a metal surface calculated theoretically , (a) The intensity of fluorescence emitted from a fluorophore undergoes a periodic modulation depending on its distance from a metal surface (b) The fluorescence from a polymer film is dependent on the diffusion profile as well as the thickness of the polymer layer (c) Interference of back-reflected electric fields leads to a standing wave pattern of intensity which modulates the fluorescence signal emitted from a fluorophore depending on its distance from the


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substrate. In the case of a perfectly reflecting substrate (r = ±1), the interference can lead to almost complete quenching of fluorescence.

THEORETICAL MODELLING Calculation of Fluorophore Diffusion Profiles The diffusion profile , is the solution of the one-dimensional diffusion equation, ,

=

,

where D is the diffusion coefficient of the diffusing species. The nature of

the function , depends on the initial and/or boundary and conditions appropriate for the system under consideration. The fluorophores shown in Figure 1a enter into the polymer film from the bulk fluid above. We consider two models of fluorophore incorporation and diffusion into the PEM. One in which the fluorophores continuously get incorporated into the PEM surface from the bulk fluid above and diffuse into the PEM layer. We refer to this model as the continuous diffusion model. The other possibility is we considered is that the incorporation of fluorophores on the surface presents an electrostatic barrier which prevents further incorporation beyond a threshold. Diffusion is then essentially a redistribution of the initially incorporated fluorophore population within the PEM. We refer to this model as the barrier diffusion model. The co-ordinate along the thickness of the PEM is designated z and z = 0 refers to the top of the PEM and z = d refers to the metal substrate. The appropriate boundary condition for the continuous diffusion model can be written as 0, = for all values of t. The metal substrate requires a no-flux boundary condition (because it is an impenetrable barrier) at z = d and the diffusion is confined between the upper and lower surfaces of the polymer film, i..e. z = 0 and z = d respectively. Then, the solution of the diffusion equation yields19


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2 − − 1 , = 2 − √4 √4 The alternate picture of barrier diffusion model can be considered as a redistribution of an initial impulse loading of "#$%" molecules on the top surface. The boundary condition can then be

written as , 0 = "#$%" & followed by the imposition of no-flux condition at z = d. With these boundary conditions, the solution can be written as, 19 , =

"#$%" √'

-./

( )* −

-. 0/

− 2+, 2 4

Figure.1b shows the distribution of fluorphores within the PEM thickness for various values of diffusion coefficient D calculated from our theoretical model.

Modulation of fluorescence intensity The modulation of reflectance is the result of interference between the incident light field and the reflected light field which can be written as Γ = |1 + |, , where r is the Fresnel reflection coefficient of the thin film structure consisting of the metal and the PEM above it with thickness z. The total fluorescence intensity from the PEM layer is obtained by integrating the product of c(z)G(z) over the thickness of the PEM. As shown in Figure 1c, the net electric field at a point P, which is at a distance z above the substrate, is given by the interference of the incident and back-reflected wave components. The net electric field at point P can be written as, 4 = 45 678 91 + 67: ;

(3)

Where is the transmission coefficient between the air and polymer film interface, is the

reflection coefficient between the film-substrate interface, 45 is the amplitude of the incident


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field, ?

@A

(4)

The interference between these two components creates a standing wave pattern of the electric field and consequently the light field intensity as a function of distance from the substrate surface will be given by, B = |4|,. From Eq. (3) a distance dependent modulation factor, C, corresponding to the standing wave created, can be defined as, ,

=>

C = D1 + 67: D = 1 + , + 2 cos H ? @A I

(5)

The fluorescence excitation process is related to the number of photons available for excitation, which in turn is proportional to the light field intensity at the spatial location of the fluorophore. Therefore the standing wave patterns of the intensity fields corresponding to excitation and emission wavelengths will modulate the fluorescence yield due to the modulation in the photon number densities corresponding to the spatially varying intensity fields. The combined modulation factor Γz is the product of the modulation factors corresponding to

excitation, γLM z and emission,γLN z, i.e. Γz = γLM zγLN z.

