Antibacterial And Protein-Selective, -Repelling Plasma Nanofilm For
Different Materials Commonly Used In Medical Applications
M. Bergmann, G. Dame, F. Olcaytug, G. Urban
University of Freiburg - Department of Microsystems Engineering - IMTEK
Abstract: Adhesion of bacteria as well as adsorption of proteins on surfaces plays an
important role in the development of biocompatible and antibacterial materials. In this
study a low-pressure magnetron-enhanced 15 kHz discharge polymerization is used to
deposit a biocompatible nanofilm which is applicable for different sorts of materials e.g.
silicon, polystyrene, polymethyl methacrylate and glass. The used process gases are
methane and oxygen, excited between two titanium electrodes with a power of 60 W.
Surface Plasmon Resonance (SPR) and fluorescent microscopy were used to
characterize the biological properties and abilities in case of antibacterial and
biocompatible behavior. By fluorescent microscopy we demonstrated a reduction in
bacteria adhesion up to 99.9 %. The protein-surface interaction primary defines the
antibacterial and biocompatible property. For that reason, SPR-studies were applied to
show the impeding character of this coating in matters of protein adsorption. By
variation of the process gases surfaces were tailored with very low protein-surface
interaction.
Keywords: antibacterial plasma coating, protein repellent, biocompatible nanofilm
1. Introduction
Adhesion and growth of bacteria on surfaces is a
widespread topic. Bacterial adhesion contaminating
surfaces causes a lot of issues not only in medical
applications [1] but also in food industry [2] and in
water transport systems [3].
Another topic in surface interaction is the attachment
of proteins playing a role in the development of
biocompatible coatings. For example the function of
implanted biosensors can be disturbed by
encapsulation of the functional part by proteins or
other blood constituentsts. The materials used in
medical applications often require a high
biocompatibility. There is a need for deposition
systems being able to produce coatings on a wide
variety of materials without disturbing their inherent
properties. In case of polymers a low temperature
process is needed. A low-pressure magnetronenhanced 15 kHz discharge polymerization is
presented, permitting to deposit a biocompatible
nanofilm which is applicable for different sorts of
materials.
Different types of materials were coated and studied.
Previous studies with silicone contact lenses [4]
showed that low pressure plasma polymerization
processes are economically advantaeous [5].
2. Experimental Section
2.1 Plasma-Coating System
The coating process of the samples is carried out in a
bell jar type reactor, see Figure 1. The reactor has a
total volume of 115 liter and is equipped with two
parallel titanium electrodes. The electrodes are
supported by two circular shaped magnet structures
which are embedded in a cooled holder. The process
is driven at pressures between 1 and 10 Pa with flow
rates of 1 to 10 sccm. Plasma polymerization uses
methane and oxygen as precursors, which are
introduced in the reactor and excited between the
electrodes at a power of 60 W. This apparatus makes
it possible to produce nanofilms with different
compositions and thicknesses.
bell jar
gas inlet
electrode
magnetrons
sample wheel
Figure 1. schematic drawing of the plasma reactor
2.2 Measuring The Protein Adsorption By
Surface Plasmon Resonance (SPR)
Surface Plasmon resonance measurements were
performed to study the protein-surface interaction of
different nanofilms. For this purpose a commercial
SPR-instrument from ResTec was used. Based on
the Kretschmann configuration a He-Ne-laser with a
wavelength of 632nm, directs monochromatic, ppolarized light through a prism onto a LaSFN9 glas
slide which is coated by an evaporated gold layer of
approximately 50 nm. Surface plasmons are excited
at the metal-liquid interface, which are affected by
the adsorbed protein layer as well as by the dielectric
constant of the interface region. The angle between
the laser and the glass slide was varied, using a
goniometer, while the reflectivity was recorded.
Before and after protein adsorption these plots were
performed. An angle shift of the plasmon resonance
indicates an adsorbed layer, which was determined
by a software tool called WASPLAS. This tool is
based on the Fresnel equations and helps to
determine the system of different layers. Also time
resolved measurements were done, which acted as a
control of the adsorption process. For this purpose
the incident angle of the laser onto the glass slide
was fixed to an angle slightly lower than the
resonant angle to reach the best sensitivity.
This experiment began with the measurement of the
gold layer in the dry state. Afterwards the glass
slides were plasma coated. A further measurement in
the dry state was applied to get the exact thickness of
the nanofilm. A prerun with buffer solution was
carried out for 45 min to reach equilibrium. After
flushing with deionized water and a drying by
nitrogen the individual layer thickness was
measured. Thereupon the adsorption measurement
begins with flushing with bufferthen with proteincontaining buffer and with buffer again to remove
unbound protein. Each of the three steps lasted 20
min. The intensity change of the reflectivity was
recorded. Another deionized water flushing and
nitrogen drying made the sample ready for the final
angle-reflectivity measurement.
2.3 Bacterial Adsorption Tested With Fluorescent
A test-strain E. coli XL1 was used, which is
transformed with a GFP (green fluorescent protein) cassette, excitable with blue light. Bacteria were
grown in LB-medium with 100 µg/ml ampicillin.
