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TRANSCRIPT
Polyelectrolyte Multilayers: an Investigation into their
Assembly and Behavior with Diffraction Based Sensing
by
Hannah Howard
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Department of Chemistry
University of Toronto
© Copyright by Hannah Howard 2015
ii
Polyelectrolyte Multilayers: an Investigation into their Assembly
and Behavior with Diffraction Based Sensing
Hannah Howard
Master of Science
Department of Chemistry
University of Toronto
2015
Abstract
A diffraction based sensing set-up was evaluated for use in monitoring the assembly of
polyelectrolyte multilayers (PEMs). PEMs are thin films assembled via the layer-by-layer
method, which consists of exposing a substrate to polyanions and polycations in sequence.
Understanding the fundamental behavior of PEMs is instrumental to designing their properties to
fulfill unique functions. In this work, diffraction based sensing is explored as a method to
monitor the build-up of multilayers. The initial polymer layer is printed as a diffraction grating
pattern and the PEM is assembled on top resulting in a change in height of the grating. This
change in height can be monitored by the change in intensity of the diffracted spots. To
demonstrate diffraction sensing is sensitive to PEM assembly, experiments testing its response to
salt concentration, polymer concentration, and post assembly processing were carried out.
iii
Acknowledgments
First of I want to thank the other students in the lab. The other students were invaluable to me for
their support. Calvin Cheng and Nari Kim, thanks for all of the guidance you’ve given me.
Nicholas Kotoulas was the best person to have in my program for how unrelentingly positive he
was. Lastly I wanted to thank Cynthia Goh for the opportunities she gave me in her lab.
Of course need to thank my family for their love and making sure I was fed while writing. And
Kevin thanks for putting up with two years of a girlfriend in Canada.
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Table of Contents
Acknowledgments ............................................................................................................................... iii
Table of Contents .................................................................................................................................. iv
List of Figures ......................................................................................................................................... vi
Polyelectrolyte Multilayers(PEM) ............................................................................................ 1
1.1 Layer by layer .......................................................................................................................................... 2
1.1.1 Attraction and adhesion .............................................................................................................................. 2
1.1.2 Choice of polyelectrolytes .......................................................................................................................... 2
1.1.3 Interpenetration of Layers ......................................................................................................................... 3
1.1.4 Polyelectrolyte conformation and condensation.............................................................................. 4
1.1.5 Salt effects on PEMs ...................................................................................................................................... 5
1.1.6 Charge compensation ................................................................................................................................... 7
1.2 Other techniques to characterize PEMs ......................................................................................... 7
1.2.1 Quartz crystal microbalance ..................................................................................................................... 8
1.2.2 Atomic Force Microscopy ........................................................................................................................... 8
1.2.3 Ellipsometry ..................................................................................................................................................... 9
1.2.4 Neutron reflectometry ................................................................................................................................. 9
Diffraction based sensing ............................................................................................................ 9
2.1 Theoretical background of diffraction gratings ....................................................................... 10
Materials and Methods .............................................................................................................. 12
3.1 Diffraction Sensing .............................................................................................................................. 12
3.1.1 PEI Stamping .................................................................................................................................................. 13
3.1.2 Flow Cell Assembly ..................................................................................................................................... 14
3.1.3 Diffraction Sensing Set-up ........................................................................................................................ 15
3.2 In situ Monitoring of Polymer Deposition .................................................................................. 16
3.2.1 Reagents .......................................................................................................................................................... 16
3.2.2 Polymer Deposition and Measurements ............................................................................................ 17
3.2.3 Data Capture and Data Analysis ............................................................................................................. 18
3.3 Discussion of factors that would increase of decrease signal ............................................. 19
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Results and Discussion .............................................................................................................. 20
4.1 Idealized scenario of PEM signal in diffraction optics signal ............................................... 20
4.2 0 M NaCl polymer results .................................................................................................................. 22
4.2.1 Procedure ........................................................................................................................................................ 22
4.2.2 0 M NaCl PSS addition behavior ............................................................................................................. 22
4.2.3 0 M NaCl PDDA addition behavior ........................................................................................................ 24
4.2.4 High concentration PDDA in 0 M NaCl ................................................................................................ 26
4.2.5 0 M NaCl reproducibility ........................................................................................................................... 28
4.2.6 Summary of 0 M NaCl results .................................................................................................................. 28
4.3 Effects of salt concentration on PEM assembly ......................................................................... 29
4.3.1 Theory summary .......................................................................................................................................... 29
4.3.2 Procedure ........................................................................................................................................................ 29
4.3.3 Results and discussion ............................................................................................................................... 30
4.3.4 Spread of values for changing salt concentrations ........................................................................ 32
4.3.5 Trends during deposition ......................................................................................................................... 35
4.3.6 Summary of the effects of salt concentration on PEM assembly .............................................. 41
4.4 PEM conformation change under environmental stimulation............................................ 41
4.4.1 Summary of Theory .................................................................................................................................... 41
4.4.2 Procedure ........................................................................................................................................................ 42
4.4.3 Results and discussion ............................................................................................................................... 42
4.5 Determining backfilling of the grating ......................................................................................... 45
4.5.1 Procedure ........................................................................................................................................................ 45
4.5.2 Results and Discussion .............................................................................................................................. 45
4.6 Signal interpretation .......................................................................................................................... 46
4.7 System troubleshooting .................................................................................................................... 48
4.7.1 Bubbles ............................................................................................................................................................. 48
4.7.2 Flow cell assembly ....................................................................................................................................... 52
Conclusions .................................................................................................................................... 52
References ............................................................................................................................................. 54
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List of Figures
Figure 2.1 Two slit experiment
Figure 2.2 Schematic of the diffraction grating pattern used for this experiment
Figure 3.1 Overview of the diffraction set-up
Figure 3.2 Top and side view of the flow cell
Figure 3.3 A photo of the diffraction set-up
Figure 3.4 Diagram of polymer addition order
Figure 3.5 Sample image of the diffraction spot
Figure 4.1 Ideal graph of signal for PEM assembly
Figure 4.2 Assembly of 4 bilayers in 0 M NaCl
Figure 4.3 PSS1 0 M NaCl
Figure 4.4 0 M NaCl PSS1-PSS4
Figure 4.5 0 M NaCl PDDA1-PDDA4
Figure 4.6 Comparison of high and low concentration PDDA in 0 M NaCl
Figure 4.7 Full data set for 0 M NaCl build-up of 4 bilayers
Figure 4.8 A comparison of the build-up of 4 bilayers in various salt concentrations
Figure 4.9 The average endpoint values of each layer
Figure 4.10 Overlay of the data sets for all salt concentrations
Figure 4.11 The data sets for 0 M, 0.1 M, and 0.5 M NaCl are shown graphed on separate axis.
Figure 4.12 PSS1-PSS4 for all salt concentrations
Figure 4.13 PDDA1-PDDA4 for all salt concentrations
Figure 4. 14 Comparison of 4 bilayers in various salt and polymer concentrations
Figure 4.15 Swelling response of 4 bilayers to salt
Figure 4.16 An experiment with back filling of the diffraction grating
Figure 4.17 Example of signal from an air bubble in the flow cell
Figure 4.18 Beads of moisture in an air bubble with 1 bilayer
Figure 4.19 Beads of moisture in an air bubble with 2.5 bilayers
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1
Polyelectrolyte Multilayers (PEM)
A polyelectrolyte multilayer (PEMs) is a matrix of alternately charged polymers assembled on a
substrate. A polyelectrolyte is a polymer whose monomer has a charged side group called an
electrolyte. PEMs came about from observing polyelectrolyte complexes in solution (Michaels et
al, 1965) and modifying the condensation procedure so that the complex was built up in layers
on a surface instead of aggregates in a solution. The most commonly used assembly procedure is
the layer-by-layer method. For this method, the cleaned surface is exposed to a solution of
polyanions. When the surface is exposed to the polyanions, the polyanions adhere to the surface
through electrostatic forces. After the first polyanion layer the surface will be exposed to a
polycation solution. The previously deposited polyanions will attract the polycations and the
latter will attach to the former. This process of exposure to alternatingly charged polymers can be
repeated to form a uniform thin film. Decher was a pioneer in this field with the first papers
describing this process (Decher et al, 1997). Since then PEMs made with the layer-by-layer
method have become widely used with applications for drug delivery capsules (Rathodl et al,
2014), membranes for separation (Joseph et al, 2014), and biomedical applications (Boudou et
al, 2010). The reasons for the wide range of applications are the ease of fabrication, the ability
to modify the properties of the film to suit the specific application and the ability to control the
surfaces in a replicable manner. The behavior and morphology of PEMs can be modified with
simple adjustments in the adsorption procedure such as rinsing, drying between layers,
concentration of polymer, salt concentration, identity of polymer, pH (Shiratori et al, 2000),
among many others. The wide range of applications has shifted the focus of current research
from the fundamentals of the PEM build-up to applications. In order to have full control over the
behaviors of PEMs, the fundamentals of the interactions that lead to their formation must be
studied. This study will go on to describe using diffraction based sensing as a method to monitor
the fabrication of PEMs and their response to environmental stimulus.
