# Study and Optimization of Self-Assembled Polymeric Multilayer Structures with Neutral Red for pH Sensing Applications

Authors: Javier Goicoechea; Francisco J. Arregui; Jesus M. Corres; Ignacio R. Matias
Journal: Journal of Sensors
 (2008)

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## Abstract

The characterization of nanostructured thin films is critical in the design and fabrication of optical sensors. Particularly, this work is a detailed study of the properties of layer-by-layer electrostatic self-assembled multilayer (LbL) structures fabricated using poly(allylamine hydrochloride) (PAH) and Neutral Red (NR) as cations, and poly(acrylic acid) (PAA) as polyanion. These LbL films, due to the colorimetric properties of the NR, are suitable for sensor applications such as pH sensing in the physiological range. In the (PAH+NR/PAA) LbL structure, it has been observed a very important influence of the pH of the solutions in the properties of the resultant films. Different techniques such as spectroscopy and atomic force microscopy (AFM) are combined to characterize the films, and the results are analyzed showing coherence with previous works. The LbL structure is finally optimized and dramatically improved nanostructured films were fabricated, showing good sensing properties, short response times, and good stability.


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## Body

## 1. Introduction

Optical sensing is
becoming a real alternative to the conventional sensors in some applications
because of their important advantages as electromagnetic immunity,
biocompatibility, multiplexing, miniaturizing capability, and so forth.
Nanostructured thin-films have demonstrated to be one of the most promising
approaches for optical sensor solutions as far as it is possible to tailor
their properties and sensitivity, and they can be fabricated onto a lot of
optical instruments, for example, lenses, prisms, even optical fibers. One
powerful feature of nanostructured materials is the tunability of the
molecular-scale composition, leading to new composite materials with very
interesting properties resulting from a smart design. In fact, the number of
scientific publications and patents in this topic has risen exponentially since
1985 [1]. Several techniques are commonly used in the fabrication of these
nanostructured thin films, like CVD [2], thermal vacuum deposition [3],
spin-coating [4], Langmuir-Blodgett (LB) technique [5], layer-by-layer electrostatic
self-assembly method (LbL), and so forth. More specifically, the LbL technique,
first reported by Decher [6], is a very versatile tool useful in creating multilayer
structures in the nanometric range. With this technique it is possible to tune
the composition of the layers at molecular scale, fabricate interesting
composite materials in a simple and inexpensive way onto substrates composed by
almost any material and with almost any size or shape. This technique has been
successfully demonstrated in many applications such as conductive coatings [7], antireflective coatings [8],
sensitive coatings [9–14], organic light emitting diodes [15],
photovoltaic cells [16], and so forth.In the past, several
approaches have been reported for pH optical sensing like the use of lenses or
optical components coated with pH indicators [17], sol-gel matrices with fluorescent
dyes [18], absorbance pH indicators [19], or polystyrene microspheres [20, 21], just
to give some examples. This study is focused on the characterization of
polymeric LbL multilayer coatings for optical fiber sensors. These LbL sensors
offer some advantages like high quality of the optical films, reproducibility,
miniaturization of the probes, fast response, biocompatibility, and dynamic
range within the physiological range. These properties make them suitable for
in vivo applications, for example. This work is an optimization of the
properties of LbL films fabricated using poly(allylamine hydrochloride) (PAH)
as polycation and poly(acrylic acid) (PAA) as polyanion, adding to this
polymeric matrix neutral red (NR), a cationic pH indicator. These LbL films
follow the structure PAH+NR/PAA. Previous works of Rubner and his group on the
PAH/PAA LbL multilayer films were used as reference [22–25]. The addition of a
third material, NR, during the self-assembly process modifies the properties of
the LbL films, so the resultant films are suitable for sensing applications in
the physiological range, as it was experimentally demonstrated in [13]. Moreover,
some fabrication parameters have a dramatic influence in the thickness and
morphology of the LbL films, for example, the immersion time [26], the
temperature [27], the polyelectrolyte concentration, and ionic strength of the
solutions [28], the pH of the solutions when working with weak polyelectrolytes
[23], and so forth. The aim of the present work is to characterize the
resultant PAH+NR/PAA LbL films, and to find optimal experimental conditions for
achieving the higher concentration of the pH indicator, NR, in the sensitive
films.The experimental study involves several steps that will be communicated below.
In a first approach of the analysis, it is important to know the pKa value of
the materials involved in the LbL process in order to understand how the
molecules behave during the assembly process. Afterwards, some LbL films were
fabricated at different pH values and their thickness and composition were
measured in order to characterize the PAH+NR/PAA structures. The discussion is
structured into two main parts: the first one is an AFM study of the films as
the pH of the solutions is varied, which gives information about the thickness
and roughness of the films, and the second part is a spectroscopy study of the same
films which give information of their composition. Finally, the analysis of the
experimental results will lead us to an optimization of the NR concentration of
the LbL films. To our knowledge this is the first experimental study performed
to optimize this kind of polymeric structure towards sensing applications.