(6)

γLM z and γLN z can be calculated by substituting the value of the excitation and emission

wavelengths respectively in Eq. (4). The distance dependent modulation of fluorescence from fluorophores diffusing through a porous polymer film is schematically shown in Figure 1a. One can see from Eq. (4) that a strongly reflecting substrate with a high value of , can lead to significant modulation in the fluorescence intensity. For e.g. a perfect metal, with a reflection coefficient of = −1, should lead to complete quenching of fluorescence at the surface, (i..e.

= 0). For weakly reflecting substrates such as glass, this effect is not prominent and may be ACS Paragon Plus Environment

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below the resolution of ordinary CCD cameras with limited dynamic range. This modulation effect has previously been used to enhance fluorescence signal by engineering substrates to have high reflectivity (for e.g. multilayer dielectric mirrors with = 1.21,22 In the case of absorbing dyes such as Methylene Blue, the modulation term of Eq. (6) will only contain a single term equal to γPQR z corresponding to the peak absorption wavelength of the dye.

The net fluorescence (or color output in the case of absorbing dyes) intensity BST from the PEM thickness will then be given by,

.W

BST , ∝ V .5 , Γ

(7)

where c(z,t) is obtained as described in the previous section.

EXPERIMENTAL Materials The polymers poly(allylamine hydrochloride) (PAH, Mw∼15 kDa) and poly(acrylic acid) (PAA, Mw∼200 kDa), poly(ethylenimine) (PEI, Mw∼25kDa), poly(L-glutamic acid)(PGA Mw∼50100 kDa), poly(L-lysine) (PLL Mw∼30 kDa,), fluorescein-labeled PLL (PLL-FITC, Mw∼30 kDa) and other chemicals fluoerescein labelled bovine serum albumin (FITC-BSA), fluoerescein conjugated

biotin,

1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide

(EDC),

N-

hydroxysuccinimide (NHS), were purchased from Sigma Aldrich. Sodium chloride (NaCl), sodium hydroxide (NaOH), and hydrochloric acid (HCl) were purchased from Merck. Methylene Blue (MB Mw∼319) was purchased from Rankem Industries limited. All solutions were prepared by using ultrapure water from Millipore with a resistivity of 18.2MΩ. Aqueous solutions of polyelectrolytes PAH and PAA (0.01 M) (based on repeat unit molecular weight) were prepared by DI water of ionic strength 0.1 M NaCl. The solution pH was adjusted to 5.5


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using 0.1 M NaOH and 0.1 M HCl. For interdiffusion experiments, PGA/PLL (1mg/ml) polyelectrolyte solutions were made by water using 0.1M NaCl at pH 4.4. FITC-BSA (100µg/ml) and FITC-biotin (100µg/ml) were prepared by phosphate buffered saline solution (PBS) and MB solution of 100µg/ml was prepared in ultrapure water. All chemicals were used without any further purification.

Multilayer Film Deposition For metal distance-dependent fluorescence measurements, 100nm aluminium layer was deposited on piranha cleaned (3:1 H2SO4: H2O2) microscopic glass slides by homemade thermal evaporator. The thickness was measured by Bruker, XT Stylus Dektak Profileometer. Then polyelectrolyte multilayer films were deposited on Al-coated substrates by dip-assisted Layerby-layer (LbL) self assembly process where PEMs were constructed mainly due to electrostatic interactions, by alternate deposition into solution of polycations and polyanions. Dip time in each polyelectrolyte solution was 1 minute followed by three rinsing with agitation in water and drying in pure nitrogen. The desired number of polyelectrolyte bilayer stack ranging from 0.5BL upto 20.5 BL at an interval of 2 BL has been made repeating the LbL cycle with (PAH/PAA)n and PEI(PGA/PLL)nPGA polyelectrolytes. Thickness measurements of PEMs were carried out using spectroscopic ellipsometer from J.A. Woollam Co. Inc. (SI text: section 2 and Figure S2)