The addition of 0.5 % arabinose activated the GFPvector making the bacteria visible. The samples were
incubated in sterile Falcon-tubes for 24 h at 37 °C
with 100 rpm. A washing procedure was applied to
remove nonadherent bacteria from the surface.
Samples were dried and investigated in a fluorescent
microscope detecting the fluorescence of the GFP.
This method allows the inspection of the bacteria
which form the first layer, also called “first
colonizers”. For a quantitative analysis the pictures
were analyzed by ImageJ, which allows counting of
adherent bacteria. For statistically more confident
data at least five different spots on the surface were
investigated.
3. Results
3.1 Protein-Surface Interaction
The protein-surface interaction was tested using
Surface Plasmon Resonance. Two proteins with
different behavior were used to determine the
interaction between the proteins and the nanofilm
coating.
The first tests were performed with lysozyme which
is a globular, cationic protein with 129 amino acids
and a mass of 14 kDa. This protein plays an
important role in the field of contact lenses (CL),
because it adsorbs on CL material brought into
contact with the tear fluid. The lysozyme
concentration was held at 2 mg/ml, which is
consistent with the concentration in tear fluid.
The second protein tested is fibrinogen which is
categorized between linear and globular proteins.
The length is 3410 amino acids and it has a mass of
340 kDa. Fibrinogen is used in many studies as a
reference for biocompatibility. It prefers to attach to
hydrophobic surfaces inducing in implants unwanted
inflammatory reactions at the surface.
In the next diagram a coating produced under a gas
ratio of 3:1 sccm (methane:oxygen) was
investigated. The layer thickness of the nanofilm is
varied between 12-36 nm and the thickness of
adsorbed protein layer is shown.
Figure 2. The adsorbed protein layer on three nanofilm
thicknesses and two references (gold, polystyrene) is shown. In
the case of lysozyme a reduction of adsorbed protein could be
demonstrated. The adsorption of fibrinogen could not be
avoided compared to the references
Hydrophobicity is often used to predict whether the
surface is repellent or attractive concerning protein
surface interaction. To enhance the nanofilm in case
of fibrinogen adsorption the ratio of the process
gases was changed. Higher oxygen concentrations
lead to more hydrophilic coatings. The different
deposition rates were figured out and the SPR
samples were coated with a constant thickness of 24
nm. Adsorption measurements were performed and
show the following result (Figure 3). Nanofilms
produced under a gas ratio of 2,5:1,25 sccm show no
fibrinogen adsorption.
Figure 3. The interaction of fibrinogen with different nanofilms
produced under different gas ratios is shown. The more oxygen
the coating contains, the lower is the fibrinogen interaction.
3.2 Bacteria-Surface Interaction
The antibacterial behavior of the nanocoating was
tested using E. coli GFP. Different types of materials
were coated and examinated: polystyrene,
polymethyl methacrylate, glass and silicon. These
samples were inspected after 24 h via fluorescent
microscopy. Figure 4 shows the immense reduction
of attached bacteria.
Acknowledgments
This study was supported by the Aif in a “Zentralen
Innovationsprogramm Mittelstand” (ZIM).
References
[1] Brian J. Nablo: Nitric oxide-releasing sol-gels
as antibacterial coatings for orthopedic implants.
Biomaterials, Volume 26, Issue 8
Figure 4. Bacteria adhesion on coated and uncoated substrates.
Average number of bacteria on inspected spots showing the
antibacterial behavior of the plasma nanofilm. A Reduction of
bacteria adhesion up to 99.9 % could be reached.
First experiments were performed to test the long
term performance of this coating. Thus coated and
a)
b)
uncoated samples were incubated for 32 days. An
inspection by light microscope showed huge
differences between the coated and uncoated
samples (Figure 5).
a)
b)
[2] Stefania Quintavalla: Antimicrobial food
packaging in meat industry. Elsevier, Meat Science
62 (2002) 373-380
[3] MW LeChevallier: Full-scale studies of factors
related to coliform regrowth in drinking water. Appl.
Environ. Microbiol. 07 1996, 2201-2211, Vol 62,
No. 7
[4] H. Yasuda: Biocompatibility of Nano-filmEncapsulated Silicone and Silicone-Hydrogel
contact lenses, Macromol. Bio-sciences 2006, 6,
121-138.
[5] Hirotsugu Yasuda: Economical Advantages of
Low-Pressure Plasma Polymerization Coating.
Plasma Process. Polym. 2005, 2, 507–512
Figure 5. Long term experiment with uncoated (a) and coated
(b) contact lens material analyzed with a light microscope
showing the reduction of adsorbed material
4. Conclusion
This study clearly shows the antibacterial behavior
of the plasma coating. Reduction in bacteria
adhesion up to 99.9 % could be reached, regarding
“first colonizers”. Even on different substrate
materials the coating was applicable with remarkable
results. Also the long term experiments showed a
promising achievement. In case of protein-surface
interaction the experiments showed the dependency
of the coating composition in matters of protein
interaction.