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1.1 Layer by layer
1.1.1 Attraction and adhesion
The attraction and adhesion of the polyelectrolytes to the surface are prompted by electrostatic
and entropic forces. The electrostatic forces involved in the PEM build-up are the most easily
understood. The surfaces most often used for PEMs are glass and silicon, which are negatively
charged when clean. The clean substrate is exposed to the polycations which are attracted to due
electrostatic forces. The substrate is then rinsed to remove the excess poorly adsorbed
polycations. The substrate with its first polymer layer is then exposed to the polyanion. The
polyanion will be attracted to the polycations and bind to the surface. This process of exposure to
a polyelectrolyte, rinsing, and consequent exposure to an oppositely charged polyelectrolyte can
be repeated as many times as needed to achieve a film of a desired thickness. At each polymer
addition the surface charge of the PEM switches charge completely allowing the next layer to
adsorb.
The entropic forces that lead to PEM assembly are loss of hydration waters surrounding the
polymers and the switch from extrinsic charge compensation to intrinsic charge compensation
(Schlenoff et al, 2001). There is a system entropy gain from the loss of the hydration waters of
the polymers as they adsorb to the surface. The polymers also release salt that had been
surrounding them in solution. The polyelectrolytes in solution have a high concentration of ions
around them due to counter ion concentration. When the polymers adsorb to the surface of the
PEM the charges on the newly adsorbed polymers interact and are compensated by the polymers
already present in the film. This means that the system gains entropy both by the loss of
hydration waters of the polymer and the release of ions into the solution.
The processes that drive the adsorption of the next layer cannot be exhausted after a distance
from the layer has been exceeded because the forces are not dependent on the initial surface.
1.1.2 Choice of polyelectrolytes
Polyelectrolytes fall into two broad categories, weak polyelectrolytes and strong polyelectrolytes.
The charge on the side chains of weak polyelectrolytes is pH dependent. Therefore when weak
polyelectrolytes are used in PEMs, the pH has to be closely monitored. PEM films made of the
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same weak polyelectrolyte pair at a different pH will have different characteristics. Strong
polyelectrolytes will have a strongly charged side group regardless of changes in pH. The
polymers used in these experiments are all strong electrolytes, which means that pH will not
need to be a consideration for our system. Polyethylenimine (PEI), and
poly(diallyldimethylammonium chloride) (PDDA) were used for the polycations and
poly(styrenesulfonate) (PSS) was used for the polyanion. The PSS/PDDA polymer pair has been
used extensively and the interaction widely characterized (Guzman et al, 2009)(Soltwedel et al,
2012)(Nestler et al, 2013). This makes them a good choice to use to characterize the response of
our diffraction sensor. A high molecular weight version was chosen for each polymer. A high
molecular weight of PEI was chosen due to better transferring when stamping with PDMS. For
PSS and PDDA a high molecular weight were chosen as high molecular weight polyelectrolytes
most consistently adsorb to the underlying polymer layer. The use of low weight polyelectrolytes
can sometimes lead to PEM film degradation (Sui et al, 2003) (Ghostine et al, 2013).
1.1.3 Interpenetration of Layers
While the assembly of PEM is talked about in terms of layers it is important to recognize that the
boundaries of these layers are “fuzzy” at best (Decher et al, 1997). This means that the layers of
polymer do not have discrete boundaries with strands and loops of polymer from one layer
reaching into the next. At the start of the first few additions these layers are extremely patchy.
The deposition of these first few layers have been characterized by a previously by our lab
(Eisenstein et al, 2015). The layers were characterized with Frequency Modulated-Kelvin Probe
Force Microscopy, FM-KPFM, to provide topographic data and localized surface charge data. It
was found that the PEM exhibits patchy coverage until the fourth layer after which a
homogenous surface is seen both in topography and surface potential. This indicates that until
the fourth layer was reached interpenetration of polymer layers was due not just to relaxation and
interdiffusion of polymers but adsorption of incomplete layers of polyelectrolytes.
Interdigitation and penetration of the polymers has been more widely studied in the bulk
of the PEM than in the first few bilayers. This is because measuring movement within the first
layers would be difficult due to the small thickness of the films and the irregularities that occur
before a large number of bilayers have been built up. Interpenetration of the layers past the first
layers occurs due to multiple mechanisms including relaxation and diffusion of the polymers.
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Interdigitation of the polymers on addition of each layer occurs throughout the film(Losche).
While it was understood that the boundary between layers was fuzzy, it was assumed due the
mechanism of PEM assembly hinging on irreversible adsorption that polymers incorporated into
a layer did not move unless the salt concentration surrounding it was altered. Recent work has
shown that this assumption is untrue and was due to PSS being less mobile in films than the
polycations it is most often paired with (Ghostine et al, 2013). Many of the neutron scattering
studies used deuterated PSS incorporated into a specific layer in order to measure the movement
of polymers in the bulk of PEMs (Jomaa et al, 2005) (Soltwedel et al, 2012) (Losche et al,
1998). Due to monitoring only the PSS and not the PDDA, the movement of the PDDA in the
bulk was not noticed.
1.1.4 Polyelectrolyte conformation and condensation
It is a well-established phenomenon that flexible polyelectrolytes in solution will take on a
multitude of conformations in response to the environment. The conformations stem from the
interplay of the polymer’s charged side chains long-range Coulombic forces with the attractive
hydrophobic interactions of the monomer. These forces are then mediated by the solvent, the
temperature and counter ions in solution. The conformation of the polymers range from an
extended linear conformation to a range of intermediary semi-collapsed forms to collapsed
globular conformations (Manning, 1969). One of the theories to explain this collapse processes is
counter ion condensation. The case for the counter ion condensation of PSS and PDDA will be
examined. When in solution, ions at equilibrium will be at a distance such that the electrostatic
energy is equal to the thermal energy. This is also called the Bjerrum radius, rB. The Bjerrum
radius for a monovalent ion in water at room temperature is
where εο is the vacuum dielectric constant, e is the electron charge, ε=80 is the dielectric
constant of water, and T is the temperature. The charge side chains of the polymers can be
treated as ions. The distance in-between monomer groups, b, for PSS is 2.6 Å and for PDDA is
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5.2 Å. Considering a solution of polymers with no added salts, the polymer will take a linear
conformation because the distance between monomer group is less than the Bjerrum radius the
charges will repel each other. When we consider a strong polyelectrolyte solution with ions the
conformation will change. The distance between ions in a strong polyelectrolyte solution
increase to the Bjerrum radius due to counterion condensation (Manning,1969). The potential of
the charged side chain will decrease with distance due to the screening from the surrounding
ions. The Debye radius, rD, is the distance at which the potential is 1/e of the original value and
can be calculated as
where zi is the charge and ni the number of ions of a type summed over the different types of ions
in solution. For water at room temperature this can be simplified to rD=3.04√C Å. When the
concentration of ions is such that rB �rD then the side chains will not be shielded and will repel
each other giving a linear conformation. When solved so that rB=rD the concentration is 0.2 M.
This means that at 0.2 M or above the side chains will be shielded and not repel each other
providing a globular conformation. As such it will be sufficient to examine 0 M, 0.1 M and 0.5
M NaCl to see the effects of linear, semi collapsed, and collapsed polyelectrolytes on PEM
assembly.
1.1.5 Salt effects on PEMs
The factor that affects the behavior and morphology of PEMs the most is salt
concentration. Polyelectrolytes take on different conformations in solution in the presence of salt.
When there is no salt present the charges on the monomers of the polyelectrolyte repel each
other, and the polyelectrolyte takes on a linear conformation (Manning, 1969). Salt provides
charge screening for the polyelectrolyte and the conformation becomes globular. High
concentrations of polyelectrolytes can also provide charge screening and affect conformation in
solution. When PEMs are assembled from solutions with salt the resulting layers are much
thicker (Wu et al, 2012) and rougher (McAloney et al, 2001). Although this is partly attributed to
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the added polymer added being in a globular formation, the layer are also found to be thicker
because each layer has more polymer overall (Ladam et al, 2000). This change in morphology is
apparent on the surface of the films. Films made in the absence of salt have a smooth flat surface.
The morphology of the surface made in high salt environments take on a vermicular appearance
(McAloney et al, 2001).
When brought into contact with solutions whose ionic concentrations are different than
that the films were made in, there are two reactions. The first is swelling of the film. The second
is film remodeling that can include nanopores (Fery et al, 2001), smoothing(Ghostine et al,
2013) (Jomaa et al, 2005), thinning and complete film degradation(Schlenoff et al, 2001). The
difference between the two reactions can be explained by whether the change in salt
concentration is large enough or exposure was for long enough to interrupt polymer-polymer
charge interactions. Polyelectrolytes in solution collect ions around them as available, which
leads to counter ion condensation and a variety of conformations in solution. When these
polyelectrolytes are incorporated into multilayers they can change from having their charge
compensated by ions in solution to other polymers in the multilayer. A change in ion
concentration in the environment around the already assembled film can lead to these polymers
being extrinsically compensated again and the polymers will then be more mobile to rearrange.