## 2. Experimental

### 2.1. Materials

The polyelectrolytes
in this work are poly(allylamine hydrochloride) (PAH) as the polycation and
poly(acrylic acid) (PAA) as the polyanion. The active dye used in this work is neutral
red (NR). All the chemicals were purchased from Sigma-Aldrich Quimica (S.A.)
and were used without any further purification. Since NR is a cationic small
molecule, it will be added to the polycationic solution (PAH+NR) rather than to
the PAA to avoid aggregation with the polyanion, which could led to
inhomogeneous layers.


### 2.2. LbL Process

Dilute aqueous
solutions of PAH, NR, and PAA (10−2M based on the repetitive unit
molecular weight) were prepared. Afterwards, the PAH and NR solutions were
mixed so the molar ratio of NR is fixed to a 6.5% respect to the PAH. The pH
value of the solutions was adjusted to the desired value by adding a few drops
of HCl or NaOH. The pH of the polyelectrolyte solutions was varied throughout
all the experiments, sweeping from 2.5 to 9.5, keeping the pH of both
polycationic and polyanionic solutions equal in each experiment. For pH values
lower than 2.5 and higher than 9.5 the polymers are not stable and the final
films showed serious flaws and inhomogeneities. The substrates used in this
work are standard glass microscope slides with a pretreatment of immersion in a
solution of H2SO4:H2O2(7:3 vol.)
and afterwards thoroughly rinsed in ultrapure water. Then the substrates were
immersed for 2 minutes in the polyelectrolyte solution, they were rinsed in two
separate beakers of ultrapure water for 1 minute each, and afterwards the same
protocol was performed using the counter-polyelectrolyte solution, completing a
bilayer (a polycation/polyanion layer block). The final structure can be
written as (PAH+NR/PAA)n, where n is the number of bilayers. For
this experiment, 25 bilayers were chosen unless otherwise stated. The
fabrication of the multilayer films was carried out using a 3-axis cartesian
dipping robot (R&K GmbH) with the dipping protocol detailed before.


### 2.3. Thickness and Roughness Measurements

An AFM digital
instruments NanoScope IIIa Multimode in tapping mode was used. The thickness
measurements were performed making a scratch in the films with a razor blade,
and then measuring the depth of the line. The roughness of the films was also
measured with the AFM in tapping mode, with silicon cantilever (RTESP, Veeco)
in a region near the central zone of the sample to avoid border effects.


### 2.4. Absorbance Measurements

The absorbance spectra of the films were measured using a typical transmission
fiber optic setup with an OceanOptics DH-2000-S white light source (from 200 nm
to 1500 nm), and an OceanOptics USB4000 Spectrometer.


## 2.1. Materials

The polyelectrolytes
in this work are poly(allylamine hydrochloride) (PAH) as the polycation and
poly(acrylic acid) (PAA) as the polyanion. The active dye used in this work is neutral
red (NR). All the chemicals were purchased from Sigma-Aldrich Quimica (S.A.)
and were used without any further purification. Since NR is a cationic small
molecule, it will be added to the polycationic solution (PAH+NR) rather than to
the PAA to avoid aggregation with the polyanion, which could led to
inhomogeneous layers.


