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"Protein and cell adsorption: topographical dependency and adlayer viscoelastic properties
determined with oscillation amplitude of quartz resonator"
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Remark: for lisibility, all references have been omitted.
Introduction
Presentation of the work
The understanding of protein and
cell adsorption is of high importance for the production of biocompatible
implants as well as for the biosensor field, where a protein layer has
to be designed as a functional coating. It is well-known that a living
body brought into contact with a surface will induce protein adsorption,
on which further proteins or cells will adsorb. The foreign body reaction
is composed of a succession of events. The first protein adsorption occurs within
minutes. During the first day cells (neutrophils and macrophages) will
attack the foreign body. When the macrophages find they cannot digest
the implant, they fuse into giant cells to engulf the object in the following
next 4 days. However the implant is too large to be completely ingest,
therefore inducing cytokines (messengers) secretion to alert other cells.
In response fibroblasts arrive and begin synthesizing collagen during the
next 10 days. After 3 weeks the implant is completely encased in an acellular
and avascular collagen bag. The properties of the first protein adlayer
can therefore play a crucial role in the integration of implant in the
body.
Depending on the application, varying
adhesion qualities are required. For example bones or tooth implants
demand good protein adhesion to allow the growth of the bone and the
formation of a stable and strong interphase between the implant and the
bone. In contrast the protein adhesion has to be poor for catheters or
contact lenses, where a low contact with the tissue is required. The interactions
between proteins and a solid surface can be influenced by modifying the
surface properties, which are the chemistry, the surface charge and the
topography. A number of studies have shown that cells "sense" the topography
of the surface at the micrometer scale, growing
along grooves of determined width and depth.
Because proteins are always present
between the cells and the substratum, the first part of my work (see
Paper I and II) has been devoted to study the role of topography on protein
adsorption, which had never been addressed as only parameter. Created
structures had to be scaled down to reach sizes accordingly to proteins,
i.e. in the nm range, which is smaller than the lithography resolution.
The Scanning Probe Microscopy (SPM) permits manipulation of molecules,
measuring surfaces at the atomic scale or modifying the surface at the
nm scale. An Atomic Force Microscope (AFM) has been used to create nanostructures
applying the Local Anodic Oxidation (LAO), which allows us to change the
topography (figure 2)
without modifying the surface chemistry.
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Figure 2: Nanostructures created by LAO.
A: The first two letters of my university FR.
B: Wire netting structure
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On the created nanostructures proteins
have been adsorbed, and the new surface can be measured by AFM. These
experiments were static giving no information on the kinetics of protein
adsorption.
The second part of my work (see
Paper III to V) has therefore been focussed on the building of a Quartz
Crystal Microbalance (QCM), which allows us firstly to follow the adsorption
kinetics, and secondly to get information about the viscoelastic properties
of the adlayer. In comparison to commercial QCM a new parameter has been
introduced, which is the maximal oscillation amplitude of the quartz
crystal (see Paper III). The adsorption kinetics of different systems has
bee measured. Protein A-BSA-IgG is a system well-known in immunology and
fibronectin is viscoelastic in solution. For both systems the mechanical
properties of the adsorbed layers have been investigated on Ti and Au surfaces
(see Paper IV). Cell adsorption and spreading has also been performed with
different cell types, where the cytoskeleton has been disturbed with drugs
(see Paper V). This system range has allowed us to improve the interpretation
of QCM measurements, opening new applications in biotechnology research.
top
The QCM
The Quartz
Crystal Microbalance (QCM) is a powerful technique to follow the adsorption
kinetics of quite different systems, such as atoms, molecules, proteins
or even living cells. The physical phenomenon of the sensor is based on
the piezoelectric effect, whose discovery is attributed to Pierre and Jacques
Curie. In 1880 they showed that crystals of Rochelle salt could produce electricity
when pressure is applied in certain crystallographic directions. One year
later they showed the reverse effect, which consists of the production of
strain by the application of electricity. Nevertheless it is only at the end of the 1950s
that this phenomenon has been investigated. AT-cut quartz crystal is the
most used cut for frequency control applications, because it has nearly
zero frequency drift with temperature.