Fluorescence Measurements for Transverse diffusion For diffusion measurements FITC-labelled -BSA and FITC-Biotin were allowed to adsorb on (PAH/PAA)n multilayers for 35 minutes incubation time followed by three washing with PBS or water. Similar incubation procedure has been used for studying interdiffusion of FITC-PLL


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(1mg/ml) in PEI(PGA/PLL)nPGA films. For time based saturation experiments at fluorescence node and antinode the incubation time are varied from 5 minutes to up to 75 minutes. For enhancing diffusion experiments, the 1M NaCl has been used instead of normal 0.1M NaCl in preparation of FITC-PLL solution. To capture the fluorescence signatures in non-diffusive environment, EDC-NHS cross-linking chemistry has been utilized to form the chemical crosslinks between the PEI(PGA/PLL)nPGA films. PEI(PGA/PLL)nPGA films been kept in freshly prepared solution of EDC(400mM)-NHS(100mM) dissolved in 0.15M NaCl solution for 16 hours followed by rigorous washing with 0.15 NaCl. Fluorescence imaging was done by using Olympus BX51M inverted microscope with DP 71 CCD camera with magnification of 5X objective and 400ms exposure time. The quantitative estimation of temporal variation of fluorescence intensity was done by using “Image J" software to extract the transverse distribution of fluorophores in PEMs.

RESULTS AND DISCUSSION We developed a mathematical model to calculate the fluorescence modulation as a function of PEM thickness for various values of diffusion constants. The details of the model are already explained. Briefly, we used the analytical solution of the one-dimensional diffusion equation23 to calculate the distribution of fluorophores through the thickness of the PEM, referred to as c(z) in Figure 1a. Then we calculated the modulation of fluorescence intensity, referred to as Γ (z) in Figure 1a, and calculated the total intensity from the PEM layer by integrating the product of c(z)G(z) over the thickness of the PEM. This theoretical model reveals a diffusion dependent periodic modulation of the fluorescence from the PEM as a function of layer thickness. The origin of this periodic modulation is the interference of the incident light field with the one


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reflected from the metal surface. This interference leads to periodic constructive and destructive effects for the fluorescence emission20 as a function of the distance of the fluorophore from the metal surface. The presence of diffusion maintains this periodic nature but shifts the position of the minima laterally (different thickness compared to zero diffusion case) and vertically (minima occurs at higher values compared to zero diffusion case). Figure 1b shows the effect of diffusion coefficient on the fluorescence intensity as a function of PEM thickness according to our theoretical model. We see from Figure 1b, that very low values of diffusion coefficient, for e.g. D = 1.6e-16 cm2/s (black curve in Figure 1b), exhibit a nearly perfect destructive interference leading to zero intensity for certain PEM thicknesses. This happens because nearly all the fluorophores are sitting at a thickness corresponding to destructive interference between incident light and the light reflected from the metal. However, when the diffusion coefficient is increased, for e.g. D = 5e-14 cm2/s (blue solid curve in), the distribution of the fluorophores within the PEM is broadened and there is a significant fluorophore population which sits at positions away from complete destructive interference. This increases the minima and also produces a small lateral shift in the position of the minima. This is the physical basis for the observations related to fluorescence intensity. The thickness positions corresponding to constructive and destructive interferences would be of the order of 1/4th of the wavelength used (~100 nm for green fluorescent FITC corresponding to an excitation and emission of 488 nm and 525 nm respectively). Such a short spatial range corresponding to a large dynamic range in fluorescence intensity implies that very small values of D can be measured easily with this technique. With diffraction limited measurements such as FRAP, one would need to wait for long times, t for the diffusion length YW which scales as YW ~ √ to be measurable. In contrast small measurement times would produce a sufficient optical contrast in our technique because the diffusion length,