## 2.2. LbL Process

Dilute aqueous
solutions of PAH, NR, and PAA (10−2M based on the repetitive unit
molecular weight) were prepared. Afterwards, the PAH and NR solutions were
mixed so the molar ratio of NR is fixed to a 6.5% respect to the PAH. The pH
value of the solutions was adjusted to the desired value by adding a few drops
of HCl or NaOH. The pH of the polyelectrolyte solutions was varied throughout
all the experiments, sweeping from 2.5 to 9.5, keeping the pH of both
polycationic and polyanionic solutions equal in each experiment. For pH values
lower than 2.5 and higher than 9.5 the polymers are not stable and the final
films showed serious flaws and inhomogeneities. The substrates used in this
work are standard glass microscope slides with a pretreatment of immersion in a
solution of H2SO4:H2O2(7:3 vol.)
and afterwards thoroughly rinsed in ultrapure water. Then the substrates were
immersed for 2 minutes in the polyelectrolyte solution, they were rinsed in two
separate beakers of ultrapure water for 1 minute each, and afterwards the same
protocol was performed using the counter-polyelectrolyte solution, completing a
bilayer (a polycation/polyanion layer block). The final structure can be
written as (PAH+NR/PAA)n, where n is the number of bilayers. For
this experiment, 25 bilayers were chosen unless otherwise stated. The
fabrication of the multilayer films was carried out using a 3-axis cartesian
dipping robot (R&K GmbH) with the dipping protocol detailed before.


## 2.3. Thickness and Roughness Measurements

An AFM digital
instruments NanoScope IIIa Multimode in tapping mode was used. The thickness
measurements were performed making a scratch in the films with a razor blade,
and then measuring the depth of the line. The roughness of the films was also
measured with the AFM in tapping mode, with silicon cantilever (RTESP, Veeco)
in a region near the central zone of the sample to avoid border effects.


## 2.4. Absorbance Measurements

The absorbance spectra of the films were measured using a typical transmission
fiber optic setup with an OceanOptics DH-2000-S white light source (from 200 nm
to 1500 nm), and an OceanOptics USB4000 Spectrometer.


