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# I>In Situ Spectroscopic Ellipsometry Study of Protein Immobilization on Different Substrates Using Liquid Cells

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The influence of substrate materials on protein adsorption was studied by spectroscopic ellipsometry (SE) and atomic force microscopy. For model proteins fibrinogen and flagellar filaments were chosen and their kinetics of adsorption, surface coverage and adsorbed amount on virgin and chemically activated SiO(2) and Ta(2)O(5) thin films were investigated. In case of flagellar filaments the SE data were analyzed with an effective medium model that accounted for the vertical density distribution of the adsorbed protein layer. Adsorption was measured in situ using flow cells with various fluid volume. Compared to commercially available cells, a flow cell with significantly smaller volume was constructed for cost-effective measurements. The development of the flow cell was supported by finite element fluid dynamics calculations.
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RESEARCH ARTICLE
Printed in the United States of America
SENSOR LETTERS
Vol. 8, 1–6, 2010
In Situ Spectroscopic Ellipsometry Study of
Protein Immobilization on Different Substrates
Using Liquid Cells
Andrea Németh1, Péter Kozma12, Tímea Hülber1, Sándor Kurunczi1, Róbert Horváth1,
Péter Petrik1, Adél Muskotál2, Ferenc Vonderviszt12, Csaba H˝
os3, Miklós Fried1,
József Gyulai1, and István Bársony1
1Hungarian Academy of Sciences, Research Institute for Technical Physics and Materials Science,
P.O. Box 49, Budapest H-1525, Hungary
2Department of Nanotechnology, Research Institute of Chemical and Process Engineering, Faculty of
Information Technology, University of Pannonia, Veszprém, Hungary
3Department of Hydrodinamic Systems, Budapest University of Technology and Economics, Budapest 1111, Hungary
(Received: xx Xxxx xxxx. Accepted: xx Xxxx xxxx)
The inﬂuence of substrate materials on protein adsorption was studied by spectroscopic ellipsom-
etry (SE) and atomic force microscopy. For model proteins ﬁbrinogen and ﬂagellar ﬁlaments were
chosen and their kinetics of adsorption, surface coverage and adsorbed amount on virgin and chem-
ically activated SiO2and Ta2O5thin ﬁlms were investigated. In case of ﬂagellar ﬁlaments the SE
data were analyzed with an effective medium model that accounted for the vertical density distri-
bution of the adsorbed protein layer. Adsorption was measured in situ using ﬂow cells with various
ﬂuid volume. Compared to commercially available cells, a ﬂow cell with signiﬁcantly smaller volume
was constructed for cost-effective measurements. The development of the ﬂow cell was supported
by ﬁnite element ﬂuid dynamics calculations.
Keywords:
1. INTRODUCTION
Protein adsorption at liquid/solid interfaces is funda-
mental for several diverse areas of biotechnology, e.g.,
biosensing.1–4 In situ measurement of adsorption and
immobilization of proteins in liquid cells is a complex task
inﬂuenced by numerous parameters.5–8 Most of these fac-
tors (like temperature of the cell, purity of water, rate of
injection) are known to have little effect on the repeatabil-
ity of the experiment.5On the other hand, there are various
possible causes of uncertainty that need to be investigated
and as far as it is possible the experimental system needs
to be insulated from these undesired inﬂuences. Our pri-
mary aim is to increase our modeling and characterization
knowledge (thickness, surface coverage and kinetics) of
the adsorbing protein ﬁlms. By monitoring in situ pro-
tein deposition we have more insight into the mecha-
nisms of protein layer formation. The adsorbed proteins
Corresponding author; E-mail: nemetha@mfa.kfki.hu
can be applied for instance as speciﬁc receptors for ligand
binding.34The ﬁrst protein applied in our experiments
was ﬁbrinogen (Fgn), a model protein that is compact but
large enough to perform optical measurements using SE.
Fgn was selected as one of the plasma proteins, since these
(HSA, Fgn, IgG, etc.) are well-characterized model and
test proteins.910 The second protein we studied was the
ﬂagellar ﬁlament (FF). The bacterial ﬂagella are tail-like
structures responsible for locomotion. Their extracellular
parts are the FFs. Every FF is composed of thousands of
protein subunits, called ﬂagellin, by polymerization con-
stituting 5–20 m long helical ﬁlaments, the diameter of
which is 23 nm. By genetic engineering or directed evo-
lution, speciﬁc binding sites can be created in the central
parts of ﬂagellin subunits which form the outer surface
of the ﬁlament. Flagellar ﬁlaments polymerized in vitro
from these receptor subunits are stable structures with very
high binding site density on their surface.11–13 Flagellin
(molecular weight 51.5 kDa) was gained from Salmonella
typhimurium wild type strain SJW1103 and puriﬁed as
Sensor Lett. 2010, Vol. 8, No. 5 1546-198X/2010/8/001/006 doi:10.1166/sl.2010.1338 1
RESEARCH ARTICLE
In Situ Spectroscopic Ellipsometry Study of Protein Immobilization on Different Substrates Using Liquid Cells Németh et al.
previously reported.14 Due to the procedure of producing
FFs the long ﬁlaments break up to shorter parts with a
length distribution of 300–1500 nm.