When PEMs are exposed to high salt after assembly a reorganization of polymers can occur to
form smoother, thinner films (McAloney et al, 2003). This is referred to in the literature as
annealing. The accepted theory behind annealing is that due to the high salt concentration the
polymers become extrinsically compensated instead of intrinsically compensated. The links
between polymers in the film lessen and this increases the mobility of the polymers as they are
now compensated by salt ions instead of other charged polymer groups. The polymers can then
reorient to make the PEM solution interface as small as possible and the roughness of the film
lessens. Thinning of the films is due to the hydrophobic nature of many of the polymers used.
The added mobility the salt annealing provides allows the films to reconfigure to exclude more
water, becoming thinner. The speed of reorganization and extent scales exponentially with the
salt concentration.
When the timescale is on the order of minutes the films swell when in contact with salt
solutions. The extent of swelling increases with concentration (Dubas et al,2001) (Koehler et al,
2014) (Dodoo et al, 2011) (Volodkin et al, 2014). The extent of swelling also depends on the
7
concentration of salt the films were assembled in. When films are prepared in higher salt
concentrations they are “spongier” and swell more than those made with lower concentrations
(Dodoo et al, 2011).
1.1.6 Charge compensation
Charge compensation can help to explain many of the properties of the films. The
charges of the polyelectrolytes can be compensated intrinsically by the ionic interactions of an
oppositely charged polymer or extrinsically by counterions from the solution. When a polymer is
intrinsically charge compensated it is less mobile and less prone to swelling (Volodkin et al,
2014). Extrinsic charge compensation also affects the mechanical properties of a PEM (Lehaf et
al, 2011). Polymer pairs that exhibit a linear growth have their excess charge at the surface
(Schlenoff et al, 2001) while those that exhibit an exponential growth have their excess charge
located throughout the bulk (Picart et al, 2002).
The original accepted model of charge overcompensation at surface/solution interfaces
was seen as paramount to how PEMs assembled. It was believed that each layer completely
reversed the charge on the surface and overcompensated (Schlenoff et al, 2001) and that this
could explain the assembly of the multilayers (Netz et al, 1999). The Schlenoff study used radio
labeled counterions to probe the surface charge of the PEM. It was found that there was charge
reversal and over compensation at each layer for a PSS/PDDA polymer pair. Recent work that
uses the same radio labeled counter ion system to probe surface charge refutes these findings.
For a PSS/PDDA pair, charge overcompensation was found to only occur when PDDA is
adsorbed, the PSS merely compensates the PDDA (Ghostine et al, 2013). The theory supports a
complete reversal at each layer to explain polymer adsorption.
1.2 Other techniques to characterize PEMs
The most common methods for monitoring the assembly of PEMs are atomic force microscopy,
quartz crystal microbalance, neutron reflectometry and ellipsometry.
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1.2.1 Quartz crystal microbalance
Quartz crystal microbalances (QCM) use the changing frequency of a quartz crystal to
monitor the change in mass adsorbed to it. Quartz crystals can be made to oscillate by applying
an alternating current with a very high ratio of frequency to bandwidth. This allows for precise
calculation of resonant frequency. Because the frequency of oscillation is dependent on mass,
minute changes in mass can be tracked. QCM can be used in liquid, providing in situ
measurements. The combination of in situ measurements and very small mass sensitivities has
made QCM widely popular in monitoring thin film growth (Lvov et al, 1999) (Elzbieciak-
Wodka et al, 2014). Quartz crystal microbalances offer the advantage of real-time in situ
measurements. The limitations of this technique are that while it is highly sensitive to mass
adsorptions, that mass is averaged over the entire exposed surface and it is not sensitive to
conformation changes in the films.
1.2.2 Atomic Force Microscopy
Atomic force microscopy (AFM) is a type of scanning probe microscopy that can provide
topological information, mechanical information and thickness measurements (Meyer et al,
1988). In AFM the sample is approached by a cantilever on which a very small tip extends
downward towards the sample. The cantilever is coupled to piezoelectric elements that allow for
precise control of the forces and movement of the tip on the sample. A laser is reflected off of the
end of the cantilever and focused onto a photodiode to monitor the movement of the cantilever
and in turn the tip. The movement of the cantilever is recorded as a movement of the laser spot
on the photodiode and software can be used to interpret this data. For information on the
mechanical properties the tip can be used to indent the surface and the response of the sample
will be measured. For topography the tip is scanned across the surface to obtain topographical
data that can be oriented spatially to provide an image of the surface. AFM is a valuable tool for
topography (Lavalle et al, 2002) but it has a drawback when it comes to obtaining data in real
time. The scanning of the AFM tip means that it is limited in how quickly it can collect data over
an area after a change in environment. As a result, data on soft surfaces can be difficult to obtain
without damaging the surface of interest (McAloney et al, 2003).
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1.2.3 Ellipsometry
Ellipsometry is an optical technique that uses dielectric properties of a film to determine its
thickness (Jasperson et al, 1969). Linearly polarized light is directed at a surface and either the
reflection or transmission of that light is monitored. The change in polarization of the initial light
versus the light after it interacts with the sample is used to determine thickness. In order to
calculate thickness a model for the film must be used. The drawback of ellipsometry is the
requirement of a model and known parameters such as refractive index of the film (Harris et al,
2000).
1.2.4 Neutron reflectometry
Neutron reflectometry is a reflectometry method based on neutron diffraction from a stratified
thin film. A collimated beam of neutrons is aimed at a flat surface and the reflected neutrons are
measured in term of their diffraction angle. The neutrons are scattered on the nuclei of the
sample without causing damage to the sample. This allows data to be collected on the thickness,
density and position of layers of the film relative to the substrate without altering the sample. In
the case of PEMs, PSS is often deuterated before being incorporated into layers of the PEM to
increase the contrast of the nuclei (Losche et al, 1998). This allows measurement of the position
of the deuterated PSS layer relative to the substrate providing information about thickness and
the movement of polymers relative to the layer they were deposited in (Ghostine et al, 2013).
This method is very useful for obtaining information about the internal structure of the PEMs.
Because this is a specular reflection technique the roughness of the top of the film can decrease
signal and increase noise. In addition, a large sample is needed in order to obtain data.
Diffraction based sensing
Diffraction based sensing monitors reactions and binding processes by observing the
change in height of a printed diffraction pattern. The diffraction grating is a printed pattern of
one half of a binding pair. When it is exposed to the analyte, the analyte will bind to the grating.
The binding will result in an increase in the height of the grating and an increase in the intensity
of the diffracted spot. If the intensity of the diffraction spot is monitored as this binding process
10
is happening the process will be observed as an increase in the intensity of the spot. This process
can be used both qualitatively and quantitatively if a standardized curve has been created for that
binding pair. This is the basis for our diffraction based sensing.
2.1 Theoretical background of diffraction gratings
Amplitude diffraction gratings function by diffracting light off of a surface that has a
regular pattern of raised features. Previous work from our lab has shown how amplitude
diffraction gratings can explain how our diffraction sensor behaves (Anderson, 2005). It begins
by examining the pattern and intensity of a model for diffraction from a one-dimensional slit.
Huygens Principle is an important foundation for understanding diffraction. It states “each point
on a wavefront is considered to be a source of secondary spherical wave known as a Huygens’
wavelet; the envelope of all the Huygens’ wavelets emanating from a given wavefront at any
instant of time is then used to describe that same wavefront at a later instant in time” (Gaskill,
1979). This means that light reflecting from a surface can be treated as a set of point light
sources along the entire length of that surface. When phrased in this manner the pattern of
constructive and destructive interference seen when a plane wavefront impinges on a slit makes
more sense. The diffraction intensity at a far field point from two infinitesimal slits P and Q is
discussed and the intensity can be described by
where I0 is the original intensity, d is the distance between the two slits and θ is described
on figure 2.1 below.
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Figure 2.1 Two slit experiment
Andersons’ work is very thorough and, after establishing the intensity equation for two
infinitesimally small slits, applies the same approach to model an infinite lamellar grating.
The intensity can be written as
where
and N is the number of grating lines that the laser samples.
12
From this is it possible to see proof that the height of the grating will increase the
intensity of the diffracted spots. The spacing of the diffraction spots is related to the pattern of
the diffraction grating (Anderson, 2005). As long as the spacing of the diffraction grating does
not change the spacing of the diffraction spots will not move.
The grating used for this experiment is based off a beam splitter pattern with a 15 um
periodicity shown in figure 2.2.
Materials and Methods
3.1 Diffraction Sensing
The general schematic of the diffraction sensing setup is seen in Fig. 3.1. The system at its
simplest is a laser, a prism, a flow cell and a detector. The flow cell contains a stamped
diffraction pattern with the pattern described in figure 2.2. A thorough description of the flow
cell and its assembly is in section 3.1.2. The flow cell is coupled with a prism. The prism is used
for two reasons. One it makes for easier alignment for total internal reflection, and two it helps to
Figure 2.2 Schematic of the diffraction grating pattern used for this experiment.