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even if it is as small as 10 nm, would be a significant fraction of the spatial range (~ 100 nm as mentioned before) corresponding to the maximum dynamic range of our measurement. By measuring the fluorescence from PEMs with different thicknesses and fitting them to this model, the diffusion coefficient can be extracted, as we show in subsequent examples. To experimentally confirm the theoretical calculations described above, we measured the fluorescence intensity of FITC labelled BSA and Biotin diffusing through PAH/PAA multilayers deposited over Aluminium coated glass slides Figure 2b. We use a nomenclature scheme (PAH/PAA)n, where n refers to the number of bilayers. Periodic fluorescence intensity modulation was observed as predicted by the model. The periodic intensity modulation was also observed in the case of the dye MB according to our theoretical model Figure 2a. Diffusion coefficients of 5e-15 cm2/s and 1.3e-14 cm2/s fitted the experimentally observed behaviour of BSA and Biotin respectively. The higher diffusion coefficient of Biotin could be due to the smaller size of Biotin (Mw = 500 Da) compared to BSA (66 kDA). Previous measurement of BSA diffusion has concluded a very low value of diffusion coefficient below the measurement limit of the experimental system reported previously.24 The diffusion coefficient of MB in PAH/PAA multilayer system was estimated to be around 1e-15 cm2/s which is close to that previously reported value 25.


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Figure2. Periodic fluorescence intensity modulation on (PAH/PAA)n films deposited on metal (a) with methylene blue ,(b) with FITC-Biotin and FITC-BSA

We then used this technique, to probe inter-diffusion of PEs in a PEM. We used the poly(Llysine)/poly(L-glutamic acid) (PLL/PGA) system to study interdiffusion phenomena as there are several reports in the literature describing this effect in the PLL/PGA system.15,26 To probe interdiffusion, we examined the distance-dependent fluorescence intensity of FITC labelled PLL as it diffused through the PEM matrix, which consisted of PEI(PGA/PLL)nPGA deposited on Aluminium. Here, poly(ethylenimine) PEI is used for promoting adhesion over aluminium coated surface and n refers to the number of bilayers.

We used various post assembly

modifications of the PEM matrix to enhance or suppress the diffusivity of PLL as shown schematically in Figure 3a. Figure 3a represents the strategies we employed to control interlayer diffusion in PEMs, namely, enhancing diffusion by using high ionic strength and suppressing diffusion using cross-linked layers. By increasing the ionic strength of the solution containing FITC-PLL to 1M of NaCl, we were able to demonstrate a higher diffusivity compared to an ionic


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strength of 0.1M NaCl. From Figure 3b, we can see that the minimum intensity of the fluorescence modulation in the case of high salt (1M, red curve) is much higher compared to the low salt (0.1M, black curve). The extracted D values from these curves from best fits are 1.6e-14 cm2/s and 1.6e-11 cm2/s for the 0.1M and 1M cases respectively. There is a 3 order of magnitude increase in diffusivity in the presence of high salt, which we attribute to near total screening of electrostatic effects in the PEM. Previous reports on the effect of ionic strength on diffusion27-34 have described an increase in diffusivity which could be due to the swelling of the films caused by extra salt ions. Although there are no reports to directly compare our data for the PGA/PLL system, an increase in diffusivity between 1-4 orders of magnitude between 0.1M and 1M ionic strength may be expected.33,34 High salt condition favors diffusion by decreasing the density of polyanion-polycation complexes and by increasing the salt ion-polyion combinations and thus. Effect of high concentration of salt on film morphology and stability is provided in (SI text: section 2 and Figure S2-S3). In order to further probe the connection between diffusivity and observed fluorescence intensity modulation, we cross-linked the PEI(PGA/PLL)nPGA films using a well characterized method

involving

1-Ethyl-3-(3-dimethyl

amino

propyl)-carbodiimide

(EDC)/

N-

hydroxysuccinimide (NHS) chemistry.35-37 Cross-linking is expected to make the PEM much denser resulting in lower diffusivity. Specifically, we wanted to investigate to what extent the enhanced diffusivity in high salt conditions can be reversed by cross-linking. In the cross-linking method used, NHS aids the formation of amide bonds between the carboxylic groups of PGA and amine group of PLL. EDC catalyzes the formation of amide bonds between carboxylic groups of PGA and amine groups of PLL. The formation of amide bonds was revealed by FTIR spectroscopy (Figure S7) and refractive index measurements using an Ellipsometer which