In this study, spectroscopic ellipsometry (SE) was
liquid cells with various ﬂuid capacities on activated and
non-activated tantalum pentoxide (Ta2O5and SiO2sub-
strates. Final surface mass densities and kinetics of deposi-
tion were determined and compared for the different cases.
After SE measurements the samples were dried and ana-
lyzed using atomic force microscopy (AFM).
2. EXPERIMENTAL DETAILS
2.1. Spectroscopic Ellipsometry (SE)
The possibility of performing in situ and non-destructive
measurements is one of the major advantages of ellipsom-
etry in life sciences.161720 Ellipsometry detects changes
in polarization of an electromagnetic wave reﬂected from
the sample. A Woollam M2000DI multichannel rotating
compensator ellipsometer was used with different ﬂow-
through cells for in situ investigations. The ﬁxed angle of
light incidence was 75for both cells, a value close to the
Brewster’s angle of silicon.
Typical measured and ﬁtted (CompleteEASE, Woollam
Co.) (, spectra are shown in Figure 1 as a func-
tion of wavelength (). In our optical models, additionally
to the refractive index Refs. [20, 21], effective medium
approximation (EMA) and Cauchy dispersion function
were used.22
2.2. Sample Preparation
Thermally oxidized silicon substrates were used, and
onto half of the sample pieces a 190 nm thick Ta2O5
Fig. 1. Typical measured and ﬁtted ellipsometric spectra over the wave-
length range of 360–1000 nm in the case of silicon substrate with thermal
oxide and Ta2O5ﬁlm. The spectra were recorded during in situ SE mea-
surement in the small ﬂow cell during PBS ﬂow.
ﬁlm was evaporated. Before the measurements all wafers
were immersed for 10 minutes in “Piranha” solution (1:3
mixture of 30% hydrogen-peroxide and concentrated sul-
furic acid), rinsed with Milli-Q water and dried by nitro-
gen blow. In order to compare activated and non-activated
surfaces only half of the sample pieces with SiO2and
Ta2O5were activated with 3-aminopropyl-triethoxy-silane
(APTES) and glutaraldehyde crosslinker (GA).52324 Due
to the silanization, the surface changed from hydrophilic
to hydrophobic.25–27
2.3. Flow Cell Design with Ultra-Small Capacity
The capacity of the commercially available ﬂow cell of
Woollam Inc. is 5 ml. In order to minimize the amount
of the solutions needed, we designed a smaller liquid cell
with the capacity of 0.2 ml. Reducing the sample volume
has two major advantages. First, the time duration of the
transient when switching from pure buffer to protein solu-
tion can be reduced, allowing a more detailed characteri-
zation of the initial phase of the layer build up. Secondly
the measurements are much more cost-effective (smaller
amount of proteins and chemicals are needed), which is
important for several expensive proteins and reagents. The
properties of both ﬂow cells are compared in Table I.
During the design process several important aspects had
to be considered,17 like the constructible lowest capacity,
applicable ﬂow rate, and ﬂow cell’s UV-grade window
properties. The effect of ﬂow rate on the interaction
between surface and macromolecules had to be analyzed
to determine whether the ﬂow is laminar or turbulent
within the liquid cell. On the account of the complexity
of the ﬂow cell geometry Computational Fluid Dynamics
(CFD) by the commercial software ANSYS CFX 11.0 was
applied. Numerical simulations were performed to study
water ﬂow dynamics in the small liquid cell at 25 C. The
analysis was the following:
(1) computational model of the ﬂow ﬁeld was constructed,
(2) the ﬂow ﬁeld was divided into discrete cells,
(3) the boundary conditions were deﬁned and the equa-
tions of ﬂow dynamics were solved for the ﬁnite elements,
(4) the result of the evaluation was visualized by stream-
lines (Fig. 2).
Table I. Comparison of the large and small ﬂow cells.
Attributes Large ﬂow cell Small ﬂow cell
Capacity 5 ml 0.2 ml
Angle of incidence 7575
Delta offset (window effect) Negligible Small
Flow rate 2.5 ml/min Below 0.5 ml/min
Solution amount for 150 ml 30 ml
1 hour (at least)
Windows diameter 12.5 mm 4 mm
Sample size (O-ring) 50 ×13 mm 27 ×7mm
2Sensor Letters 8, 1–6, 2010
RESEARCH ARTICLE
Németh et al. In Situ Spectroscopic Ellipsometry Study of Protein Immobilization on Different Substrates Using Liquid Cells
Fig. 2. Visualization of the ﬂow ﬁeld within the small ﬂow cell rep-
resented by streamlines. The picture shows the results of dynamic ﬂow
simulations in case of 0.5 ml/min volume ﬂow. The liquid ﬂows from the
right-hand side to the left.