13
space out the diffraction spots, which decreases noise in the signal. The flow cell and prism are
mounted in a holder and aligned with the laser such that total internal reflection is attained. Total
internal reflection is chosen because it decreases noise and increases signal in diffraction based
sensing (Anderson, 2005). The diffraction spot from the flow cell is positioned onto the detector.
This set-up allows the build-up of the diffraction grating to be monitored in situ and in real time.
Figure 3.1 A brief overview of the diffraction set-up at it’s simplest: a laser impinges on a
prism coupled to the flow cell, the diffraction spots are then reflected off of a mirror onto a
digital camera.
A note on the procedure: all of the glass vials used to hold components of flow cells and stamps
need to be thoroughly cleaned prior to use. Failure to do so results in flow cells that provide
nothing but noise.
3.1.1 PEI Stamping
Glass slides (VWR Goldline) were cut to size (25mm X 9.5 mm) and cleaned via sonication in
30% ethanol, rinsed with deionized (DI) water (18.2 MΩ-cm) then dried with N2 gas. Separately,
PDMS stamps were made following the instructions in the kit (Sylgard 184 elastomer kit) using
14
a hard plastic master with the pattern seen in figure 2.2. Prior to use, the PDMS stamps were cut
to size (5mm X 10mm) and sonicated in 30% ethanol for 10 minutes, rinsed with DI water and
dried with N2 gas. The stamps were placed with the pattern facing up and 50 uL of PEI (5
mg/mL in 0.5 M NaCl prepared with DI water) sitting on the pattern for 45 minutes. The stamps
were then rinsed with a stream of DI water for 5 seconds and dried with N2 before being brought
into contact with the center of one of the cleaned glass slides for 4 minutes before removal.
3.1.2 Flow Cell Assembly
After the slides have been stamped, the rest of the flow cell was prepared. Glass slides (VWR
Goldline) were cut to 37.5 mm X 12.5 mm and cleaned via sonication in 30% ethanol for 10
minutes. The slides were then rinsed with DI water and dried with N2. 3M carpet tape was cut
into strips 3.175 cm X 2 mm. These strips were placed on the glass slides stacked 2 high, parallel
and 1 cm apart. The smaller glass slides that had been printed with PEI were placed face down
on the slides with tape and pressed to form a seal. This results in a flow cell that is approximately
1 cm X 3.175 cm with a volume of 15-20 uL and the printed PEI is facing the interior of the flow
cell. Figure 3.2 shows the top view and side view of the assembly of a flow cell.
15
Figure 3.2 The top view and side view of the flow cell assembly. The small slide that has been
printed with PEI is placed on the larger slide with double stick tape. The prism is then coupled
to the small glass slide that has the stamped PEI.
3.1.3 Diffraction Sensing Set-up
The set-up used can be seen in figure 3.3. The flow cell was coupled with the prism and mounted
on a stationary post. The laser used was 670 nm, 3.5 mW, World Star Tech. The laser path
included two mirrors before the flow cell, and a mirror after, before arriving at the CCD detector.
The mirrors are included in the set-up to be able to position the laser spot. The setup is arranged
so that the sample is in total internal reflection. The laser hits the prism, travels through the back
of the flow cell, interacts with the stamped diffraction grating and travels out the opposite side of
the prism. As a result of the stamped polymer pattern, a line of spots emerges from the prism.
The diffraction spot that was monitored was the second from the main beam.
16
Figure 3.3 A photo of the diffraction sensing set-up.
3.2 In situ Monitoring of Polymer Deposition
3.2.1 Reagents
Polyethylenimine (PEI, MW 60 kDa, Sigma Aldrich), Poly(sodium 4-styrene
sulfonate)(PSS, MW 200 kDa, Sigma Aldrich), and Poly(diallyldimethylammonium chloride)
(PDDA, MW 400-500 kDa Sigma Aldrich) were each dissolved in DI water at concentrations of
17
5 mg/mL. Various concentrations of sodium chloride (Sigma Aldrich) solid were dissolved in the
polymer solution.
3.2.2 Polymer Deposition and Measurements
Using a pipette, 25 μL of DI water was added to the flow cell. If an air bubble was in the area
of the stamped PEI the flow cell was flushed with another aliquot of 25 uL of DI water to make
the air bubble smaller or flush it out completely.
The sequence of polymer addition is illustrated in figure 3.4. The printed PEI is the first
polymer layer. The polymers are then added so that their charges are alternating with PSS first
followed by PDDA, PSS, PDDA and so forth.
Figure 3.4 Diagram of polymer addition order. The flow cell begins with stamped PEI and
then is exposed to PSS, then PDDA.
Data acquisition begins with 1 min of DI water in the cell. A P200 pipet is used to add 25 uL of
PSS on one side of the Flow cell, while a Kim wipe is held so that the corner of it is in contact
18
with the opening of the flow cell on the opposite side to wick out the excess solution. The PSS is
let sit in the flow cell for 2 minutes. After waiting 2 minutes 25 uL of DI water is adding to the
flow cell while wicking solution out of the opposite side with a Kim wipe. The flow cell is rinsed
with 25uL of DI water and let sit with for 1 minute with water in the flow cell. 25 uL of PDDA is
then added and let sit for 2 minutes. The flow cell is then rinsed with 25 uL of DI water and let
readjust for 1 minute. This pattern (PSS for 2 minutes, 1 minute water, PDDA for 2 minutes, 1
minute water) is repeated 3 more times for a total of 4 bilayers.
3.2.3 Data Capture and Data Analysis
A Silicon Video 2112C CMOS Camera was used to capture the diffraction spot. Exposure for
each image was set to between 0.3 and 1.4 ms in order to prevent saturation of the image before
the data for 4 bilayers was collected. Images were taken at 0.5 s intervals and processed once all
of the data was collected.
The XCAP imaging application was used to process the collected images. The images were
collected as 200 x 200 pixel tif image files. In each image, the bright diffraction spot was
selected as the area of interest. An example image is shown in figure 3.2.3 with the area for the
signal and area considered background shown outlined. The intensity of the area of interest was
then averaged and binned. The selected spot size depended on the size of the diffraction spot, and
tended to be around 25 x 75 pixels. The value of the diffraction spot was exported as the raw
signal versus time. A region without diffracted light but of the same size as the diffraction spot
was averaged and exported as the background and noise. The background was subtracted from
the signal and then the signal was normalized to the initial signal from water in the flow cell
(Io), as shown in the equation below:
Normalized signal=����
��
The signal was then graphed.
19
Figure 3.5 A sample image of the diffraction spot with the area of interest and background
3.3 Discussion of factors that would increase of decrease signal
A change in signal whether it is an increase or a decrease can be the result of multiple factors in
our experiment. As stated an increase in signal can most simply be attributed an increase in the
height of the grating. This can either be due to an addition of more polymer, or a swelling of the
existing PEM because our set-up will not be able to differentiate between the two. Additionally
20
the signal can increase from better coverage of the diffraction lines. The first few layers of
polyelectrolyte deposit as islands that converge to form a continuous layer as the number of
layers increase (Eisenstein, 2015). This means that our grating could be patchy. Our grating will
still be functional if the lines are patchy, the intensity of the signal will be lower though.
Babinet’s principle dictates that grating made of small particles that does not have complete
coverage will give the same discrete diffraction pattern as a completely opaque diffraction
grating but the intensity will be dependent on the coverage. The intensity scales as the square of
the coverage so that an opaque grating that would give a diffraction spot an intensity of 100%
when replaced with a grating with 50% coverage of the same diffraction grating pattern will give
a spot with 25% intensity (Meyer, 1887).
The factors that would decrease the signal are a decrease in height and polymers adhering to the
troughs of the gratings. A decrease in height either through polyelectrolyte desorption or the
PEM compacting will decrease the signal. When interpreting the data desorption of
polyelectrolyte will not be considered as the timescales used for PEM assembly in our
experiment are consistent with irreversible polymer adsorption (Schlenoff et al, 2001). Another
possibility is that the polymer does not adhere to the plateau of the grating but rather to the
trough. Previous experiments in our lab with diffraction gratings have not had to take this into
consideration due to the ability to block glass when dealing with proteins (Goh et al, 2003).
Blocking the glass eliminates random protein adsorption to the troughs without interfering with
the specific interactions that lead to the protein adsorption onto the plateaus to make a grating.
The adsorption of polymers is charge driven and nonspecific. This means that the glass cannot be
blocked because any agent charged enough to adhere to the glass in the troughs will be charged
enough to interact with the subsequent polymers. Deposition of polyelectrolyte to the troughs of
the grating will decrease the signal by making the apparent height as measured from the newly
filled in troughs less.
Results and Discussion
4.1 Idealized scenario of PEM signal in diffraction optics signal
In order to better understand the experimental results an idealized version of the diffraction
signal will be discussed. A mock up of what an ideal signal result would be is shown in figure
21
4.1. As explained in section 3.2.3 the baseline for our experiments will be the signal when there
is water in the flow cell. This has been normalized to 0%. At 60 seconds the PSS solution is
introduced. It is expected that a binding curve similar to what has been seen with the anti-body
work done with this set-up will be seen for PSS. At 180 seconds water will be added to the flow
cell to rinse out the excess polymer solution. The signal is not expected to increase or decrease
with the introduction of water. At 240 seconds PDDA solution will be added to the flow cell.