## 3. Results and Discussion

As it has been previously introduced, the degree of ionization of the
polyelectrolytes depends on the pH of the surrounding medium. This degree of
ionization affects the proportion of free ionic binding sites, and consequently
to the morphology of the polymer chains.
Therefore, the thickness and roughness of the LbL films are strongly
dependent on the pH of the polyelectrolyte solutions [20–25]. In the present
work, the self-assembly of PAH and PAA processes is altered by the addition of
the neutral red (NR) to the polycationic solution. The thickness results of the
(PAH+NR/PAA)n structure were studied to  characterize the films, and
the previous work with (PAH/PAA)n reported by Shiratori and Rubner [23] was
taken as reference in the following analysis.In the PAH/PAA system, it has been demonstrated [20–25] that the
thickness of the bilayers is affected not only by the conformation of the
adsorbing polyelectrolyte, but also by the density of electrostatic charge and
the thickness of the previous layer. Therefore, there is a complex interaction
between the two-weak polyelectrolytes depending on the pH value of the
solutions. In the analysis of the PAH/PAA system, the pKa of the
polyelectrolytes is a key factor because it gives information about the degree
of ionization of the electrolyte. As reported in [25] the pKa of PAH is
approximately 8.8, and the PAA has a pKa of 6.5. That means that PAH will be
fully ionized at low pH values (lower than pH 6) and completely de-ionized at
pH 12. In the case of the polyanion, a reverse behavior is observed; the PAA is fully
ionized at high pH values (pH 9.5 or pH 10), and almost completely deionized at
pH 4. As the pH value of polyelectrolyte solutions is varied, different
incremental thicknesses per bilayer are achieved. The (PAH/PAA) bilayer
thickness versus pH plot shows a characteristic shape [23], with a relative
maximum around pH 4.5, followed by a dramatic decrease in thickness between pH
6 and pH 7.5, and then another drastic increase for pH values higher than pH 8.
The thicker films occur when one of the polyelectrolytes is not completely
ionized while the counter-polyelectrolyte is in its fully ionized form. On the
other hand, when both polyelectrolytes are almost fully ionized, the thinnest
films were achieved.In this work, the PAH is mixed with NR, so during the immersion of the
substrate into the positive solution, both PAH and NR will compete for the free
binding sites in the PAA. It is very important to know the pKa value of the NR
in order to analyze the role that NR plays in the new PAH+NR/PAA system. In
order to calculate the pKa of NR, a similar procedure as in [25] is adopted and
the absorption spectrum of NR diluted in water at different pH values is
measured, see Figure 1.Figure 1
Absorption spectra of NR solutions at different
pH values.In this plot, there is three remarkable points, around 450 nm and 525 nm where
the basic and acid species absorption peaks are around 470 nm where the
isosbestic point is located. From these spectra, the deprotonated and
protonated species of NR were taken as references for the absorbance
measurements, thus the absorbance at 450 nm and 525 nm was taken for calculating
the degree of ionization as is described below. We considered that at very low
pH values (pH 2.5), the NR molecules were fully charged, whereas at very high
pH values (pH 9.5) the NR is completely deprotonated. Then, the ratio(A525nm/A450nm)
at pH 2.5 is taken as the 100% ionization rate, and   (A525nm/A450nm)
at pH 9.5 is taken as the 0%. Therefore the degree of ionization (DI) is
defined according to [25]
(1)DI=(A525nm/A450nm)pH−(A525nm/A450nm)pH=9.5(A525nm/A450nm)pH=2.5−(A525nm/A450nm)pH=9.5×100%.Figure2 displays the degree of ionization versus the pH of the NR
solution, and the pKa can be calculated as the pH in which the NR is half
ionized, giving an approximate value of pKa = 6.5. This value of pKa is
consistent with  other values found in the bibliography [29]. According  to the
weak electrolyte nature of the NR and its pKa value, it is predictable that it
would be more likely to be adsorbed in a LbL process at pH values under 6.5,
because in more acidic solutions it would be in its protonated state, and
consequently it would show affinity for the free negative binding sites
available at the PAA. This hypothesis is confirmed by the experimental data, as
will be shown later.Figure 2
Ionization degree of the NR in aqueous solution
versus the pH.After this previous study about the pKa of NR, the (PAH+NR/PAA)n coatings were built on microscope glass slides and the pH of the cationic and
anionic solutions used for the fabrication of the films was varied from 2.5 to 9.5 in steps of 0.5 pH
units. Then, in order to calculate the amount of NR molecules added to the LbL
films, the resultant thickness of the (PAH+NR/PAA)n films were studied
by AFM and spectroscopy.In Figure3, AFM pictures of two different LbL assemblies fabricated at
pH 2.5 and 3.5 are displayed as an example of the results  obtained. From these