2.4. Experiments in Flow Cells
Before any kind of treatments, all of the substrates were
measured by SE in their original state in the wavelength
range over 190–1700 nm. It is important to note that every
time the sample changed, a new ellipsometric spectrum
was taken and this spectrum was analyzed in order to
follow the evolution of the surface and investigate our
procedure. Birefringence of the cell windows can be com-
pensated by ﬁtting the delta offset measuring a reference
sample in the cell without liquid.
Protein deposition was followed in situ by SE. The con-
centration of the dissolved FF and Fgn was 100 g/ml
in 20 mM phosphate buffered saline (PBS: pH 7.4) solu-
tion. A measurement consists of two steps: (1) the cell
is washed through by PBS, and the PBS base-line is
recorded, (2) the protein solution is injected, the protein
deposition proceeded until saturation. The ﬂow rate was
below 0.5 ml/min and 2.5 ml/min in the case of the small
and large cells, respectively.
In order to compare the results, the deposition of the
model protein (Fgn) was injected in both ﬂow cells. Fgn
adsorption181925 was investigated at ﬁrst within the small
and large ﬂow cells to make sure that the extremely low
capacity of the small cell doesn’t affect the mechanism
of the adsorption. After reproducing and comparing the
results of Fgn adsorption in both ﬂow cells, we began to
investigate the FF adsorption in the small cell.
In the test period of the small cell the effect of the
ﬂow rate on Fgn adsorption onto silica wafers was also
investigated. An extremely low (0.13 ml/min) and high
(5 ml/min) ﬂow rate was chosen for the analysis of the
The most relevant information of the deposition of pro-
tein layers is the surface mass density () as a function
of time. is a robust measure (from the thickness and
refractive index at the wavelength of 632 nm) using de
Feijter’s equation eliminating the uncertainties of the deter-
mination of both layer thickness and refractive index.28
The kinetics of protein deposition was quantitatively char-
acterized by ﬁtting exponential functions to the adsorption
curve.29
3. DISCUSSION
3.1. Optical Model
The (, spectra were ﬁtted in the wavelength ranges of
360–1000 nm and 250–1000 nm for depositions on Ta2O5
and SiO2, respectively, due to the absorption of light in
water and in the Ta2O5layer. To construct the most proper
optical model for the multilayer sample structure refer-
ence refractive indices were applied from the literature for
the substrate and for the thermal oxide layer. For ﬁtting
the Ta2O5layer the Cauchy dispersion function was used.
The surface roughness of the deposited Ta2O5ﬁlm and the
APTES, GA layers were ﬁtted commonly with a subse-
quent Cauchy function. The protein layers were modeled
with EMA (single in case of Fgn and mulitlayers for FF)
combining the refractive index of protein (Cauchy) and
ambient (the refractive index of PBS has been determined
by a measurement on a reference wafer in PBS, taking
into account the small dispersion of PBS using a Cauchy
ﬁt). The schematic graph of a multilayer structure with the
typical thickness values is shown in Figure 3.
To ﬁnd a suitable optical model for FF is more compli-
cated, because it does not form a well deﬁned and dense
layer due to the size distribution of the ﬁlaments and their
multiple-binding to the substrate. In contrast to FF, Fgn
adsorbs in a nearly monolayer formation, thus a simpler
Fig. 3. Schematic graph of the measured multi-layer structure (typical
values of thicknesses are given in parentheses).
Sensor Letters 8, 1–6, 2010 3
RESEARCH ARTICLE
In Situ Spectroscopic Ellipsometry Study of Protein Immobilization on Different Substrates Using Liquid Cells Németh et al.
optical model can be constructed. Therefore Fgn was used
as a model protein to test the ﬂow cells.
3.2. Numerical Results and Test of the Small
Flow Cell
At seven different values of volume ﬂow, the ﬂow ﬁeld was
determined by the ﬁnite element calculations. The results
of these calculations indicated that the Reynolds-number
is below 1 (near the surface, below 1 m) in every investi-
gated case and the ﬂow ﬁeld is no longer symmetric above
the volume ﬂow of 0.5 ml/min (mass ﬂow: 8.3 mg/s). Near
the surface, along the adsorbed FFs the ﬂow is laminar.
offset caused by birefringence of the UV-grade win-
dows of the Woollam ﬂow cell was negligible. In case of
the smaller cell, offset was calibrated by a measurement
on a reference sample without liquid.