Again it is expected that a binding curve will occur. At 300 seconds the flow cell will be flushed
with water. This will be repeated until 4 bilayers have been built up. The term bilayer refers to a
layer of polyanions and the following polycation layer. It is expected that with each subsequent
exposure of the flow cell to a polymer solution the signal will increase as the PEM on the grating
sensor builds in height and increases coverage. This is the behavior that has been seen when
using anti-bodies and proteins with diffraction based sensing (Goh et al, 2003). PEMs have
stepwise adsorption that terminates, so the same type of behavior was expected.
Figure 4.1 Idealized graph of signal for PEM assembly. Water is blue, PSS is red and PDDA
is green
22
A few terms need to be introduced to make discussion of the later results easier. When referring
to a polymer layer the terms PSS1 will be used to denote the first PSS layer, PSS2 the second
and so on. Likewise PDDA1 will refer to the first PDDA layer. When talking about the end point
the numerical value is the average from 60 seconds after the polymer solution is added to the
flow cell to 120 seconds after the polymer solution is added. By this time the signal should have
plateaued.
4.2 0 M NaCl polymer results
4.2.1 Procedure
The experiments were run as described in 3.2.2 with alternating additions 25 uL of 5 mg/mL PSS
and 5 mg/mL PDDA with 0 M NaCl in each solution. Images were collected over the course of
the deposition of 4 bilayers on the initial polymer pattern in the flow cell. As said in the methods
section 3.2.3 the timing of the 25 uL solution additions were one minute DI water, 2 minutes
polymer, 1 minute water rinse, repeated until all 4 bilayers had been deposited. The data was
collected as images and then processed as described in section 3.2.3 to achieve the graphs shown
in later sections.
4.2.2 0 M NaCl PSS addition behavior
In order to better compare the behaviors of each polymer layer and the thickness of that layer, the
data was normalized with the water rinse directly preceding the polymer addition being observed.
This allowed much easier comparison of the behavior of the polymer additions across various
conditions. Figure 4.3 is a close up of the first addition of PSS to the flow cell. Behavior is
similar to what is expected to see from a binding curve. A smooth quick increase that plateaus
once all of the binding sites have been occupied. The addition of water to the flow cell at 180
seconds does not change the signal. When multiple iterations of this experiment are shown on the
same graph a few issues become clear. Figure 4.4 shows repetitions of PSS1. The increase in
signal with the addition of PSS has a wide spread of values. But the time it takes for the signal to
plateau is the same regardless of the endpoint value of PSS1. Figure 4.4 show PSS2, PSS3, and
PSS4. The shape of the curve and the time until the signal plateaus stay the same for each layer.
23
The endpoint was largest for PSS1. The endpoints of PSS2, PSS3 and PSS4 are relatively similar
and have a wide spread of values.
Figure 4.4. Repetitions of 0 M NaCl PSS1, PSS2, PSS3 and PSS4. Multiple iterations of the
same experiment shown on the same graph. For each the signal was normalized to that layer
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24
4.2.3 0 M NaCl PDDA addition behavior
The PDDA additions were treated in the manner as the PSS in order to compare the adsorption
behavior. It was expected that the PDDA adsorption would lead to a binding curve similar to that
seen with PSS. The PDDA additions did not have a standard binding curve shape. The PDDA
binding curve also changed shape as the number of layers increased. The PSS layers did not have
a change in behavior as the layers increased but the curves did become larger. As can be seen in
figure 4.5 PDDA1 has a small increase followed by a shallow decline in signal that continues
when the flow cell was flushed with a water rinse. PDDA2 in figure 4.5 had a much less sharp
initial bump and the decrease after had a much smaller slope. PDDA3 in figure 4.5 did not have
the sharp initial bump. The beginning of the graph looked similar to the PSS additions in that
there was a first initial steep slope that gradually reached a maximum. Like the previous PDDA
additions, the water rinse that follows had the same shallow slope as the PDDA addition before.
The fourth PDDA addition in figure 4.5 was very different. When looking at the behavior across
the full data set PDDA4 either had a very small increase from the water rinse preceding it that
plateaus very quickly, or not a very discernable difference from the water rinse preceding it. The
signal during the water rinse after PDDA4 did not follow any pattern. On some runs there was a
marked increase , on others a decrease, and some had fluctuations. The runs that had a higher
signal increase over the entire 4 bilayer experiment were also those that had a bump at the
beginning of the PDDA addition.
25
Figure 4.5 Repetitions of 0 M NaCl PDDA1, PDDA2, PDDA3 and PDDA4. Multiple iterations
of the same experiment shown on the same graph. For each the signal was normalized to that
layer
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26
4.2.4 High concentration PDDA in 0 M NaCl
A few experiments were conducted with 50 mg/ml PDDA instead of 5 mg/ml PDDA. Figure 4.6
shows the build up of 4 bilayers following the procedure laid out in methods 3.2.2 with the high
concentration PDDA compared to a low concentration PDDA in 0 M NaCl. The behavior of
both the PSS and PDDA additions were affected even though the PSS concentration remained
the same. The PSS additions in the high concentration PDDA are much larger than those in the
low concentration PDDA. When in high concentration the PDDA additions have much more
exaggerated trends than what was seen in the low concentration PDDA. PDDA1 has a large
decrease. PDDA2, PDDA3, and PDDA4 have similar behavior to what was seen for low
concentrations but more exaggerated. PDDA2, PDDA3, and PDDA4 all have a sharp addition
that reaches a peak within 5 seconds that then a shallow linear decrease. As seen with the low
concentration layers, as the number of layers increases the decrease after the peak levels out. The
slope after the peak of PDDA2 is angled downward, where as PDDA4 is nearly level.
27
Figure 4.6 A comparison of the buil-up of 4 bilayers in 0 M NaCl with high(50 mg/mL) or low
(5 mg/mL) PDDA
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Comparison of 0 M added salt with high (50mg/ml) or
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water % high [PDDA]
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PDDA % high [PDDA]
water % low PDDA
PSS % low [PDDA]
PDDA% low [PDDA]
28
4.2.5 0 M NaCl reproducibility
Up to this point the data has been shown either for individual layers or only one experiement
with all 4 bilayers. When the entire data set for 0 M NaCl is graphed together the problem of
reproducibility becomes apparent, figure 4.7. It was established in figure 4.3 that PSS1 had an
endpoint with a very large variability, and that each individual polymer layer after also has a
large variability. When the assembly of all 4 bilayers is graphed together the spread of value
compounds so that the range of values for PDDA4 endpoints is greater than that of PSS1
endpoints. Although the reproducibility of the values of each run is poor, the behavior of the
polymer is consistent when compared for each layer.
Figure 4.7 The full data set for the build-up of 4 bilayers in 0 M NaCl is shown on the same
axis
4.2.6 Summary of 0 M NaCl results
A diffraction set-up was used to monitor the build-up of 4 bilayers of a PSS/PDDA polymer
multilayer. The actual data varied from the ideal in the adsorption behavior of the polymers. The
0
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29
shape of the PSS adsorption curve was similar to what was expected. The PDDA adsorption
curve was not. The PDDA exhibited a sharp peak followed by a decline. The shape of the
adsorption curve also changed from layer to layer. For PSS as the number of layers increased the
signal change became smaller. For PDDA as the number of layers increased the initial peak
became smaller, and the decline following it became shallower. The reproducibility of the values
for each layer is poor, but the shape of the curve for each experiment is similar. An experiment
with 50 mg/mL PDDA was carried out. The layers for both polymers were much thicker and the
trends for polymer adsorption as the layers increased where the same as seen before but more
pronounced.
4.3 Effects of salt concentration on PEM assembly
4.3.1 Theory summary
Salt concentration during assembly of PEMs is the factor that will have the most influence on
film morphology and characteristics. A high salt concentration will lead to polymer condensation
and collapse in solution. This conformation change means that films assembled in high salt
concentrations will be rougher and taller than a similar number of layers assembled in lower salt
concentrations. This is due to the polymers adsorbed being in the globular conformation and that
each of the layers also has a larger mass added when the salt concentration is higher. In
diffraction based sensing this means that when PEM assembly is monitored the endpoint value of
each layer should be larger when there is a higher salt concentration.
4.3.2 Procedure
The procedure was very similar to that outlined in section 3.2.2. The only difference was the salt
concentration of the polymer solutions was changed to either 0.1 M NaCl or 0.5 M NaCl. The
timing and the DI water rinse were kept the same.
30
4.3.3 Results and discussion
Changing the salt concentration of the polymer solutions should change the signal for the
PEM assembly. Increasing salt concentration will increase the shielding of the charged side
chains of the polyelectrolytes leading to more globular conformations in solution. This means
that when compared to the experiments run with 0 M NaCl, experiments run with salt in solution
should have higher signal values as the layers will be thicker. Figure 4.8 shows a comparison of
the buildup of 4 bilayers in 0 M, 0.1 M and 0.5 M NaCl. Each of the signal traces shown were
picked because they had the median value of their data set for PDDA4 endpoint. From looking at
figure 4.8 it appears that the signal does scale with salt concentration. This is consistent with
others finding when looking at films made in similar environments by the layer-by-layer method.
