CONCLUSION AND OUTLOOK
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.
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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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