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showed an increase in the RI value from 1.52 to 1.67 measured for 9BL, presumably indicating the formation of crosslinks between PGA and PLL making the PEM stack denser and consequently increasing the refractive index. Fluorescence modulation data from crosslinked PEMs shown in Figure 3b indicates that the enhanced diffusivity of PLL due to high salt condition could be suppressed to that of low salt values by cross-linking. This observation implies that diffusion in the crosslinked condition is dominated by the film morphology and not by electrostatic interactions. We did not observe any significant difference in diffusivity between cross-linked and non-cross-linked PEMs for the low salt condition. This may be because the diffusivity was already quite low for low salt case and further suppression due to cross linking was not significant in a relative sense.


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Figure3. Probing Interlayer diffusion of FITC-PLL in PLL/PGA films (a) Schematic depicting the different experimental conditions to probe interdiffusion in PLL/PGA films, (b) Fluorescence modulation showing enhanced diffusion by using high salt concentration, (c) Cross-linking arresting diffusion even in high salt environment. (Fluorescence micrographs provided in (SI text: section 3 and Figure S4)

In addition to enabling the extraction of diffusion coefficients, this technique can also help us develop a physical picture of molecular diffusion in PEMs. To illustrate this, we consider two models of molecular diffusion as described in the theoretical section (Figure 4). One referred to as a continuous diffusing model where the molecules continuously diffuse into the PEM matrix from a constant reservoir above (fluid containing the labelled molecule) and the other where initial adsorption of the charged molecules (such as proteins or PEs) near the PEM surface presents an electrostatic barrier to further incorporation of molecules at the surface. The continuously diffusing model predicted a monotonic increase of the FL intensity at the nodal and anti-nodal points with respect to incubation time (Figure 4a) whereas the barrier model predicted an increase of the FL intensity at the nodal point and a decrease at the anti-nodal point as shown in Figure 4b. The nodal (N) and antinodal (AN) points refer to the bilayer thicknesses producing the minimum and maximum FL intensity, respectively. This lead us to conclude that the measurement of temporal evolution of the fluorescence signal will throw some light on the adsorption process.


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Figure 4 (a) Theoretical model showing the continuous diffusion time evolution of fluorescence intensities at anti-nodal (AN) and nodal (N) points, (b) same for barrier diffusion model.

We experimentally measured fluorescence intensity as a function of time at the nodal and antinodal points of the fluorescence modulation curves. The data is shown in Figure 5. We observed that in the case of diffusion of proteins through PAH/PAA system and the interdiffusion of FITC-PLL in PEI(PGA/PLL)nPGA system under low ionic strength (0.1M), Figure 5(a-c) the fluorescence intensity of the anti-nodal samples decreased as a function of time as predicted by the barrier diffusion model. The continuous diffusion model, on the other hand, predicted an increase. Therefore, our experimental data provides support for the barrier diffusion model to be operating under the regimes investigated in the present work. The robustness of the experimental observation leading to this conclusion was further confirmed by examining the histograms of fluorescence images as shown in Figure S6. It was found not only the mean values but the entire distribution of fluorescence intensities shifted to lower values as a function of increasing incubation time for anti-nodal samples as predicted by the barrier diffusion model. All these experimental observations concludes that the barrier diffusion model as the dominant


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mechanism for incorporation of charged species and their diffusion into the PEM. Diffusion of FITC-PLL under high salt condition showed a flat temporal response (Figure 5d) as predicted by either of the two models.

Figure 5. Temporal Intensity modulation with different incubation time (a) for FITC-Biotin on (PAH/PAA) at 8.5BL and 12.5 BL, (b) FITC-BSA on (PAH/PAA) at 8.5BL and 12.5 BL, (c) FITC-PLL(0.1M NaCl) on PEI(PGA/PLL)nPGA at 9 BL and 11 BL and (d) FITC-PLL(1M NaCl) on PEI(PGA/PLL)nPGA at 9 BL and 11 BL, all (a-d) with representative fluorescence micrographs taken at 5min, 30min, 60 min and 75 min.