A more quantitative comparison of the endpoint values of each layer also supports this, figure
4.9. For each layer the average endpoint value scaled with salt concentration, with the exception
of PDDA1 where the endpoint of the 0.1 M NaCl was the smallest.
31
Figure 4.8. The build-up of 4 bilayers in 0 M, 0.1 M, and 0.5 M NaCl are shown.
32
Figure 4.9 The average endpoint values of each layer with error bars.
4.3.4 Spread of values for changing salt concentrations
The issue of reproducibility of the value of each experiment has been discussed in section 4.2.5
When the signal of an experiment is examined over the 4 bilayer build-up the effects of the
inconsistencies of each layer multiply. Figure 4.10 shows the signals from 0 M, 0.1 M, and 0.5
M NaCl experiments displayed together. The data set from each salt condition sit within the
spread of values for the 0.5 M NaCl experiments. An experiment with a low value PDDA4
endpoint from the 0.5 M NaCl data set would be similar in value to anything within the 0 M
0
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1st PSS 2nd PSS 3rd PSS 4th PSS
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33
NaCl data set. This will make using diffraction based sensing for qualitative measurements of
PEM thickness difficult until the inconsistency can be resolved. When the different salt
concentration data sets are graphed separately, figure 4.11, and compared it is visible that the
range of endpoint values are distinct if overlapping.
Figure 4.10 The graph shows the data sets for 0 M, 0.1 M, and 0.5 M NaCl all on the same
axis where green is 0 M NaCl, blue is 0.1 M NaCl and red is 0.5 M NaCl.
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34
Figure 4.11 The data sets for 0 M, 0.1 M, and 0.5 M NaCl are shown graphed on separate
axis.
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35
4.3.5 Trends during deposition
One of the advantages of this method is that differences in the adsorption of each polymer layer
can be easily monitored. The adsorption process for each polymer changes as the number of
layers increase and respond differently to varying salt concentrations. There are two different
characteristics to examine: the endpoint value, and the shape of the curve. The endpoint value
will give information about the change in adsorbed mass and conformation. The shape of the
curve provides information about the kinetics of the adsorption.
4.3.5.1 Trends in PSS deposition with change in salt concentration
Each layer at each salt concentration is shown in figure 4.12. The PSS adsorption changes mostly
in magnitude, not in shape, with an increase in layers and salt concentration. When comparing
the PSS1 across salt concentrations, the signal increases with an increase in the salt
concentration. The curve also becomes sharper and reaches a flatter plateau faster. This pattern
of a higher salt concentration leading to a larger signal and faster plateau does not hold for the
later PSS layers. The PSS1 has the largest endpoint of any of the layers for each of the salt
concentrations. For the 0 M NaCl and 0.1 M NaCl experiments there is no trend among the
layers endpoints, PSS1 has the largest endpoint and the next are much smaller with no distinct
trend. The endpoint of PSS1 in 0.5 M NaCl is the largest, and as the layers increase each
subsequent layer is smaller. The kinetics of the polymer adsorption follow in a similar fashion.
The 0 M and 0.1 M NaCl experiments don’t show a marked change in the slope of the adsorption
curve as the numbers of layers increase, or between the two salt concentrations. The 0.5 M NaCl
experiments on the other hand have a decline in slope of the curve as the layers increase. The
PSS1 curve has a sharp increase at the beginning. The initial slope of the curve decreases as the
layers increase until PSS4 is in some cases a straight line. In summary the PSS behaved the
manner expected. Each layer added increased signal value, and when looking at the values
normalized to each layer the value added increased with increasing salt concentration except for
PSS4 in 0.5 M NaCl. This is consistent with the findings that PEMs made in higher salt
environments have layers that are thicker and that thickness scales with concentration of salt.
36
Figure 4.12 Each PSS1-PSS4 a shown for each salt condition, normalized to the water rinse
preceding it.
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37
4.3.5.2 Trends in PDDA deposition with change in salt concentration
The PDDA adsorption curve is a very different shape then the PSS curves which can make it
difficult to examine the endpoint values. The adsorption curve for each PDDA layer for all the
salt concentrations are shown in figure 4.13. The 0 M NaCl PDDA1 has a sharp peak with a
declining slope after it. The decline continues during the water rinse as well. As the layers
increase the height of the peak decreases slightly and the slope of the decline after the peak
lessens. Both of these trends lead to a curve that looks more and more like PSS as the layers
increase. If you compare the adsorption behavior of PDDA across salt concentrations there does
seem to be concentration dependent behavior, but not easily summarized trends. As the layers
increase for both 0.1 and 0.5 M NaCl experiments the decline after the initial peak becomes more
a of flat line as opposed to a curve and the slope becomes more shallow. One of the main
differences is the behavior of the signal for the water rinse of the 0.5 M NaCl experiments. The
water rinse following PDDA4 in 0.5 M NaCl showed step signal response to the introduction of
the water rinse. This behavior is strongest in PDDA2 and seen to a lesser degree in PDDA3 and
PDDA4. This type of response is most likely due to swelling and will be discussed later.
38
Figure 4.13 Each PDDA1-PDDA4 a shown for each salt condition, normalized to the water rinse
preceding it.
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t in
cre
ase
Time (seconds)
-5
0
5
10
15
900 1000 1100
Pe
rce
nt
incre
ase
Time (seconds)
-20
-10
0
10
1260 1360 1460
Pe
rce
nt
incr
ea
se
Time (seconds)
0 M NaCL 0.1 M NaCl 0.5 M NaCl
PDDA1
PDDA2
PDDA3
PDDA4
39
Comparing the endpoint values of PDDA layers to the endpoint values that have been
normalized for each layer show some interesting things. When looking at the endpoints where
each layer has been normalized the PDDA1 endpoint is negative for each salt concentration.
Comparing this to the values for PSS1 and PDDA1 we can see that PDDA1 is a lower value so
the makes sense. All of the layers follow this trend where if the change in the normalized value is
consistent with the endpoint values that have not been normalized to the layers. The odd one out
is PDDA4 in 0.5 M NaCl. The endpoint normalized to the layer shows that PDDA4 has a
decrease of -7.35%. If one compares the values of PSS4 (190.72%) and PDDA4 (207.74%) when
it is not normalized to the layer there is an increase seen of 17%. This would seem to indicate
that there is an adsorption of PDDA as the signal does increase between PSS4 and PDDA4, but
that the water rinse in between leads to swelling in the film. The swelling behavior will be
discussed in section 4.4
4.3.5.2.1 High concentration PDDA in 0.5 M NaCl
Two of the factors that affect the behavior of polymer adsorption are salt concentration and
polymer concentration. Both increase the thickness of the layers because they both make the
polymer take on a more globular conformation in solution. So far these factors have been
examined separately. Figure 4.14 shows a comparison of the build-up of 4 bilayers in 0 M and
0.5 M NaCl for both low and high PDDA conditions. Both polymer concentrations exhibit a 4
fold increase when comparing the PDDA4 endpoint of the 0 M to the 0.5 M NaCl. The behavior
of each of the polymers also changes. The adsorption behavior of the PSS changes mostly in
value. For the low concentration PDDA, in both salt concentrations as the layers increase the
layers become smaller. This can be seen in figure 4.12. The PSS addition with high PDDA does
not exhibit this decrease for either salt concentration. The PDDA has a large behavior shift in 0.5
M NaCl with high polymer concentration. It exhibits a much sharper increase, and a much
steeper decrease after the peak then the experiments run with 0.5 M NaCl at 5 mg/mL PDDA.
Both high salt and a higher concentration have been known to increase the mass of polymer
adsorbed at each layer. This in turn means that each adsorbed polymer will be more crowded on
the surface. This crowding would effect relaxation and could account for the difference in
adsorption curve shapes. The high polymer, high salt experiments also had a new behavior with
the later water rinses. After PDDA3 whenever there was water in the flow cell the signal would
40
drop significantly. This was not seen with other experiments with low salt and low polymer
concentrations.
Figure 4.14 A comparison of The build-up of 4 bilayers in 0 M and 0.5 M NaCl with 5mg/mL PDDA
or 50 mg/mL PDDA.
0
50
100
150
200
250
300
0 240 480 720 960 1200 1440
Pe
rce
nt
incr
ea
se
Time (seconds)
A comparison of 4 bilayer build-up with 5
mg/mL PDDA in 0 M and 0.5 M NaCl0.5 M NaCl
0 M NaCl
0
200
400
600
800
0 240 480 720 960 1200 1440
Pe
rce
nt
incr
ea
se
Time (seconds)
Comparison of 4 bilayer build-up with 50
mg/mL PDDA at 0 M and 0.5 M NaCl
0.5 M NaCl
0 M NaCl
41
4.3.6 Summary of the effects of salt concentration on PEM assembly
The assembly of 4 bilayers of PSS/PDDA was carried out in 0 M, 0.1 M, and 0.5 M NaCl in
order to see whether the effects of salt concentration would be visible to the diffraction set-up.