To compare the cells, Fgn adsorption onto thermally
oxidized silica substrates was investigated within the
smaller and the larger ﬂow cells, simultaneously. The sur-
face mass density of the adsorbed protein layer was found
to be the same for both cases (0.27 g/cm2. Calculating
the surface mass density as a function of time the curves
were ﬁtted by the following exponential function: =−A·
expt/ +0, where Ais the amplitude, tis the time,
and is the time constant. The value was also found to
be the same for both ﬂow cells (1.5 min).
The effect of ﬂow rate on protein adsorption was also
investigated in the small ﬂow cell using silicon substrates
and Fgn. Comparing the values we have quantitative
information about the relation between the ﬂow rate and
the kinetics: the higher the ﬂow rate the faster the kinet-
ics (is smaller) and the lower the ﬁt quality. The results
are represented numerically in Table II.). Note that ﬁtting
the Fgn adsorption curves by double exponential func-
tion resulted in higher ﬁt quality than the ﬁtting of the
single exponential suggesting that probably two processes
(transport limited and adsorption limited) occur during
deposition.3031 In case of FF adsorption ﬁtting by one
exponential function revealed good agreement showing
that the adsorption limitation is the dominant process. The
authors intend to perform more detailed investigations in
Table II. Comparison of the surface mass densities (, the exponential
time constants ( and the correlation coefﬁcients for a ﬁt using one
exponential function (R2, denoting the ﬁt quality) for the ﬂow rates. The
exponential functions were ﬁtted in the same range of time. The values
were determined at the end of the ﬁtted curves, and the saturation was
probably reached later.
[g/cm2][min] R2(ﬁt quality for 1 exp.)
Flow rate Fgn FF Fgn FF Fgn FF
0.1 ml/min 0.33 — 1.83 0.984
0.5 ml/min 0.26 — 1.50 0.983
5 ml/min 0.30 0.74 — 0.924
Fig. 4. Surface mass densities as a function of time for two protein
adsorptions, FF and Fgn. The circles and triangles mark the deposition
of FF and Fgn, respectively. The lines denote the results of the ﬁtted
exponential functions. In case of Fgn double exponential and for FF one
exponential ﬁt was needed for a good agreement.
the future to reveal the theoretical background of the Fgn
3.3. Adsorption onto Activated and Non-Activated
Surfaces
The testing procedure was followed by Fgn and FF adsorp-
tion experiments on activated and non-activated SiO2and
Ta2O5substrates. The adsorbed protein surface mass den-
sity was found to be consistently higher and the satura-
tion is reached later in the case of FF (around 1 g/cm2,
more than one hour) than in the case of Fgn (around
0.35 g/cm2, 20 minutes). A typical measurement can be
seen in Figure 4. The activation has propulsive effect on
Fig. 5. Surface mass densities during FF depositions onto activated and
non-activated silicon substrates, with thermal oxide ﬁlm. The circles and
the triangles mark the increase of surface mass density as a function of
time and the lines denote the ﬁtted exponential functions.
4Sensor Letters 8, 1–6, 2010
RESEARCH ARTICLE
Németh et al. In Situ Spectroscopic Ellipsometry Study of Protein Immobilization on Different Substrates Using Liquid Cells
Table III. Comparison of the surface mass densities (, the exponen-
tial time constants ( and the correlation coefﬁcients for a ﬁt using one
exponential function (R2, denoting the ﬁt quality) for the materials. The
exponential functions were ﬁtted in the same range of time. The values
were determined at the end of the ﬁtted curves, and the saturation was
probably reached later.
[g/cm2][min] R2(ﬁt quality for 1 exp.)
Material Fgn FF Fgn FF Fgn FF
Activated
SiO20.34 0.39 0.83 32 0.902 0.998
Ta2O50.27 0.58 1.62 101 0.940 0.996
Not activated
SiO20.29 0.36a1.58 128 0.991 0.999
Ta2O50.45 0.26a4.18 145 0.972 0.998
aThe optical signal (also the adsorbed amount) increased constantly.
the FF adsorption kinetics. By Fitting the exponential func-
tion mentioned above to (t, the value of Aobtained is
more than two times higher and the value obtained is
more than four times lower in the case of activation. In
other words a faster FF deposition and higher adsorbed
amount was observed (Fig. 5). In case of Fgn this rela-
tion is not so clear. The results for different materials are
compared in Table III.
