IWC Annual Report 2008 - Institut für Wasserchemie und chemische ...

1.3.3 Analysis of biofilm matrix by Raman Microscopy and Surface Enhanced
Raman Scattering (SERS)
Funding: DFG
Cooperation: Prof. Horn, TUM
Biofilms present a ubiquitous form of microbial life in natural environment and
can occur at solid–liquid, solid–air, liquid–liquid, and liquid–air interfaces. They are
structured communities of microorganisms, which are embedded in a matrix formed
by extracellular polymeric substances (EPS, such as polysaccharides, proteins, nucleic
acids and lipids). Detailed information about chemical composition and structure of
the EPS matrix is relevant e.g. for the optimization of biocides, of antifouling strategies
and for biological wastewater treatment. Raman Microscopy (RM) is a nondestructive
spectroscopic analytical technique which is based on the effect of inelastic light
scattering by molecules. RM provides “whole-organism fingerprints” for the characterization
and identification of different biological systems with spatial resolution of
optical microscope. RM requires no or limited sample preparation. Raman spectra
are characterized by a high specificity, generally revealing sharper and clearer bands
than IR spectra, and low water background.
The study of multispecies biofilms showed that RM provides
detailed chemical information about different constituents
of a complex biofilm matrix, and can correlate
variations of the chemical composition to different structural
appearances within the EPS matrix. The results of
the RM analysis of biofilms are in good agreement with
data obtained by Confocal Laser Scanning Microscopy
(CLSM) that was applied to study the distribution of microorganisms
and EPS glycoconjugates in biofilm matrix.
However, the sensitivity of RM is limited. Thus, collection
times of minutes are needed to record spectra with a
high signal-to-noise ratio, even when applying high laser
power.
The Raman effect can be dramatically enhanced if a
molecule is attached or in the immediate proximity to
metallic substrate (usually Ag or Au). This technique is
known as SERS. We obtained reproducible SERS spectra
from different constituents (aggregates and protozoa) of
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SERS-Spectra of biofilm
multispecies biofilm. The use of colloidal silver nanoparticles for in situ SERS measurements
by RM allowed us to achieve an enhancement factor of up to 2 orders of
magnitude (see Figure). Comparison of normal Raman (NR) and SERS spectra, both
obtained with an excitation wavelength of 633 nm, revealed significant differences in
the positions and the relative intensities of the bands. The SERS spectra are characterized
by higher number of discriminable peaks. The good reproducibility of the SERS
spectra obtained from different biofilm constituents suggests a great potential of SERS
for a detailed and sensitive chemical characterization of different biofilm components
and the analysis of their relative abundance in the biofilm matrix.
Thus, RM and SERS can be efficient tools for a label-free chemical analysis of
biofilms. Moreover, the combination of RM/SERS with CLSM can provide new knowledge
about the complex structure/function-correlations in biofilms matrix.
(N. P. Ivleva)
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