An increase in salt concentration during assembly of a PEM will increase the thickness of the
polymer layers. This was observed and the data was consistent with layer thickness scaling with
salt concentration. The adsorption behavior of the polymers was affected by the salt
concentration. Lastly the effects of salt concentration and polymer concentration were examined
together. It was seen that by increasing salt and the PDDA concentration the layers of both
polymer thickened and the decrease seen in layer thickness with increasing layers was no longer
present.
4.4 PEM conformation change under environmental stimulation
4.4.1 Summary of Theory
The concentration of salt around a PEM is the most important factor in determining it’s behavior.
The effect of salt on PEM assembly and it’s characteristics have been discussed in section 4.3.
Now the effects of salt on PEM’s post assembly will be summarized. There are two distinct
responses to a change in the environment that are short term and long term. The short term
response to a change in salt concentration is that the multilayer matrix will swell in response to
an increase in salt in the environment. Long term there will be film remodeling that will lead to
smoother, thinner films, and sometimes to film degradation. The film remodeling is due to the
polymers charge being compensated by the more mobile salt ions as opposed to the polymers in
the film, which would act as tethers. Having a smoother film leads to a smaller interface at the
liquid so it is energetically favorable. The response of a PEM to a change in the environment is
also dictated by what salt condition the PEM was assembled in. PEMs assembled in higher salt
conditions are found to be spongier and more likely to swell. With this background taken into
consideration we should see that the signal from our PEM increases when the salt concentration
42
increases in the flow cell. Likewise a film that was assembled in a higher salt environment
should show more swelling and a higher signal for that same concentration of salt.
4.4.2 Procedure
Samples that were assembled from previous experiments were used for the swelling experiments
following the procedure in 3.2.2. Flow cells that had 4 bilayers were allowed to equilibrate with
DI water for 10 minutes. Data acquisition started after the PEM had been allowed to equilibrate.
Data recorded for 2 minutes with water in the flow cell. Then 25 uL of 0.1 M NaCl was added to
the flow cell. After 2 minutes 25 uL of 0.5 M NaCl was added to the flow cell.
4.4.3 Results and discussion
One of the advantages to diffraction based sensing is the ability to observe the changes in
conformation of the film in real time due. To watch the swelling of the layers in response to salt
concentration changes, after 4 bilayers were assembled the solution in the cell was cycled
between 0, 0.1 and 0.5 M NaCl solutions. Figure 4.15 shows the response of a PEM assembled in
0 M NaCl. When the water in the flow cell is replaced with 0.1 M NaCl the signal has in
immediate step response. The signal has the same immediate step response when 0.5 M NaCl is
introduced to the flow cell. The change in signal between the differing salt concentrations is
present but not significant with the noise in the system. PEMs assembled in different salt
concentrations have different characteristics and so their response to a change in the environment
will also change. Figure 4.15 is the response of 4 bilayers of a PEM assembled in 0.1 M NaCl. It
has a step function in response to the change in salt concentration. The response is mush larger
than that seen with the sample assembled in 0 M NaCl. This could be due to two reasons. First
the film assembled in 0.1 M NaCl will be thicker than that assembled in 0 M NaCl. Therefor if it
swells there is more of it to expand. The second reason is that PEMs assembled in higher salt
concentrations have been shown to have a greater capacity to swell and take up water, often
termed spongier. This increase in signal with increased salt concentration was reproducible both
in terms of signal value and speed of response when repeated multiple times on each sample. It
this was repeated multiple times without a decrease in quality of the signal response, but over
time the film should degrade with repeated cycling of the salt concentration. The increase in the
signal with increase in salt concentration helps to describe another quality of our system. When
43
the film swells and takes up water it will become less dense and the index of refraction will
decrease and become closer to that of water. Decreasing the difference of the refractive index
should decrease the signal. At the same time the swelling of the PEM will increase the height of
the film, which will increase the signal. This has shown that the overall effect on the signal will
be positive and that the effect of the height change contributes more to the signal than the
refractive index.
44
Figure 4.15 Swelling response of 4 bilayer films made in 0 M, and 0.1 M NaCl when exposed
to 0 M, 0.1 M and 0.5 M NaCl.
460
480
500
520
540
560
580
180 240 300 360 420 480 540
Arb
itra
ry U
nit
s
seconds
Salt swelling of 4 bilayers made in 0.5M
NaCl
water
0.1M
0.5M
120
122
124
126
128
130
132
134
0 60 120 180 240 300
Arb
itra
ry U
nit
s
seconds
Salt swelling of 4 bilayers made in 0M
NaCl
water
0.1M
0.5M
45
4.5 Determining backfilling of the grating
One of the problems of using diffraction based sensing for PEMs is trying to figure out exactly
what is going on at the grating. Remember the glass is negatively charged and PDDA is
positively charged. It would stand to reason that on addition of PDDA to the flow cell the PDDA
could adhere to the glass and eradicate the diffraction grating by filling in the troughs. To
investigate this flow cells were first exposed to PDDA and then PSS and observed.
4.5.1 Procedure
The procedure is similar to that outlined in section 3.2.2 except that the order of polymers is
changed. All of the polymer solutions used are in 0 M NaCl. The first polymer solution added to
the flow cell is PDDA, and the second is PSS. Only one bilayer is added to the flow cell.
4.5.2 Results and Discussion
In order to see if exposing the flow cell to PDDA will result in backfilling of the troughs of the
grating the order of polymer exposure was changed. If the flow cell is exposed to PDDA first and
the signal decreases this will indicate that there is back filling of the trough. Figure 4.16 shows
the data set for the backfilling experiments. For some of the runs there was a decrease with the
introduction of PDDA, for some there was no reaction, and others showed an increase. A
decrease in signal would indicate that there is some back filling of the troughs. An increase
indicates that more polymer filled in the peaks of the grating. What’s more interesting is that
after exposure to PDDA introduction of PSS showed no change in signal for every experiment in
the data set. This means that either the PSS is not adsorbing to the surface at all, or more likely is
adsorbing equally across the surface. The latter is much more likely. This simple experiment
indicates that the PDDA will fill in the troughs of the grating. What’s interesting is that when the
grating is exposed to the polymers in the normal order (negative PSS first, then positive PDDA)
although this backfilling of troughs is probably happening the signal still increases with
increasing layer.
46
Figure 4.16 An experiment to test the back filling of the grating troughs was done where the
order of the polymers was reversed. The flow cell was exposed to PDDA and the PSS.
4.6 Signal interpretation
One of the main questions of this thesis was whether or not diffraction based sensing was able to
monitor the build-up of PEMs and what advantages this provided. In order to test this we
investigated the diffraction signal response to a build-up of 4 bilayers in various conditions. In
the process of PEM assembly with the layer by layer technique each layer adds mass and
thickness to the growing film (Schlenoff et al, 1998). As such it should be visible in our signal as
an increase in endpoint value for each polymer addition. We see that in figure 4.9 the average
endpoint does increase with each added layer. Another known change in thickness is an increase
in layer thickness with an increase in the salt concentration of the assembly solution (Dubas et al,
1999). We have shown that the thickness of each layer does scale with the salt concentration of
the assembly conditions. Like salt, an increase in the concentration of the polymer will increase
47
the thickness of each layer which we have observed in figure 4.12. Lastly PEM films are
sensitive to changes in their environment (Koehler et al, 2014). A change in the salt
concentration of the surrounding solution will result in a swelling of the film. We observed that
an increase in the salt concentration resulted in increase in signal consistent with film swelling
figure 4.15. The response of the films was also consistent with the films made in higher salt
concentration being spongier (Dodoo et al, 2011) that would result in more response to a change
in environment. Overall the response of the signal was consistent with conditions in which PEMs
would be expected to increase in mass or thickness.
The adsorption behavior that each polymer exhibited was unique. Firstly the PSS showed a more
consistent and normal adsorption curve. PDDA exhibited an overshoot behavior. This behavior
lessened as the layers increased, but increased with salt concentration and polymer concentration.
Both of those conditions lead to an increase in the globular conformation of PDDA in solution.
There are two things that could be happening: the decrease from the peak PDDA could be due to
a desorption of polymer, or a rearrangement of the polymer already adsorbed. Desorption of the
polymer is unlikely due to the concentration of polymer used, a desorption of polymer is seen
only when using smaller concentrations (Pefferkorn et al, 1990).That leaves rearrangement of the
polymers as a possible source of the decrease in signal. When the polymers are in solution the
concentration of salt and polymer will dictate the conformation of the polymer. Once the
polymer adsorbs to the surface it’s possible that the polymer will relax the conformation of the
polymer will be dictated by it’s interaction with the surface.
