Using nature's genius for pollutant detection

In all environments single cell organisms such as bacteria are directly affected by changing and sometimes extreme environmental conditions. This includes not only pH, temperature and salinity but also the presence of toxic ions, compounds and complexes. It is thusly essential for these microorganisms to possess a robust and selectively permeable cell surface. For this purpose, many bacteria form a proteinaceous cell envelope, the so called surface layer (S-layer). This cell envelope has different functions in different organisms for example the binding of toxic metals and metalloids to protect cells from being damaged by these elements. On other cells, S-layers may act as immobilization matrix for exoenzymes, as molecular sieve or as ion and molecule trap or they protect the cell from being affected by other bacteria or by lytic enzymes. S-layers are composed of identical protein or glycoprotein monomers, which are able to self-assemble to highly ordered monomolecular layers. They form para-crystalline sheets in suspension, on interfaces and on surfaces. Furthermore, on the surface of such a layer different functional groups are available which can be modified without the loss of its structure. The protein layers are in general mechanically and chemically highly stable. These properties make S-layers very interesting building blocks for the construction of new bioinspired nanomaterials and nanocoatings. Using the two-dimensional protein arrays in combination with layer-by-layer technique different kinds of technical surfaces can be functionalized.
This technique is used to design a new kind of sensory layers which will allow detecting small amounts of analytes with high selectivity. This sensory device will consist of an S-layer coating, a selective receptor and two fluorescence dyes. Aptamers were used as compound specific receptors . These are short, single stranded nucleic acid oligomers that meet the requirements for a more selective and sensitive detection of pollutants in nature, medicine and industry. Aptamers are able to recognize almost all classes of substrates and bind them in analogy to antigen-antibody interactions. By using an in vitro selection and amplification technique aptamers can be developed for pollutants like heavy metals, pharmaceuticals but also for proteins and complex targets like viruses and microbial spores. Currently, several aptamers for different antibiotics have been selected and experiments confirmed their high selectivity for single antibiotics or groups of antibiotics. As third component two fluorescence dyes allowing a FRET as signal transducer system will be coupled onto the S-layer lattice. This setup can be combined in different ways to optimize the sensitivity and selectivity of the sensor. Comparable to the binding of exoenzymes on S-layer carrying cells the three sensor components can also be linked to S-layer coated technical surfaces. In first experiments we coupled the model aptamer, anti-thrombin-aptamer, on S-layer proteins and proved its functionality after being linked to the protein. Furthermore, we modified S-layer proteins with a FRET pair and proved the energy transfer between them. We used a FRET pair containing a green and red fluorescence dye and succeed to detect a FRET between the S-layer linked fluorescence dyes. The FRET efficiency was 40 %. Because of the regular arranged functional groups on the S-layer lattice, coupling of the sensor components can be done in a defined and reproducible way.
In further work we will combine all components, aptamers and fluorescence dyes, on the S layer proteins. The aptamer will bind the specific analyte, affecting also the fluorescence dyes and disturbing FRET because of their close proximity to each other. In result a sensory layer is developed which uses the high specificity of aptamers and fluorescence dyes for an easy detection due to an optical signal. Additionally, in the future other techniques such as phage surface display will be used to select also peptide based binding molecules. Hopefully, this will allow detecting even more and also other kinds of pollutants not being bound by aptamers.