The increase of phase shift ( as a function of time
during protein adsorption from base-line to saturation was
around 7 degrees for SiO2. In contrast, the phase shift for
Ta2O5increased by 2 degrees from base-line to satura-
tion (at 600 nm). This means that better sensitivity can be
reached in case of SiO2(mostly because using this layer
structure the angle of incidence of 75used in the cell is
closer to the pseudo Brewster angle). The effect of activa-
tion on the adsorbed protein amount seems to be more sig-
niﬁcant for Ta2O5than for SiO2, but further investigations
are needed for better understanding. Without activation the
Fig. 6. AFM image about a non-activated silicon substrate covered by
FF. The protein was adsorbed in the small ﬂow cell. After SE mea-
surement the substrate was dried with nitrogen blow and the image was
recorded by AFM in air.
optical signal increased constantly even after 100 minutes,
while in case of activation a plateau has been reached after
80 minutes.
To reveal the morphology, the samples were measured
in air using AFM after adsorption (Fig. 6). The structure
of the FF layer under buffer is like grass, after drying with
nitrogen blow, the strains lie down. This is in agreement
with previous ﬁnding for shorter ﬁlaments.32
4. CONCLUSIONS
Well-controlled in situ measurements were performed in
a ﬂow cell of extremely low capacity. The dynamic ﬂow
simulations for the small ﬂow cell revealed that the ﬂow is
laminar for the applied ﬂow rates and showed that above
0.5 ml/min the proﬁle of the ﬂow ﬁeld is no longer sym-
metric. The increase of surface mass density of FF was
analyzed during SE measurements using multilayer effec-
tive medium models. Good repeatability of the adsorbed
amount of Fgn was found by subsequent measurements.
The effect of the surface activation for immobilization and
the substrate material was also analyzed, and a higher
adsorbed amount and a faster saturation were observed
in the case of activation. The adsorbed FF layer can be
divided into sublayers with a higher adsorbed amount near
to the surface (100 nm, around 0.5 g/cm2and another
thicker one with lower mass density (around 500 nm,
around 0.3 g/cm2.
Acknowledgments: Support from the Hungarian Sci-
entiﬁc Research Fund (OTKA Nos. K61725, K81842,
PD 73084, and T046238) is greatly acknowledged.
This work was also supported by the European
Commission–Research Infrastructure Action, under the
FP6-Program “Structuring the European Research Area,”
through the Integrated Infrastructure Initiative “Euro-
pean Integrated Activity of Excellence and Network-
ing for Nano and Micro-Electronics Analysis,” contract
no. 026134(RII3)ANNA and by the OPTIBIO 231055
FP7-Program.
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6Sensor Letters 8, 1–6, 2010
... Although spectroscopic ellipsometry has a lower sensitivity, it measures in a wide wavelength range. The increased number of measured data allows the use of sophisticated optical models with numerous parameters [11,18]. Consequently, the advantage of a combined tool is the complex modelling capability supported by the excellent sensitivity. ...
... In case of the second measurement, the optical model was completed with a subsequent Cauchy model to describe the protein adsorption. The A, B and C parameters of this model were fixed at 1.45, zero and zero, respectively, where A, B and C are the parameters of the Cauchy dispersion formula [11,13,18]. During the evaluation of the in situ measurements, only the thickness of the adsorbed protein layer was fitted. ...
... The dn/dc ratio is the derivative of n with respect to the protein concentration (c) that can be taken from the literature (0.18 cm 3 /g measured at the wavelength of 632.8 nm) [3,26]. The value of n a was fixed at 1.45 [11,13,18]. ...
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... Moreover, it provides spectroscopic information which helps to build complex optical models and to measure complex structures in a more quantitative way. 4 Ellipsometry has mainly been used in a configuration measuring the surface through the liquid using flow cells. 4,5 There have been further configurations proposed and demonstrated, which measure from the substrate side, mainly in order to utilize plasmon enhancement. [6][7][8][9][10][11] We used a semi-cylindrical lens with the Kretschmann geometry to use spectroscopic ellipsometry in a broad wavelength range at different angles of incidence, and studied the performance of the tool for different gold layer thicknesses. ...
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A semi-cylindrical lens in Kretschmann geometry combined with a flow cell was designed for a commercial rotating compensator ellipsometer to perform internal reflection spectroscopic ellipsometry measurements, while allowing the use of multiple angles of incidence. A thin glass slide covered with a gold film was mounted between the half-cylindrical lens and a small-volume flow cell ensuring an improved sensitivity for protein adsorption experiments. The performance of the system was investigated depending on the angle of incidence, wavelength range and thickness of the gold films for surface plasmon resonance enhanced ellipsometric measurements, and a sensitivity increase was revealed compared to ellipsometric measurements with standard flow cells, depending on the measurement parameters and configuration. The sensitivity increase was demonstrated for fibrinogen adsorption.