results it can be deducted that  the roughness of the resultant layers can also
be optimized by simply varying the pH of the solution, which is critical in
certain applications as in long period grating (LPG) based on nanostructured
coatings [30]. The experimental results of the AFM analysis are shown in Figure 4, where it is displayed the evolution of thickness and roughness as the pH of
the fabrication pH is varied. This useful information allows us to determine
the optimal conditions of thickness or roughness for the final sensitive films.
Nevertheless, in this particular application, the focus is placed in the
thickness of the multilayer coatings rather than in their roughness as far as
the concentration of NR respect to the polymeric matrix is being optimized. The
thickness versus pH profile shown in Figure 4 is very similar to the (PAH/PAA)n
structure reported by Shiratori and Rubner in [23] due to the weak polyelectrolyte nature of
the materials involved in the LbL process.
However, some important differences are found here with respect to that
structure. In the (PAH+NR/PAA) structures there is a relative thickness maximum
around pH 5.5 which is slightly right-shifted compared to the (PAH/PAA)
structure [23]. In addition, a
thickness minimum region between pH 6.5 and pH 7.0, but layers, are thicker
than that in the (PAH/PAA) case. This is logical since the presence of NR
implies a higher ionic strength of the polyionic solutions and, therefore a
higher thickness of the resulting coatings [28].AFM pictures of two (PAH+NR/PAA)25 structures
built up at different pH values.
(a)(b)Figure 4
Left: the bars show the bilayer thickness as
the pH of the polyelectrolyte solutions was varied. Right: the dotted line is
the square roughness of the surface of the (PAH+NR/PAA)25.The spectroscopy study performed on the films is shown in Figures5 and
6. Figure 5 displays the absorbance spectra of different LbL films as the pH
value of the polyelectrolyte solutions is varied. This plot shows that the
absorption spectra of the films from pH 6 to pH 9.5 are almost flat, so the
films are very transparent, revealing no presence of NR in the films. From pH
2.5 to pH 5.5 the spectra show a clear peak around 520 nm, which corresponds to
the protonated NR absorption peak. A detailed observation of these spectra
shows a slight displacement of the NR peak to lower wavelengths as the pH value
is increased from pH 2.5 to pH 5.5. This left shift of the peak is due to the
color change of the NR indicator itself as consequence of the increasing of the
pH. As far as the objective of this spectroscopic study is to calculate the
amount of NR contained in the LbL films, the absorbance at the isosbestic point
of the NR spectrum (around 470 nm) is taken as indicator in order to avoid any
interference of the peak shift. In Figure 6 it is shown the evolution of the
absorbance at 470 nm as the pH of the solutions is varied which gives an idea of
the amount of NR in the polymeric films. This absorption at the isosbestic
point of NR showed in Figure 6 suggests that NR molecules are being
incorporated to the LBL structure only at low pH values of the solutions, which
hold well with the hypothesis previously formulated keeping in mind that NR has
a pKa around 6.5.Figure 5
Absorption spectra of (PAH+NR/PAA)25 fabricated
with polyelectrolyte solutions at different pH values.Figure 6
Comparison between the average bilayers
thickness and the absorbance at 470 nm. This comparison shows evidence that the
composition of the (PAH+NR/PAA)25 changes as pH is increased.The absorption at the isosbestic point of the NR gives information about
the total amount of NR contained in the films, regardless of their thickness.
It has to be noticed that the film which presents a highest absorbance at 470 nm
is not the thickest one. Comparing the data shown in Figures4 and 6, it can be
seen that the maximum thickness in Figure 4 happens at pH 5.5, while the maximum
absorbance in Figure 6 is at pH 4.5. This is because a thicker bilayer does not
imply  necessarily a higher adsorption of NR and it is due to the difference
between the pKa values of the PAH and the NR. Taking as starting point pH 2.5
and increasing the pH, the number of binding sites in the PAA is increased,
leading to thicker layers. In this pH range the PAH and the NR compete for the
charged sites in the PAA. The NR molecules probably due to their size seem to
be more likely to be adsorbed onto the PAA leading to slightly thinner layers
with high concentration of the dye. When the pH reaches values higher than 4.5,
the degree of ionization of NR molecules drops abruptly (see Figure 2). Consequently, the concentration of NR in respect
to the PAH decreases while PAH is still almost fully ionized and it still forms
thick layers. The result is a variation in the NR richness of the resultant
film. In order to have a measurement of the NR adsorbed per unit of volume it
is necessary to calculate the ratio between absorption at 470 nm and the
thickness of the film. This ratio is equivalent to the linear absorption