Two electrodes are evaporated on
each side of the thin quartz plate for the application of an alternative
voltage, which induces oscillation of the crystal. In 1959 Sauerbrey published
an article showing that the frequency shift of quartz crystals is proportional
to the adsorbed mass under vacuum. The QCM was born.
In 1981 the application field of
QCM is enlarged to liquid environments, opening new research insights in biotechnologies
and electrochemistry.
The general setup of our QCM is
presented in figure 8.
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Figure 8: Setup of our home-made Quartz Crystal Microbalance.
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It is composed of three different
main components, which are the sensor environment, the QCM-computer interface
and the computer with the program allowing to acquire, treat and present
the data.
The
sensor environment top
10MHz quartz crystals have been
used, whose electrodes show two different diameters. Only the larger one
enters in contact with liquids, in order to avoid perturbating effects due
to the electrical properties of the liquid. The quartz crystal is entrapped between two
plexiglas pieces, which are sealed with VITON O-rings. The liquid exchanges
are performed manually with micropipettes and a tubing system.
The sensor and the solutions are
placed in a temperature control box in order to maintain a constant temperature
of T ±0.1oC by water circulating
inside its walls. No artefacts due to thermal modification of liquid
properties can therefore occur.
It is possible to combine the QCM
with microscopy, by mounting the crystal under an optical or fluorescence
microscope.
The
QCM-computer interface top
The electronic interface controls
the relay and digitizes, stores and sends the measured data to the computer.
A frequency generator excites the
crystal near its series resonance frequency f and a second frequency
generator determines the sampling frequency fsampling. The
relation between the measured alias frequency and the quartz resonance
frequency is given by the following relation:
| falias=fsampling ·frac
( |
f
fsampling
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When fsampling » f, Delta fsampling
= Delta f, so that measured shifts of the alias
frequency are proportional to resonance frequency shifts.
The relay is closed about 3ms to
excite the crystal, and is thereafter opened. Due to energy losses occurring
in the system the crystal amplitude A begins to decrease according to
| A(t) = A0 e-t/tau sin(2 pi
f t + phi) |
|
where tau
is the decay time constant, f the measured frequency and phi the phase angle. One measurement under liquid
lasts about 1.5ms. During the measurement the amplitude signal is digitized
and stored in the RAM. It is thereafter transmitted to the computer with
the GPIB bus, and a new measurement can be performed by closing the relay.
The
program top
A program has been developed in
LabView in order to acquire, treat and show the data over hours. The measured
amplitude is transmitted to the computer via the GPIB bus, and the program
fits the data in order to determine f, tau and
A0 each 300ms. The Dissipation factor D=2/(w tau) is also calculated.
Figure 10 (see pdf file) shows the
developed Graphical User Interface (GUI). It is composed of three different
parts.
In part A the acquired data are
monitored. First of all the amplitude decrease of each measurement is shown
in the left part. It allows us to adjust precisely the frequency generator
at the resonance frequency of the crystal and to control continuously the
crystal behavior. The decay time constant is determined and the exponential
decrease is also shown in this first graph. The amplitude decrease is
transformed in a pure sinusoidal curve in order to determine the resonance
frequency. This signal is presented under the amplitude decrease. For each
measurement f, D and A0 are plotted in two different ways. The
general overview of the adsorption kinetics is visualized in the three
small graphs (middle of part A). The parameters of the last 60 measurements
are shown in the three larger graphs in the right part.
The input parameters such as the
user profile, the measurement time, the acquisition rate and the created
file name have to be given in part B.
Finally the part C allows us to
control the Start/Stop of the measurements. The user can choose to start/stop
the data acquisition directly or at a scheduled date and time.
Similarly the measurement can be
immediately stopped, or its end can be determined.
The
resolution top
Under vacuum we obtain an excellent
resolution of f ±0.03Hz, tau±1.5E-8sec, A ±3a.u. and
D ±0.5E-7.