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Environmental monitoring of Ni is needed around the WHO threshold limit of 0.34 µM. This sensitivity target can usually only be met by time consuming and expensive laboratory measurements. There is a need for cheap field-applicable methods, even if it is only used for signaling the necessity of a more accurate laboratory investigation. In this work bio-engineered protein-based sensing layers were developed for Ni detection in water. The bacterial Ni-binding flagellin variants were fabricated using genetic engineering, and their applicability as Ni-sensitive biochip coatings was tested. Nanotubes of mutant flagellins were built by in vitro polymerization. A large surface density of the nanotubes on the sensor surface was achieved by covalent immobilization chemistry based on a dithiobis(succimidyl propionate) crosslinking method. The formation and density of the sensing layer was monitored and verified by spectroscopic ellipsometry and atomic force microscopy. Cyclic Voltammetry (CV) measurements revealed a Ni sensitivity below 1 µM. It was also shown that even after two months storage the used sensors can be regenerated and re-used by rinsing in 10 mM solution of ethylenediaminetetraacetic acid at room temperature.
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Understanding interface processes has been gaining crucial importance in many applications of biology, chemistry, and physics. The boundaries of those disciplines had been quickly vanishing in the last decade, as metrologies and the knowledge gained based on their use improved and increased rapidly. Optical techniques such as microscopy, waveguide sensing, or ellipsometry are significant and widely used means of studying solid‐liquid interfaces because the applicability of ions, electrons, or X‐ray radiation is strongly limited for this purpose due to the high absorption in aqueous ambient. Light does not only provide access to the interface making the measurement possible, but utilizing the phase information and the large amount of spectroscopic data, the ellipsometric characterization is also highly sensitive and robust. This article focuses on ellipsometry of biomaterials in the visible wavelength range. The authors discuss the main challenges of measuring thickness and optical properties of ultra‐thin films such as biomolecules. The authors give an overview on different kinds of flow cells from conventional through internal reflection to combined methods. They emphasize that surface nanostructures and evaluation strategies are also crucial parts of in situ bioellipsometry and summarize some of the recent trends showing examples mainly from their research.
Chapter
The evaluation of thickness, refractive index, and optical properties of biomolecular films and self-assembled monolayers (SAMs) has a prominent relevance in the development of label-free detection techniques (quartz microbalance, surface plasmon resonance, electrochemical devices) for sensing and diagnostics. In this framework Spectroscopic Ellipsometry (SE) is an important player. In our approach to SE measurements on ultrathin soft matter, we exploit the small changes of the ellipsometry response ($$\delta \varDelta$$ and $$\delta \varPsi$$) following the addition/removal of a layer in a nanolayered structure. So-called $$\delta \varDelta$$ and $$\delta \varPsi$$ difference spectra allow to recognize features related to the molecular film (thickness, absorptions) and to the film-substrate interface thus extending SE to a sensitive surface UV-VIS spectroscopy. The potential of ellipsometry as a surface spectroscopy tool can be boosted when flanked by other characterizations methods. The chapter deals with the combined application of broad-band Spectroscopic Ellipsometry and nanolithography methods to study organic SAMs and multilayers. Nanolithography is achieved by the accurate removal of molecules from regularly shaped areas obtained through the action of shear forces exerted by the AFM tip in programmed scans. Differential height measurements between adjacent depleted and covered areas provide a direct measurement of film thickness, which can be compared with SE results or feed the SE analysis. In this chapter we will describe the main concepts behind the SE difference spectra method and AFM nanolithograhy. We will describe how SE and AFM can be combined to strengthen the reliability of the determination of thickness and, as a consequence, of the optical properties of films. Examples will be discussed, taken from recent experiments aimed to integrate SE and AFM nanolithography applied to SAMs and nano layers of biological interest. By analysing in detail the changes of the spectroscopic features of compact versus non-compact layers and correlating such changes with the post-lithography AFM analysis of surface morphology SE unravels the specific versus unspecific adsorption of biomolecules on gold surfaces functionalized with suitable SAMs.
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Bovine fibrinogen monolayers on thin gold films and glassy carbon substrate were investigated using grazing incidence X-ray fluorescence (GIXRF) and spectroscopic ellipsometry (SE). The aim was to determine the amount of protein and to develop models and references for the SE measurement. Both methods were capable of measuring protein amount in the range of μg cm−2 with a sensitivity below 10%, which suggests the use of both techniques as complementary, combined methods. To do it with a high confidence, the lateral uniformity and the stability of the layers during transportation has to be investigated in more detail in the future.
Article
Plasmon-enhanced in situ spectroscopic ellipsometry was realized using the Kretschmann geometry. A 10-μL flow cell was designed for multi-channel measurements using a semi-cylindrical lens. Dual-channel monitoring of the layer formation of different organic structures has been demonstrated on titania nanoparticle thin films supported by gold. Complex modeling capabilities as well as a sensitivity of ~40 pg/mm2 with a time resolution of 1 s was achieved. The surface adsorption was enhanced by the titania nanoparticles due to the larger specific surface and nanoroughness, which is consistent with our previous results on titanate nanotubes.