coefficient (α) at 470 nm, defined as
(2)α470nm=Absorbance470nmTotalFilmThicknessavrg.Figure7 displays the evolution of the α470nm of the films as
the pH of the polyelectrolyte solutions is varied. The maximum of this ratio is
the optimal film composition, as far as it contains the highest concentration
of NR per unit of volume, and this happens at pH 4.0. The experimental data
collected from the films built-up at different pH value are summarized in Table 1. This complete characterization of the films, taking into account not only
the total absorbance but also the thickness, is very important in the design of
many optical devices like filters or optical fiber sensors where both the
thickness of the layers and the concentration of the active molecules (NR in
this case) are necessary in order to achieve a proper design [13].Table 1
Measurements of the thickness, roughness, and absorbance at 470 nm. The absorption coefficient at 470 nm was calculated from these data.
pH of the solutionsBilayer thickness (nm)Surface roughness (nm,  rms)Absorbance at 470 nm (a.u.)Absorption coefficient  (nm−1)2.55.54.120.2460.000893.04.94.760.3460.001413.54.86.360.4250.001764.06.86.720.6290.001854.514.55.241,1790.001635.026.72.611,0790.000815.535.21.480.5790.000336.027.00.740.0420.000036.59.72.040.0030.000017.06.41.060.0590.000117.56.61.640.0360.000118.014.80.620.0550.000078.532.30.340.0340.000029.056.60.310.1080.000049.582.80.380.1570.00004Figure 7
Absorption coefficient at 470 nm versus pH of
the polyelectrolyte solutions. A maximum concentration of NR is found for the
films built-up at pH 4.0.In order to corroborate that the NR indicator still kept its sensing
properties once embedded in the polymeric matrix, the (PAH+NR/PAA) optimized
coatings were immersed into different pH buffer solutions and a dramatic change
of color was observed, as is displayed in Figure8. In fact, the coatings
showed an optical response between the acid state (red) and the basic state
(yellowish) with fast response times, in the order of just a few seconds.Figure 8
Micrograph of a (PAH+NR/PAA)25 coating fabricated
at pH 4 partially immersed into pH 3 (left) and pH 7 (right) buffer solutions.
Change of color of the coating is fast (few seconds) and reversible.Finally, some fiber optic sensor was fabricated and tested. The aim of this test is only to prove the good sensing properties of
the optimized LbL films in a real application, but the sensor setup is not
optimized and its performance can be enhanced in future works. The sensor
multilayer structures were built-up onto the end-face of cleaved standard
telecommunication multimode optical fibers (62.5 and 125 microns of core and
cladding diameter, resp.) following the procedure described in the
experimental section, and using a pH value of the polyelectrolyte solutions of 3,
4, and 7 (noted as S01, S02, and S03, resp.). Afterwards the tip of the different
fibers was immersed into pH 4 and pH 7 buffer solutions and the reflected
optical power was registered using a CCD spectrometer. In Figure9, the dynamic
response of the three different sensors is displayed. This figure clearly shows
how the sensibility and stability of the sensitive LbL coating can be optimized
when the nanostructure properties are properly adjusted. Figure 10 shows the
optical response from the optical fiber sensor fabricated at pH 4. As it can be
seen, the spectral response of the sensor shows a broad absorption band around
520 nm when it is immersed in pH4, that vanishes when the pH is changed to pH7, corresponding to the typical
behavior of NR. The response of the sensor was found to be fast and very
repetitive, suggesting good stability of the LbL sensing films.Figure 9
Dynamic response of three sensors consisting in
a (PAH+NR/PAA)25 fabricated at three different pH values. S01 was fabricated at
pH 7, S02 at pH 3, and S03 at pH 4. S03 response was found to be the most
sensitive and stable signal.Figure 10
Spectral response of an optical
fiber/(PAH+NR/PAA)25 sensor fabricated at pH 4.


## 4. Conclusions

PAH+NR/PAA LbL coatings was studied and optimized for pH sensing. As far
as all the materials are weak electrolytes, the pH of the aqueous solutions
used for building up the films plays a key role in the final properties of the
assemblies. Some conclusions about the
interaction of the molecules during the LbL process have been extracted by combining spectroscopic measurements with AFM morphological analysis of the films.	 It was found that the concentration of NR molecules adsorbed
into the LBL films and the bilayer thickness as well strongly depends on the pH
of the polyelectrolyte solutions used for the fabrication of the coatings. An
optimization of the absorption linear coefficient was performed achieving a
maximum when the pH of the polyelectrolyte solutions is fixed to 4.0. Therefore,
in order to obtain sensitive PAH+NR/PAA LbL coatings with a maximum adsorption
of NR, the nanostructured films have to be fabricated at pH4.



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*Source: 142854-2007-11-11.xml*