The measured mass sensitivity corresponds therefore to m ±0.135ng/Hz cm2 using the Sauerbrey
relation.
Under liquid loading the resolution
decreases due to damping of the liquid. When nanodeionized water is
in contact with the quartz at 25oC, we reach a resolution
of f ±2Hz, tau±0.5E-6sec,
A ±50 a.u. and D ±1E-6.
The mass sensitivity is m ±9ng/Hz cm2,
corresponding to the better resolution which is possible to obtain with
10MHz quartz crystals.
top
Protein adsorption on topographically nanostructured titanium
C. Galli, M. Collaud Coen, R. Hauert, V.L. Katanaev, M.P. Wymann,
P. Gröning and L. Schlapbach
Surface Science 474 (2001) L180-184
Abstract
We study the protein adsorption on surfaces in order to investigate their predominant
role in biocompatibility. Nanostructures are created by local anodic oxidation on titanium
using the atomic force microscope. A remarkable specificity of the actin filament adsorption
on the nanostructure height is noticed. F-actin is observed to have a low adsorption on
nanostructures of a height of 4 nm and the adsorbed proteins appear to be randomly oriented.
In contrast high protein adsorption is observed for structure height between 1 and 2 nm,
moreover the filaments adsorb preferentially parallel to the nanostructured pattern.
Keywords: Atomic force microscopy; Surface structure, morphology, roughness, and topography;
Chemisorption; Titanium oxide; Biological molecules-proteins
top
Creation of nanostructures to study the topographical dependency of
protein adsorption
C. Galli, M. Collaud Coen, R. Hauert, V.L. Katanaev, P. Groening,
L. Schlapbach
Colloids and Surfaces B: Biointerfaces 26 (2002) 255–267
Abstract
Nanostructures of sizes comparable to protein dimensions are created
on Si and Ti surfaces by local anodic oxidation (LAO) using the atomic
force microscope (AFM). The characterization of the surface by X-ray photoelectron
spectroscopy (XPS) reveals that this method assures a modification of the
topography of the surface without a change of its chemical composition.
Surfaces structured by LAO therefore represent ideal systems to study the
dependence of protein adsorption on topography. We are able to visualize
the created nanostructures with an AFM and successively adsorb the proteins
in situ, rinse and image the new surface. The densities of adsorbed proteins
on the nanostructured and neat surfaces are compared and we find that the
protein arrangement depends on the underlying nanostructures, showing that
proteins can ‘‘sense’’ the topography of surfaces at the nanometer scale.
This result can be considered as the nanoscale analogous of the adsorption
found for cell systems on micrometer structures.
Keywords : Nanostructures; Topography; Protein; Adsorption; Surface
top
The simultaneous measurements of the maximal oscillation amplitude
and the transient decay time constant of the QCM reveal stiffness changes
of the adlayer
C. Galli Marxer, M. Collaud Coen, H. Bissig, U.F. Greber, L.
Schlapbacha
Accepted for publication in Analytical and Bioanalytical Journal,
May 2003
Abstract
The interpretation of adsorption kinetics measured with Quartz
Crystal Microbalance (QCM) can be difficult for adlayers undergoing modifications
of mechanical properties. We have studied the behavior of the oscillation
amplitude A0 and the decay time constant τ of quartz during adsorption
of proteins and cells using a home-made QCM. We are able to measure simultaneously
the frequency f, the dissipation factor D, the maximal amplitude A0 and
the transient decay time constant τ each 300ms under liquid, gaseous or
vacuum environments. This enables distinguished analyses between adsorption
and modifications of liquid/mass properties. Moreover the surface coverage
and the stiffness of the adlayer can be estimated. These improvements promise
to increase the appeal of the QCM methodology for any applications, measuring
intimate contacts of dynamic material with a solid surface.