Chapter
Nowadays, a broad choice of instruments, including dedicated synchrotron radiation beamlines, allows to exploit Spectroscopic Ellipsometry (SE) to investigate the thickness and the dielectric properties of thin films, from the terahertz down to the VUV wavelength range. Instruments combining fast parallel detection, precision, accuracy, are pushing forward real time and in-situ applications, to monitor the dynamics of processes such as e.g. film growth, oxidation, polymerization, electrochemical processes, with a diverging spectrum of scientific and industrial applications in the fields of nano-electronics, coatings, solar cell materials, polymer technology, bio-sensing, photonics, just to name a few. This chapter, beyond presenting the essentials of principles and instrumentation of SE, is intended to place this thin-film technique in the perspective of the surface scientist, through the selection of applications to ultra-thin films and nanostructures. Emphasis is placed on reflection experiments, in the 190–1700 nm wavelength range covered by high-quality commercial instruments, although some infra-red (IRSE) and far UV experiments are also discussed.
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Indirect optical methods like ellipsometry or scatterometry require an optical model to calculate the response of the system, and to fit the parameters in order to minimize the difference between the calculated and measured values. The most common problem of optical modeling is that the measured structures and materials turn out to be more complex in reality than the simplified optical models used as first attempts to fit the measurement. The complexity of the optical models can be increased by introducing additional parameters, if they (1) are physically relevant, (2) improve the fit quality, (3) don't correlate with other parameters. The sensitivity of the parameters can be determined by mathematical analysis, but the accuracy has to be validated by reference methods. In this work some modeling and verification aspects of ellipsometry and optical scatterometry will be discussed and shown for a range of materials (semiconductors, dielectrics, composite materials), structures (damage and porosity profiles, gratings and other photonic structures, surface roughness) and cross-checking methods (atomic force microscopy, electron microscopy, x-ray diffraction, ion beam analysis). The high-sensitivity, high-throughput, in situ or in line capabilities of the optical methods will be demonstrated by different applications.
Chapter
This chapter describes ellipsometric characterization of thin films. The chapter illustrates that the newly activated interest is driven by the demand for rapid, nondestructive analysis of surfaces and thin films—particularly films and surfaces occurring in different device technologies. The fact that ellipsometric measurements can be performed under any ambient conditions is a definite advantage over other surface-science (electron or ion beam) techniques for industrial applications. In ellipsometry, the change in polarization state of a linearly polarized beam of light has to be measured after non-normal reflection from the sample to be studied. The polarization state can be defined by two parameters—for example, the relative phase and relative amplitude of the orthogonal electricfield components of the polarized light wave. The technique of principles of ellipsometry found its first practical use with the development of so-called rotating element ellipsometers and in computers to solve complex equations. These polarimeter-type ellipsometers measure continuously thus, wavelength scanning can be performed.
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The controlled surface placement of protein molecules represents a crucial step toward many new biotechnological devices and processes. A promising means of directing the structure and formation rate of an adsorbed protein layer is through an applied electric potential difference. We present here a method for continuously measuring the protein adsorption under a direct current voltage using optical waveguide lightmode spectroscopy. An indium tin oxide-coated waveguiding sensor chip serves as the anode and adsorbing substrate, and a platinum counter electrode serves as the cathode in a parallel plate arrangement. For (negatively charged) human serum albumin in either pure water or N-[2-hydroxyethyl]piperazine-N'-ethanesulfonic acid (HEPES) buffer, we find the transport-limited and initial surface-limited rates of adsorption to significantly increase with the applied potential. For (positively charged) horse heart cytochrome c, we observe no influence of the voltage on the transport-limited adsorption rate in either solvent and a decrease with the voltage in the initial surface-limited rate in a HEPES (but not a pure water) solvent. Interestingly, we find the rate of adsorption at moderate to high surface density to greatly increase with the voltage for both proteins; this effect is more pronounced in water than in HEPES. We attribute this enhanced adsorption to contact between electrode and protein patches of complementary charge, leading to more oriented and efficiently packed adsorbed molecules and, in the case of high voltage, to multilayer formation.
Article
Protein adsorption is an important aspect for the improvement of many applications, such as medical implants, biosensor design, etc. The density, orientation and conformation of surface-bound proteins are believed to be key factors in controlling subsequent cellular adhesion. The aim of this work is the development of a methodology in order to study in-situ and real-time protein adsorption phenomenon, and describe fibrinogen adsorption on amorphous hydrogenated carbon (a-C:H) thin films developed by rf reactive magnetron sputtering under different deposition conditions. Spectroscopic Ellipsometry (SE) in Vis–UV energy region was implemented for this purpose. SE is a non-destructive, surface sensitive technique, with the capability of performing real-time measurements in air as well as in liquid environment, with great potential in biomedical studies. An appropriate ellipsometric model has been developed, in order to describe accurately the protein adsorption mechanisms in real-time. It was found that the thickness and density of fibrinogen are larger on the a-C:H thin film deposited under absence of bias voltage application. The differences in fibrinogen thickness and transition of fibrinogen from liquid to adsorbed state are presented and discussed in the terms of the surface and optical properties of a-C:H films.