There still exist the question of what the diffraction grating actually looks like. It has been
assumed that the polymer for each layer adds to the peaks of the diffraction grating only without
depositing to the troughs of the grating. The troughs of the grating in this assumption would
therefor be bare glass at the beginning of the experiments and at the ends after 4 bilayers. This is
probably not the case. PEM are known for their ability to fill in holes in coverage with the
addition of more layers( Diziain et al, 2007). The spacing of the diffraction grating is on par with
spacing of the initial islands of polymer seen for deposition when the substrate is immersed in
the polymer solution(Eisenstein, 2015). It was seen that the islands of deposited polymer spread
until there was a smooth continuous layer. Seeing as the spacing of the diffraction grating and
the spacing of the initial islands are both under 5 um it is possible that the diffraction grating
does not keep a standard step height pattern but instead the troughs become filled in. The issue
48
then becomes how does this idea of filling in of the grating fit with the continued signal that is
still reporting behavior consistent with PEM behavior. Seeing as the spacing of the diffraction
spots did not change the periodicity and pattern of the diffraction grating did not change.
4.7 System troubleshooting
Two main troubleshooting issues were large air bubbles in the flow cell and the flow cell
leaking.
4.7.1 Bubbles
One of the problems I had in the course of this research was the appearance of large air bubbles
in the sample flow cell. Whenever one uses PDMS as a stamp for microcontact printing there is a
small and generally negligible transfer of monomers of PDMS. This leaves behind not only a
pattern of whatever was intended to be transferred but also areas of hydrophobicity due to the
PDMS monomer transferring. For my flow cells that translates into the PEI stamped region
having the possibility of added hydrophobicity. Early on in the project what would happen is that
when solution was added into the flow cell the solution would flow through without covering the
entirety of the patterned area. The solution would wet the back glass panel of the flow cell and
not come into contact with the printed area of the front panel of the flow cell. If the laser was
focused on an area that was printed with PEI but was not being wetted by the solution one of two
things would happen: either the signal would not change when the solution in the flow cell was
changed, and the random fluctuations that accompany a change in solutions would also not be
apparent, or the signal with water in the flow cell would be very high and the signal with
polymer solution in the flow cell would be very low. An example of this is shown in figure 4.17.
49
Figure 4.17 An example of the signal when a large air bubble is in front of the grating.
Each time water is added to the flow cell the signal spikes and does not reach a stable level.
Upon addition of a polymer the signal decreases sharply and levels out. This behavior can be
explained by observing the flow cell under an optical microscope with a polarizing contrast
filter. A flow cell that has been stamped with PEI but not exposed to any polymer solution is
placed in the field of view. The diffraction grating is not visible. Upon addition of water to the
flow cell a large air bubble can be seen. Within a few seconds smaller beads of moisture can be
seen growing in lines. Although the grating is not visible the beads of moisture are growing in
the spacing that indicate they are selectively growing on the diffraction grating. After
approximately a minute the beads of moisture do not appear to the eye to be growing larger or
anymore numerous. When the solution in the flow cell is changed to a polymer solution the
beads of moisture disappear. This reversal is seen for each change of solution. Figure 4.18
shows a flow cell that has had one bilayer build-up with water in the flow cell. The dark curved
line at the top of the image is the edge of the air bubble. Above that line is water and below it is
the pocket of air. Within that pocket of air small beads of moisture appear as circles. When the
flow cell is filled with water the beads of moisture appear long the lines of the printed diffraction
grating. After a time they stop growing and appearing. Then when the water is replaced with a
polymer solution the beads of moisture disappear. This follows the signal pattern discussed
earlier where the water had a sharp increase in signal value while the polymer solutions always
lead to a decrease. There is a trend with the size and number of the moisture beads following the
number of layers. There is a visible change in the size and number of the beads of moisture from
0
100
200
300
400
500
600
0 200 400 600 800 1000 1200 1400 1600
Pe
rce
nt
inc
rea
se
Time (seconds)
Example of signal for air bubble
air
water
PSS
PDDA
50
the first time water is introduced to the flow cell to when water is added after having 2.5 bilayers
added to the flow cell.
Figure 4.18 Beads of moisture in a large air bubble a flow cell. The flow cell has 1 bilayer
built up and water is filling the flow cell.
Figure 4.19 shows the flow cell filled with water after 2.5 bilayers have been built up. After 2.5
bilayers water is in the flow cell the beads of moisture are much smaller and more numerous. It
also becomes apparent that there are two sizes of lines in the diffraction grating. The beads of
moisture grow in the small channels( size 1.5um ) as opposed to only being visible in the
large(4.5 um) channels. Both of these trends, smaller beads of moisture and more of them, lead
to a denser coverage of the diffraction grating which explains the increase in signal as the
number of bilayers of polymer increase. Due to the increase in moisture beads as the PEM is
built-up it is plausible to say that the beads of water are forming on the peaks of the diffraction
grating as opposed to the troughs.
51
Figure 4.19 Beads of moisture in a large air bubble a flow cell. The flow cell has 2.5 bilayers
built up and water is filling the flow cell.
How this system works was not completely characterized. Although it is apparent that the
polymers affect the grating that the beads of moisture are forming on, it is not apparent if or how
the polymers are adsorbing on that surface. The change in the formation of the beads of moisture
as more bilayers are deposited suggests that the diffraction grating in the air bubble is affected,
but how the polymers would reach the grating is not apparent.
These large air bubbles that lead to odd data were consistently problematic until I started using a
more stringent cleaning procedure for all materials involved with making the flow cells.
52
4.7.2 Flow cell assembly
Assembling the flow cell had a few issues to remediate: the flow cell would leak at the bottom
edge where the double-sided tape was supposed to seal the cell. This interfered with collecting
data as the solution would leach into the contact area between the prism and the flow cell. When
this happened there would be visible dots and smudges passing through the signal, leading to
noise and difficult to interpret data. The leaking polymer solution would also adhere to the prism.
In order to prevent the flow cell from leaking the first thing I tried was applying more pressure
when assembling the flow cell. This improved the contact between the tape and the glass slides
which decreased the leaks, but also increased the frequency of large air bubbles. I then switched
to using two strips of tape stacked on top of each other. When using two strips of tape using
more pressure on the flow cells did not increase the frequency of air bubbles forming in the flow
cells. Being able to apply more pressure with out an increase in large air bubbles lead to less
frequent leaks of the flow cell.
Although improving the seal on the flow cell decreased how dirty the prism got, the prism still
needed to be cleaned frequently to improve the signal to noise ratio. First the prism would be
cleaned with hexanes in order to remove the oil used to couple the cells to the prism. This was
not sufficient to completely clean the prism of polymers that inevitably leak from the flow cell
and deposit on the prism. The other thing that had to be cleaned from the prism were traces of
permanent marker used to aligned the stamped area of the glass to the flow cell. When the flow
cell is coupled to the prism traces of the permanent marker will transfer to the prism. The
hexanes do not remove the traces of permanent marker from the prism. In order to clean the
polymers and marker from the prism hydrochloric acid was used. After being rinsed with water
the best results were obtained if the prism was allowed to dry overnight. If dried with nitrogen
gas and used immediately the signal will be full of noise.
Conclusions
A diffraction based set-up was examined for its ability to monitor the build-up of a PEM film by
observing its signal response under a variety of conditions. PEMs assemble at surfaces through
sequential adsorption of alternately charged polymers. Each polymer layer adds mass and height.
First, the system was used in 0 M NaCl solutions to monitor the build-up of 4 bilayers. The
53
signal correlated with the expected behavior, with each layer increasing the signal. The
adsorption curve was unique to each polymer and signal increase with each adsorption
diminishes as the number of layers increase. When the same procedure was carried out in 0.1 M
NaCl and 0.5 NaCl the layers were thicker as inferred from the larger signal increase which is
explained by the globular conformation of polymers in salt solutions. The effect of polymer
concentration was also tested. As expected, an increase in the PDDA concentration did lead to a
thicker PDDA layer, but it also lead to an increase in the thickness of PSS layers. The adsorption
curves with a high concentration PDDA was a different shape for both of the polymers compared
to the adsorption behavior in the low concentration PDDA. The PSS layers did not decrease in
thickness as the layers increased and the PDDA had a more pronounced overshoot.
At the moment this diffraction technique can provide qualitative data but in order to get
quantitative data, the reproducibility of values has to be addressed. First, the cause of the
variation in signal values for the same number of layers has to be found. One source could be the
PEI stamping, which is done by hand. Another could be the hand-assembled flow cells, which
varied slightly in size. Once the reproducibility of values has been addressed, the data from
diffraction sensing should be compared to AFM data in order to produce a standard.
A comparison with AFM data would be useful to determine how the PEM on the diffraction
grating is actually growing. As discussed previously, it is not known whether the PEM will fill in
the troughs of the grating or not. If the troughs do backfill what is the source of the diffraction?
Seeing as the diffraction spots do not move in space the diffraction pattern printed is still the
pattern causing diffraction, but how?
Polyelectrolyte multilayers are part of a thriving field of research due to their multiple
applications and the ease of tailoring to specific uses. Diffraction based sensing has been
investigated as an analytical tool for PEMs. The signal from diffraction was shown to correlate
with well-known trends for layer build-up, salt concentration, polymer concentration, and
swelling. Further research needs to be done before diffraction based sensing can be used for
quantitative analysis but it is a promising tool.
54
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