Keywords: QCM, amplitude, viscoelasticity, decay time constant, protein,
cell
top
Study of adsorption and viscoelastic properties of proteins with a
quartz crystal microbalance by measuring the oscillation amplitude
C. Galli Marxer, M. Collaud Coen, and L. Schlapbach
Journal of Colloid and Interface Science 261 (2003) 291–298
Abstract
The adsorption kinetics of protein A, BSA, IgG, and fibronectin
has been investigated using a homemade quartz crystal microbalance. Information
about the energy losses appearing in the system is measured by the maximal
oscillation amplitude and the dissipation factor. Only the maximal oscillation
amplitude allows us to distinguish the different contributions of liquid
and mass to the total frequency shift. The adsorption of proteins has been
performed on Ti and Au surfaces at different concentrations. The amount
of irreversible adsorbed protein A and IgG increases with increasing bulk
concentrations. On Au more proteins adsorb, but their biological activity
is reduced in comparison to Ti. Protein A forms a first monolayer in a few
seconds, which shows practically no energy losses, and following this a second
monolayer is formed. The adsorption rate for the second monolayer is much
smaller and energy losses are present. Fibronectin is forming a very viscoelastic
system, whose mechanical properties are affected by immersion in different
buffer solutions.
Keywords: Protein A; BSA; IgG; Fibronectin; Adsorption; QCM; Amplitude;
Dissipation factor; Viscoelasticity
top
Cell spreading on Quartz Crystal Microbalance elicits positive frequency
shifts indicative of viscosity changes
C. Galli Marxer, M. Collaud Coen, T, Greber, U. F. Greber,
L. Schlapbach
Accepted for publication in Analytical and Bioanalytical Journal,
May 2003
Abstract
Cell attachment and spreading on solid surfaces was investigated
with a home-made Quartz Crystal Microbalance (QCM), which measures the
frequency, the transient decay time constant and the maximal oscillation
amplitude. Initial interactions of the adsorbing cells with the QCM mainly
induced a decrease of the frequency, coincident with mass adsorption. After
about 80min, the frequency increased continuously and after several hours
exceeded the initial frequency measured before cell adsorption. Phase contrast
and fluorescence microscopy indicated that the cells were firmly attached
to the quartz surface during the frequency increase. The measurements of
the maximal oscillation amplitude and the transient decay time constant
revealed changes of viscoelastic properties at the QCM surface. An important
fraction of these changes was likely due to alterations of cytosolic viscosity,
as suggested by treatments of the attached cells with agents affecting
the actin and microtubule cytoskeleton. Our results show that viscosity
variations of cells can affect the resonance frequency of QCM in the absence
of apparent cell desorption. The simultaneous measurements of the maximal
oscillation amplitude, the transient decay time constant and the resonance
frequency allow an analysis of cell adsorption to solid substratum in real
time and complement cell biological methods.
Keywords: cell, QCM, adsorption, cytoskeleton, amplitude, decay time
constant
Conclusion and outlook
top
The thesis work
presents two different studies. In the first one the fundamental question
addressed was to know, whether proteins can "sense" the topography, similarly
to what had been shown for cells. It required to find a method, which allowed
us to modify the topography at the nanometer scale without any change of
the surface chemistry. Moreover it was necessary to visualize the nanostructured
surface before and after protein adsorption. The AFM is the only technique,
which allows us to modify the surface at a nanometric scale and to measure
in situ adsorbed biological molecules such as proteins, which are insulators.
Nanostructured surfaces by LAO have been characterized by XPS, demonstrating
that LAO treatment effectively induces no change of the surface chemistry.
Therefore ideal systems can be created by LAO to study the only influence
of the topography on protein adsorption. Globular protein A shows no adsorption
difference between parallel 1nm high nanostructures and neat Si. The bounded
IgG also shows no preferential adsorption on or outer the nanostructures
covered with protein A. In contrast, preferential adsorption of filamentous
F-actin has been measured on 1nm high created lines on Si, and the density
of adsorbed protein on neat Si is higher than on nanostructures. Ti nanostructures
of different heights have been created. The F-actin adsorption is the greatest
on 1-2nm high lines with a preferential orientation
along them. In contrast the adsorption density is low on 4nm high nanostructures,
and no defined orientation is noted. On neat Ti adsorbed F-actin shows a
smaller density than on the 1-2nm high nanostructures.