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The thickness resolution and in situ advantage of ellipsometry make this optical technique particularly suitable for studies of thin organic layers of biological interest. Early ellipsometric studies in this area mainly provided thickness quantification, often expressed in terms of surface mass. However, today it is possible to perform monolayer spectroscopy, e.g. of a protein layer at a solid/liquid interface, and also to resolve details in the kinetics of layer formation. Furthermore, complicated microstructures, like porous silicon layers, can be modeled and protein adsorption can be monitored in such layers providing information about pore filling and penetration depths of protein molecules of different size and type. Quantification of adsorption and microstructural parameters of thin organic layers on planar surfaces and in porous layers is of high interest, especially in areas like biomaterials and surface-based biointeraction. Furthermore, by combining ellipsometric readout and biospecificity, possibilities to develop biosensor concepts are emerging. In this report we review the use of ellipsometry in various forms for studies of organic layers with special emphasis on biologically-related issues including in situ monitoring of protein adsorption on planar surfaces and in porous layers, protein monolayer spectroscopy and ellipsometric imaging for determination of thickness distributions. Included is also a discussion about recent developments of biosensor systems and possibilities for in situ monitoring of engineering of multilayer systems based on macromolecules.
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We have used spectroscopic ellipsometry to determine the dielectric functions of thin films of γ-globulin, bovine serum albumin, and hemoglobin in the visible and near-uv photon energy range. We show that both thickness and dielectric function can be resolved for monomolecular films adsorbed on substrates with relatively low polarizability. The data which we consider to be closest to the intrinsic dielectric response of the protein films were obtained on HgTe and HgCdTe substrates. Less resolution was obtained on silicon substrates. For a density-deficient film, we were able to model the dielectric response with effective medium theories, and the void fraction could be determined.
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Hemoglobin Catonsville is a mutation of human hemoglobin (an alpha2beta2 tetramer) in which a glutamate residue is inserted into the first turn of a highly conserved 3(10) helix (the C helix) of each alpha subunit. In theory, amino acid insertions (or deletions) in protein helices can be accommodated via two distinct mechanisms. One, termed the register shift mechanism, preserves the geometry of the helix while requiring all of the residues on one flank of the insertion site to rotate by 100-degrees in the case of an alpha helix or by 120-degrees in the case of a 3(10) helix. The other, termed the bulge (or indentation) mechanism, distorts the local geometry of the helix but does not alter the helix register. High-resolution X-ray diffraction analysis of deoxyhemoglobin Catonsville shows that the inserted residue is accommodated as a bulge, demonstrating that this is a viable mechanism. (In contrast, no such evidence is yet available for the register shift mechanism.) More specifically, the insertion converts one turn of the C helix from 3(10) geometry to alpha helix-like geometry, raising the possibility that a common mechanism for accommodating insertions and deletions within helices may involve localized interconversions between 3(10), alpha, and pi helical structures.
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The quality of flagellin films in terms of thickness and homogeneity was investigated by spectroscopic ellipsometry. Flagellin films were prepared in three steps: silanization, glutaraldehyde activation and finally the coating with proteins. The process of film preparation was optimized by varying the duration of the silanization and by testing sticking on different substrates including Si wafer covered with different thickness of silicon-oxide and also covered with a thin film of tantallum pentoxide. Spectroscopic ellipsometry was applied to gain in-depth information on the film properties for the optimization of the immobilization. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
Article
The application of ellipsometry of the study of the adsorption behavior of proteins and synthetic macromolecules at the air-water interface has been investigated. It is shown that for macromolecules the amount adsorbed per unit area, Γ, as determined by ellipsometry, only has a well-defined physical meaning if the refractive-index increment remains constant up to high concentrations present in the adsorbed layer. It has been found experimentally that this conditioned is fulfilled for proteins. The ellipsometric Γ values of some protein agree satisfactorily with those obtained by two independent techniques has been used to investigate the adsorption from solution of κ-casein, bovine serum albumin, and polyvinyl alcohol. For bovine serum albumin, Γ reaches a plateau value of 2.9 mg/m2 for concentrations ≥ 0.05 wt%. The thickness of the adsorbed molecules. For κ-casein, Γ steadily increases with increasing centration and multilayers are formed. The technique provides interesting information on conformational changes in adsorbed macromolecules, on the rate of the process, and on the conditions under which these occur.