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CONCLUSIONS In summary, we have demonstrated a technique to probe the transverse diffusive behaviour of labelled molecules through ultra-thin (100 – 200 nm) polymer films. The data presented in this article provided a good physical picture related to the incorporation of charged molecules from solution into a PEM matrix and their diffusion within the PEM stack. This knowledge will be useful for developing architectures for applications ranging from the design of biomaterial surfaces to programmed drug delivery, where precise control of the chemical concentrations as well as their distribution through the matrix is desired.

Supporting Information Available The details of the model for the calculation of fluorophore diffusion profile and calculation of temporal intensity at nodal and antinodal points are provided in supporting information document. Film growth analysis and effect of salt on film thickness has been monitored by spectroscopic ellipsometer. Data validating our experiments eg. Histogram anaylsis, film stability as well as FTIR to confirm crosslinking has also been included in SI document file. This information is available free of charge via the Internet at http://pubs.acs.org/.

AUTHOR INFORMATION Corresponding author * Email [email protected]


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"For Table of Contents Use Only"

Subdiffraction-Resolution Optical Measurements of Molecular Transport in Thin Polymer Films Suman Pahal1, Ashok M Raichur2, 4 and Manoj M Varma1, 3* 1

Center for Nano Science and Engineering, Indian Institute of Science, Bangalore 2

3

4

Dept. of Materials Engineering, Indian Institute of Science, Bangalore

Dept of Electrical Communication Engineering, Indian Institute of Science, Bangalore

Dept Nanotechnology and Water Sustainability Unit, University of South Africa, Florida 1710, Johannesburg


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Figure1. Distance dependent modulation of fluorescence intensity near a metal surface calculated theoretically , (a) The intensity of fluorescence emitted from a fluorophore undergoes a periodic modulation depending on its distance from a metal surface (b) The fluorescence from a polymer film is dependent on the diffusion profile as well as the thickness of the polymer layer (c) Interference of back-reflected electric fields leads to a standing wave pattern of intensity which modulates the fluorescence signal emitted from a fluorophore depending on its distance from the substrate. In the case of a perfectly reflecting substrate (r = ±1), the interference can lead to almost complete quenching of fluorescence. 124x95mm (300 x 300 DPI)


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Figure2. Periodic fluorescence intensity modulation on (PAH/PAA)n films deposited on metal (a) with methylene blue ,(b) with FITC-Biotin and FITC-BSA 138x65mm (300 x 300 DPI)


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Figure3. Probing Interlayer diffusion of FITC-PLL in PLL/PGA films (a) Schematic depicting the different experimental conditions to probe interdiffusion in PLL/PGA films, (b) Fluorescence modulation showing enhanced diffusion by using high salt concentration, (c) Cross-linking arresting diffusion even in high salt environment. (Fluorescence micrographs provided in (SI text: section 3 and Figure S4) 127x80mm (300 x 300 DPI)


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Figure 4 (a) Theoretical model showing the continuous diffusion time evolution of fluorescence intensities at anti-nodal (AN) and nodal (N) points, (b) same for barrier diffusion model. 145x65mm (300 x 300 DPI)


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Figure 5. Temporal Intensity modulation with different incubation time (a) for FITC-Biotin on (PAH/PAA) at 8.5BL and 12.5 BL, (b) FITC-BSA on (PAH/PAA) at 8.5BL and 12.5 BL, (c) FITC-PLL(0.1M NaCl) on PEI(PGA/PLL)nPGA at 9 BL and 11 BL and (d) FITC-PLL(1M NaCl) on PEI(PGA/PLL)nPGA at 9 BL and 11 BL, all (a-d) with representative fluorescence micrographs taken at 5min, 30min, 60 min and 75 min. 124x108mm (300 x 300 DPI)


Subdiffraction-Resolution Optical Measurements of ... - ACS Publications

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