Therefore proteins are able to react to modifications of the topography
at the nanometer scale, what has to be taken into account in the design
of new biomaterials.
In order to approach in vivo conditions,
further studies have to be performed under aqueous conditions and with
the presence of protein mixtures. Moreover the next experimental step consists
on the adsorption of cells on the proteins oriented by such nanostructured
surface. In this view new methods would be required, because the work
area of most actual commercial AFM is restricted to 104 micro m2 and the method is too slow to create
nanostructures over larger areas. The lithography technique will be perhaps
the solution to produce nanostructures at large scale, because its resolution
is becoming improved and its work area is in the cm2 range. Nevertheless
further developments are required before the lithography reaches the LAO
resolution.
The second part of this work was
concentrated on the development of the QCM technique, and especially on
the improvement of the interpretation of QCM data. At the moment the QCM
is the only method, which allows us to follow the adsorption kinetics
and to get simultaneously viscoelastic information of the adlayer. Nevertheless
the interpretation of measurements performed with commercial QCM is still
difficult, because modifications of liquid and mass properties can not
be separated from the variation of adsorbed mass. We have demonstrated that
the introduction of the maximal oscillation amplitude and the decay time
constant improves the determination of the phenomena arising at the quartz
surface. Contribution of adsorbed mass can effectively be distinguished
from the one of modification of viscosity or density of the liquid or of
the mass. Using the protein A-BSA-IgG system, the biological activity of
adsorbed protein A has been investigated on Au and on Ti. At concentration
of 160 mM no difference is noted, but divergences
have been measured at higher concentrations. More proteins always adsorb
on Au surfaces and the adlayers dissipate less energy than on Ti. However
the biological activity is better on Ti. Further experiments with other protein
systems are required in order to determine if the biological activity can
be directly related to the viscoelastic properties of the protein layer.
The effect of buffer properties on adsorbed fibronectin has also been investigated.
Immersion in Citrate-Phosphate buffer induces only delamination of the
adlayer. In contrast the mechanical properties of the fibronectin are modified
in Hepes solution.
The QCM open new applications in
cell biology and biotechnologies, because the interactions occurring between
a surface and a cell can be directly investigated. We have demonstrated
that different phenomena influence the frequency, the amplitude and the
decay time constant during cell adsorption. During the first 80min the frequency
decreases due to an increase of adsorbed cells. Thereafter the frequency
begins to increase, what would correspond to mass desorption. Nevertheless
living cells are still present at the surface. We demonstrate that the
development of the cytoskeleton induces stiffening of the cell and increases
the total cell viscosity. This phenomenon influences the QCM sensitivity
in frequency, although no modification of the amount of adsorbed cells occurs.
The cytoskeleton state has also been modified with drugs, which increase
or decrease the polymerisation state of actin and microtubules. It allowed
us to reproduce rapid increasing and decreasing of frequency in less than
30min due to fast changes of the cell viscosity and cell adsorption.
The understanding of complex systems
such as cells requires nevertheless the combination of different technique.
We tried to combine a QCM with a fluorescence microscopy. The quartz
electrode has to be modified in order to avoid fluorescence bleaching
on Au. Moreover it is essential to work with oil objectives, which have
very short working distances. The building of the whole setup is therefore
complicated in order to fulfill all requirements, such as temperature
stabilization and exchange of fluid. To date no reproducible measurements
could be performed during several hours. Nevertheless this process is
the only one, which would allows us to correlate the number of developed
focal points with the measured frequency, amplitude and decay time constant.
Moreover this method combination would allow us to track modification of
cell properties during for example virus infection. In order to quantify
the variation of the frequency, the amplitude and the decay time constant
during viscoelastic modifications of adlayer, colloidal systems can be
very interesting. They are firstly much less complex than living cells
and secondly they allow us to study phase transitions. Our first tests
are very promising.
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